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... transcriptional reprograming directed by H3K4me3 or H3K27me3 modifications has been demonstrated to enable fungal pathogens to coordinate growth, metabolism, and development, thereby adapting to various host or natural environments [10][11][12][13][14] . ...
... Previous studies revealed that H3K4me3 and H3K27me3 modifications in Fg were respectively catalyzed by the conserved histone methyltransferases Set1 and Kmt6 in Fg 11,14 . We, therefore, performed ChIP-qPCR assays to determine the occupancy of Set1 or Kmt6 at BCG1 gene loci during infection using PH-1::Set1-GFP and PH-1::Kmt6-GFP strains. ...
... This is the first report illustrating the biological function of bivalent modification in fungi, which represents an important epigenetic mechanism that modulates fungal pathogenesis via fine-tuning their gene expression. Moreover, as both H3K4me3 and H3K27me3 have also been detected in different fungal species [11][12][13][14] , it can be speculated that bivalent chromatin modificationbased host immune evasion is potentially conserved in different fungal pathogens, and efforts should be made in the future to identify bivalent domains and related features in other fungal pathosystems. ...
Bivalent histone modifications, including functionally opposite H3K4me3 and H3K27me3 marks simultaneously on the same nucleosome, control various cellular processes by fine-tuning the gene expression in eukaryotes. However, the role of bivalent histone modifications in fungal virulence remains elusive. By mapping the genome-wide landscape of H3K4me3 and H3K27me3 dynamic modifications in Fusarium graminearum (Fg) during invasion, we identify the infection-related bivalent chromatin-marked genes (BCGs). BCG1 gene, which encodes a secreted Fusarium-specific xylanase containing a G/Q-rich motif, displays the highest increase of bivalent modification during Fg infection. We report that the G/Q-rich motif of BCG1 is a stimulator of its xylanase activity and is essential for the full virulence of Fg. Intriguingly, this G/Q-rich motif is recognized by pattern-recognition receptors to trigger plant immunity. We discover that Fg employs H3K4me3 modification to induce BCG1 expression required for host cell wall degradation. After breaching the cell wall barrier, this active chromatin state is reset to bivalency by co-modifying with H3K27me3, which enables epigenetic silencing of BCG1 to escape from host immune surveillance. Collectively, our study highlights how fungal pathogens deploy bivalent epigenetic modification to achieve temporally-coordinated activation and suppression of a critical fungal gene, thereby facilitating successful infection and host immune evasion.
... As for other fungal pathogens, the Moset1 or Mrkmt2 deletion mutants exhibited severe defects in hyphal growth, conidiation, appressorium formation, and virulence of M. oryzae and M. robertsii, respectively (19,26). In F. graminearum and B. bassiana, Set1 is also required for hyphal growth and virulence and functions in conidiation and conidial quality control of B. bassiana (20,27). P. expansum infects host cells dependent on conidia and invasive hyphae without other special invasive structures. ...
... Likewise, B. bassiana Set1 modulated genes involved in cell The Regulatory Role of PeSet1 in P. expansum Microbiology Spectrum wall composition (b-1,3-glucan and chitin) and CWI pathway (Slt2-Mkk1-Bck1 cascade) (27). In contrast to the hypersensitivity of DPeSet1 to Congo red, deletion of Set1 in F. graminearum and F. verticillioides reduced sensitivity to Congo red, suggesting that Set1 modulates fungal CWI in diverse regulatory manners (20,22). Taken together, PeSet1 may mediate the expression of PeFks1, PeGel1, and PeGel5 to modulate b-1,3glucan biosynthesis and structure remodeling, thereby contributing to CWI and fungal cell survival. ...
... We found Set1 played a critical role in patulin production for the first time and proposed that PeSet1 may serve as a positive regulator of patulin cluster genes. Our results are consistent with the role of Set1 in the regulation of mycotoxin biosynthesis in F graminearum and A. flavus (20,24). Set1-mediated H3K4 methylation is associated with active chromatin in diverse eukaryotic organisms. ...
Fruit blue mold disease and patulin contamination caused by Penicillium expansum lead to huge economic losses and food safety concerns worldwide. Many genes have been proven to be involved in the regulation of pathogenic and toxigenic processes of P. expansum. Histone H3 lysine 4 (H3K4) methylation is well recognized for its association with chromatin regulation and gene transcription. However, it is not clear whether H3K4 methylation is related to infection and patulin biosynthesis in Penicillium. Here, we characterized PeSet1, which is responsible for H3K4me1/me2/me3 in P. expansum. The deletion of PeSet1 caused severe defects in hyphal growth, conidiation, colonization, patulin biosynthesis, and stress responses. Moreover, we demonstrated that PeSet1 is involved in the regulation of patulin biosynthesis by mediating the expression of patulin cluster genes and crucial global regulatory factors. Likewise, PeSet1 positively regulated key genes in β-1,3-glucan biosynthesis and the reactive oxygen species scavenging process to modulate cell wall integrity and oxidative stress responses, respectively. Collectively, we have proven for the first time the function of Set1 in patulin biosynthesis and the crucial role of Set1 in colonization and stress responses in P. expansum. IMPORTANCE Penicillium expansum is one of the most important plant fungal pathogens, which not only causes blue mold rot in various fruits, leading to huge decay losses, but also produces mycotoxin patulin, posing a threat to human health. Both pathogenesis and patulin biosynthesis in P. expansum are regulated by complex and sophisticated networks. We focused on the epigenetic modification and identified a conserved histone H3K4 methyltransferase PeSet1 in P. expansum. Our work revealed the important role of PeSet1 in growth, development, colonization, patulin production, and stress responses of P. expansum. In particular, we originally described the regulation of Set1 on patulin biosynthetic pathway. These findings will provide new targets for the prevention and control of blue mold disease and patulin contamination.
... Within the SET-domain, there are four major proteins, namely KMT-COMPASS, SET2, spCLRC/NcDCDC, and PRC2, catalysing the di-and trimethylation of H3K4, H3K36, H3K9, and H3K27, respectively [23]. H3K4me and H3K36me are considered as active marks for gene expression [67], contrasting with H3K9me3 [68] and H3K27me3 [69] labelling gene expression repression. In addition, H3K9me3 has been further identified as a mark of constitutive heterochromatin, principally associated with centromeric heterochomatin, and H3K27me3 as a mark of facultative heterochromatin [69]. ...
... H3K4me is typically described as an active gene mark. The reduction of H3K4me in F. graminearum leads to severe growth defects and decreased virulence [67]. In the ascomycete Colletotrichum higginsianum, the deletion of CclA required for the deposition of H3K4me3 leads to reduced mycelial growth, asexual sporulation, and spore germination, a decreased production of secondary metabolites production associated with a strongly reduced virulence on plants [79]. ...
... In F. graminearum and F. verticilloides, H3K4me has been identified mainly as a transcriptional activator of biosynthetic gene clusters. For instance, the loss of Set-1 in F. verticillioides and F. graminearum, involved in the deposition of H3K4me3, leads to a decrease of fumonisins and DON production, respectively, coupled with a downregulation of the expression of their respective biosynthetic gene clusters [67,90,91]. Contrasting with the steady state of active gene expression linked to H3K4me3, the lack of H3K4 methyltransferase CclA in the grass endophyte Epichloë festucae, in several Aspergillus species and in C. higginsianum leads to the induction of biosynthetic gene clusters [79,[92][93][94]. ...
Chromatin structure is a major regulator of DNA-associated processes, such as transcription, DNA repair, and replication. Histone post-translational modifications, or PTMs, play a key role on chromatin dynamics. PTMs are involved in a wide range of biological processes in eukaryotes, including fungal species. Their deposition/removal and their underlying functions have been extensively investigated in yeasts but much less in other fungi. Nonetheless, the major role of histone PTMs in regulating primary and secondary metabolisms of filamentous fungi, including human and plant pathogens, has been pinpointed. In this review, an overview of major identified PTMs and their respective functions in fungi is provided, with a focus on filamentous fungi when knowledge is available. To date, most of these studies investigated histone acetylations and methylations, but the development of new methodologies and technologies increasingly allows the wider exploration of other PTMs, such as phosphorylation, ubiquitylation, sumoylation, and acylation. Considering the increasing number of known PTMs and the full range of their possible interactions, investigations of the subsequent Histone Code, i.e., the biological consequence of the combinatorial language of all histone PTMs, from a functional point of view, are exponentially complex. Better knowledge about histone PTMs would make it possible to efficiently fight plant or human contamination, avoid the production of toxic secondary metabolites, or optimize the industrial biosynthesis of certain beneficial compounds.
... In the rice pathogen F. fujikuroi and the cereal pathogen F. graminearum, the Set1 orthologs FfSet1 and FgSet1, respectively, are detrimental for H3K4 di-and trimethylation, while remaining levels of H3K4 monomethylation (H3K4me1) were observed for both species [103,110]. SM biosynthesis was de-regulated in strains lacking the catalytic subunit or other COMPASS components, i.e., Bre2/Ccl1 or Sdc1, that are essential for wildtype H3K4me3 levels [110]. This is exemplified by the deletion of FfSET1, which revealed an increase in the biosynthesis of the two red pigments bikaverin (BIK) and fusarubins (FSR), as well as fusarin C (FUS) and, to some extent, also fusaric acid (FUB), while production of the plant hormone and virulence factor GA was completely abolished in axenic culture [103]. ...
... In the rice pathogen F. fujikuroi and the cereal pathogen F. graminearum, the Set1 orthologs FfSet1 and FgSet1, respectively, are detrimental for H3K4 di-and trimethylation, while remaining levels of H3K4 monomethylation (H3K4me1) were observed for both species [103,110]. SM biosynthesis was de-regulated in strains lacking the catalytic subunit or other COMPASS components, i.e., Bre2/Ccl1 or Sdc1, that are essential for wildtype H3K4me3 levels [110]. This is exemplified by the deletion of FfSET1, which revealed an increase in the biosynthesis of the two red pigments bikaverin (BIK) and fusarubins (FSR), as well as fusarin C (FUS) and, to some extent, also fusaric acid (FUB), while production of the plant hormone and virulence factor GA was completely abolished in axenic culture [103]. ...
... Similarly, in F. graminearum, the lack of FgSet1 and associated COMPASS components, i.e., FgBre2 or FgSdc1, led to an impaired DON biosynthesis. Again, ChIP-qPCR revealed drastically reduced levels of H3K4me2 and decreased levels of me1-and me3 at the TRI cluster genes in ∆fgset1 [110]. Next to DON, biosynthesis of the red pigment aurofusarin (AUR) was impaired upon loss of FgSet1 accompanied by reduced H3K4me2 levels at AUR cluster genes [110]. ...
Fusarium is a species-rich group of mycotoxigenic plant pathogens that ranks as one of the most economically important fungal genera in the world. During growth and infection, they are able to produce a vast spectrum of low-molecular-weight compounds, so-called secondary metab-olites (SMs). SMs often comprise toxic compounds (i.e., mycotoxins) that contaminate precious food and feed sources and cause adverse health effects in humans and livestock. In this context, understanding the regulation of their biosynthesis is crucial for the development of cropping strategies that aim at minimizing mycotoxin contamination in the field. Nevertheless, currently, only a fraction of SMs have been identified, and even fewer are considered for regular monitoring by regulatory authorities. Limitations to exploit their full chemical potential arise from the fact that the genes involved in their biosynthesis are often silent under standard laboratory conditions and only induced upon specific stimuli mimicking natural conditions in which biosynthesis of the respective SM becomes advantageous for the producer. This implies a complex regulatory network. Several components of these gene networks have been studied in the past, thereby greatly advancing the understanding of SM gene regulation and mycotoxin biosynthesis in general. This review aims at summarizing the latest advances in SM research in these notorious plant pathogens with a focus on chromatin structure. Key Contribution: This review summarizes the latest advances on chromatin regulatory mechanisms with a focus on the expression of genes involved in fungal secondary metabolite (SM) biosynthesis, including potent mycotoxins in the genus Fusarium.
... A previous study revealed that the deletion of Set1, the only H3K4 methyltransferase in C. albicans, attenuated its virulence in mice [15]. H3K4 methyltransferase-deletion in other pathogenic fungi has also been shown to attenuate the virulence [16][17][18][19][20][21]. H3K4 methyltransferases positively regulate the expression of secondary metabolite genes in some fungal pathogens, thereby contributing to their virulence [17,20]; however, the mechanisms linking the effect of Set1 on virulence to transcriptional regulation by Set1mediated H3K4 methylation in C. albicans remain unclear. ...
... For instance, Set1 has been shown to be involved in the virulence of the plant pathogen Fusarium verticillioides by regulating the expression of fumonisin B1 toxin-encoded genes [17], whereas Set1-mediated H3K4 methylation activates the expression of TR1, which encodes the toxin deoxynivalenol (DON), in the plant fungal pathogen Fusarium graminearum [20]. In addition, Set1 is involved in Magnaporthe oryzae fungal virulence by activating virulence-related genes via H3K4 methylation [21], while an H3K4 methyltransferase is required to induce genes in the entomopathogenic fungus Metarhizium robertsii under host conditions and induce virulence to mosquito infection [19]. Importantly, C. albicans is the only example that the virulent effects of Set1 have been reported in a human pathogen [15]. ...
... H3K4me3 is a marker of active transcription that is enriched in actively transcribed genes; however, we found that the absence of SET1 did not change 97% of the total gene expression (Figure 1(a-b)). Similarly, recent genome-wide studies revealed that the absence of H3K4 methyltransferase did not dramatically change the overall gene expression in other fungal organisms, such as S. cerevisiae [46,47], M. oryzae [21], and Fusarium fujikuroi [18]. Because we observed that the expression of some genes increased in the absence of H3K4 methyltransferase, it is difficult to assume that the role of H3K4 methylation simply correlates with active transcription. ...
Candida albicans is an opportunistic human fungal pathogen that exists in normal flora but can cause infection in immunocompromised individuals. The transition to pathogenic C. albicans requires a change of various gene expressions. Because histone-modifying enzymes can regulate gene expression, they are thought to control the virulence of C. albicans. Indeed, the absence of H3 lysine 4 (H3K4) methyltransferase Set1 has been shown to reduce the virulence of C. albicans; however, Set1-regulated genes responsible for this attenuated virulence phenotype remain unknown. Here, we demonstrated that Set1 positively regulates the expression of mitochondrial protein genes by methylating H3K4. In particular, levels of cellular mitochondrial reactive oxygen species (ROS) were higher in Δset1 than in the wild-type due to the defect of those genes’ expression. Set1 deletion also increases H2O2 sensitivity and prevents proper colony formation when interacting with macrophage in vitro, consistent with its attenuated virulence in vivo. Together, these findings suggest that Set1 is required to regulate proper cellular ROS production by positively regulating the expression of mitochondrial protein genes and subsequently sustaining mitochondrial membrane integrity. Consequently, C. albicans maintains proper ROS levels via Set1-mediated transcriptional regulation, thus establishing a rapid defense against external ROS generated by the host.
... Some quantitative PCR-coupled chromatin immunoprecipitation (ChIP) experiments also proved that Ccl1 regulates balance between H3K4me2 and H3K4me3 independently of the transcriptional status of secondary metabolite genes (Shinohara et al., 2015;Studt et al., 2016Studt et al., , 2017. FgSet1 is a Set1 orthologue of Fusarium graminearum and is one subunit of the COMPASS-like complex, which also plays an important role in regulating fungal growth and secondary metabolism (Bachleitner et al., 2019;Connolly et al., 2013;Liu et al., 2015). ...
... In F. graminearum, FgSet1 physically interacts with multiple proteins, including FgBre2, FgSpp1, and FgSwd2. FgBre2 further interacts with FgSdc1 (Liu et al., 2015). Our biochemical studies have demonstrated that the M. oryzae complexes are very similar to their mammalian counterparts in terms of subunit composition. ...
... and Fusarium spp., respectively, resulted in hyphal growth defects, cell wall stress, and virulence reduction. The Δfgset1 strains were restricted to the inoculated spikelets after point inoculation (Liu et al., 2015;Studt et al., 2017). ...
Histone-3-lysine-4 (H3K4) methylation is catalysed by the multiprotein complex known as the Set1/COMPASS or MLL/COMPASS-like complex, an element that is highly evolutionarily conserved from yeast to humans. However, the components and mechanisms by which the COMPASS-like complex targets the H3K4 methylation of plant-pathogenic genes in fungi remain elusive. Here we present a comprehensive analysis combining biochemical, molecular, and genome-wide approaches to characterize the roles of the COMPASS-like family in the rice blast fungus Magnaporthe oryzae, a model plant pathogen. We purified and identified six conserved subunits of COMPASS from M. oryzae: MoBre2 (Cps60/ASH2L), MoSpp1 (Cps40/Cfp1), MoSwd2 (Cps35), MoSdc1 (Cps25/DPY30), MoSet1 (MLL/ALL), and MoRbBP5 (Cps50), using an affinity tag on MoBre2. We determined the sequence repeat in dual-specificity kinase splA and ryanodine receptors domain of MoBre2 can interact directly with the DPY30 domain of MoSdc1 in vitro. Furthermore, we found that deletion of the genes encoding COMPASS subunits of MoBre2, MoSPP1, and MoSwd2 caused similar defects regarding invasive hyphal development and pathogenicity. Genome-wide profiling of H3K4me3 revealed that it has remarkable co-occupancy at the transcription start site regions of target genes. Significantly, these target genes are often involved in spore germination and pathogenesis. Decreased gene expression caused by the deletion of MoBre2, MoSwd2, or MoSpp1 was highly correlated with a decrease in H3K4me3. These results suggest that MoBre2, MoSpp1, and MoSwd2 function as a whole COMPASS complex, contributing to fungal development and pathogenesis by regulating H3K4me3-targeted genes in M. oryzae.
... However, the ubiquitin ligase E3′s role in virulence regulation varies and is dependent on different pathways in diverse pathogenic fungi. For instance, ubiquitin ligase E3 is involved in virulence regulation via appressorium development in M. oryzae [9], whereas in F. graminearum, it regulates secondary metabolism [33]. Our present study emphasizes the identification of two ubiquitin ligases E3, namely, VdBre1 and VdHrd1, in relation to the pathogenicity of V. dahliae. ...
... However, the ubiquitin ligase E3 s role in virulence regulation varies and is dependent on different pathways in diverse pathogenic fungi. For instance, ubiquitin ligase E3 is involved in virulence regulation via appressorium development in M. oryzae [9], whereas in F. graminearum, it regulates secondary metabolism [33]. Our present study emphasizes the identification of two ubiquitin ligases E3, namely, VdBre1 and VdHrd1, in relation to the pathogenicity of V. dahliae. ...
Verticillium dahliae, a virulent soil-borne fungus, elicits Verticillium wilt in numerous dicotyledonous plants through intricate pathogenic mechanisms. Ubiquitination, an evolutionarily conserved post-translational modification, marks and labels proteins for degradation, thereby maintaining cellular homeostasis. Within the ubiquitination cascade, ubiquitin ligase E3 demonstrates a unique capability for target protein recognition, a function often implicated in phytopathogenic virulence. Our research indicates that two ubiquitin ligase E3s, VdBre1 and VdHrd1, are intrinsically associated with virulence. Our findings demonstrate that the deletion of these two genes significantly impairs the ability of V. dahliae to colonize the vascular bundles of plants and to form typical penetration pegs. Furthermore, transcriptomic analysis suggests that VdBre1 governs the lipid metabolism pathway, while VdHrd1 participates in endoplasmic-reticulum-related processes. Western blot analyses reveal a significant decrease in histone ubiquitination and histone H3K4 trimethylation levels in the ΔVdBre1 mutant. This research illuminates the function of ubiquitin ligase E3 in V. dahliae and offers fresh theoretical perspectives. Our research identifies two novel virulence-related genes and partially explicates their roles in virulence-associated structures and gene regulatory pathways. These findings augment our understanding of the molecular mechanisms inherent to V. dahliae.
... In Alternaria alternata, one of the most serious phytopathogenic fungi, AaSet1 also plays critical roles in cell development and is involved in the adaptation to cell wall interference agents and osmotic stress [17]. In Fusarium graminearum, FgSet1 is not only predominantly responsible for H3K4me, but it also plays an important role in response to cell wall-damaging agents via negatively regulating phosphorylation of FgMgv1, a core kinase in the cell wall integrity pathway [18]. FgSet1 has also been shown to associate components of the deoxynivalenol and aurofusarin biosynthesis pathways, indicating a regulatory role of COMPASS-mediated H3K4 methylation in secondary metabolism [18]. ...
... In Fusarium graminearum, FgSet1 is not only predominantly responsible for H3K4me, but it also plays an important role in response to cell wall-damaging agents via negatively regulating phosphorylation of FgMgv1, a core kinase in the cell wall integrity pathway [18]. FgSet1 has also been shown to associate components of the deoxynivalenol and aurofusarin biosynthesis pathways, indicating a regulatory role of COMPASS-mediated H3K4 methylation in secondary metabolism [18]. Deletion of COMPASS subunits in various Aspergillus species also leads to changes in the biosynthesis of several secondary metabolites [19][20][21][22]. ...
The Complex of Proteins Associated with Set1 (COMPASS) methylates lysine K4 on histone H3 (H3K4) and is conserved from yeast to humans. Its subunits and regulatory roles in the meningitis-causing fungal pathogen Cryptococcus neoformans remain unknown. Here we identified the core subunits of the COMPASS complex in C. neoformans and C. deneoformans and confirmed their conserved roles in H3K4 methylation. Through AlphaFold modeling, we found that Set1, Bre2, Swd1, and Swd3 form the catalytic core of the COMPASS complex and regulate the cryptococcal yeast-to-hypha transition, thermal tolerance, and virulence. The COMPASS complex-mediated histone H3K4 methylation requires H2B mono-ubiquitination by Rad6/Bre1 and the Paf1 complex in order to activate the expression of genes specific for the yeast-to-hypha transition in C. deneoformans. Taken together, our findings demonstrate that putative COMPASS subunits function as a unified complex, contributing to cryptococcal development and virulence.
... Lysine methylation has been associated with virulence of some protozoa and fungi [7,8,22,[25][26][27]. Thus, to investigate whether EhPKMT2 participates in the virulence of E. histolytica, we decided to analyze its expression and cellular localization in two events related to the virulence of this parasite: heat shock and phagocytosis [11,28]. ...
... It has also been shown that lysine methylation and, consequently, PKMTs of fungiand protozoa-parasites play an important role in many cellular processes of these microorganisms, including virulence [7,8,22,[25][26][27]. In blood stages of Plasmodiun falciparum, 570 lysine-methylated proteins were identified, indicating that this PTM is widespread in this pathogen [31]. ...
Lysine methylation, a posttranslational modification catalyzed by protein lysine methyltransferases (PKMTs), is involved in epigenetics and several signaling pathways, including cell growth, cell migration and stress response, which in turn may participate in virulence of protozoa parasites. Entamoeba histolytica, the etiologic agent of human amebiasis, has four PKMTs (EhPKMT1 to EhPKMT4), but their role in parasite biology is unknown. Here, to obtain insight into the role of EhPKMT2, we analyzed its expression level and localization in trophozoites subjected to heat shock and during phagocytosis, two events that are related to amoeba virulence. Moreover, the effect of EhPKMT2 knockdown on those activities and on cell growth, migration and cytopathic effect was investigated. The results indicate that this enzyme participates in all these cellular events, suggesting that it could be a potential target for development of novel therapeutic strategies against amebiasis.
... Methyltransferase complex Set1/COMPASS has been found to catalyze H3K4 methylation in Saccharomyces cerevisiae [137]. Elimination of the histone modification by disrupting Set1 abolished DON production in F. graminearum, with drastically decreasing the transcription levels of 8 TRI genes, including TRI6 and TRI10 [62]. Other two subunits involved in Set1/COMPASS, Bre2 and Sdc1, have been shown to physically interact with Set1 in regulating TRI genes [62]. ...
... Elimination of the histone modification by disrupting Set1 abolished DON production in F. graminearum, with drastically decreasing the transcription levels of 8 TRI genes, including TRI6 and TRI10 [62]. Other two subunits involved in Set1/COMPASS, Bre2 and Sdc1, have been shown to physically interact with Set1 in regulating TRI genes [62]. The SAGA/ADA complex is responsible for the acetylation of H3K9, H3K18 and H3K27, and is also implicated in a regulatory role in DON induction [63]. ...
Mycotoxin contamination in food poses health hazards to humans. Current methods of controlling mycotoxins still have limitations and more effective approaches are needed. During the past decades of years, variable environmental factors have been tested for their influence on mycotoxin production leading to elucidation of a complex regulatory network involved in mycotoxin biosynthesis. These regulators are putative targets for screening molecules that could inhibit mycotoxin synthesis. Here, we summarize the regulatory mechanisms of hierarchical regulators, including pathway-specific regulators, global regulators and epigenetic regulators, on the production of the most critical mycotoxins (aflatoxins, patulin, citrinin, trichothecenes and fumonisins). Future studies on regulation of mycotoxins will provide valuable knowledge for exploring novel methods to inhibit mycotoxin biosynthesis in a more efficient way.
... Transformants were grown on synthetic medium (SD) lacking Leu and Trp medium for 4 days, and then transferred to SD/−Ade−Leu− Trp−His medium and grown for 4 days at 30°C. At least three independent experiments were performed to confirm Y2H assay results (Liu et al., 2015). ...
... Protein eluted from agarose was analysed by western blotting with anti-GFP antibodies. The protein samples were also detected with monoclonal anti-actin antibody (ABclonal Technology) as a control (Liu et al., 2015). ...
The basal transcription factor II H (TFIIH) is a multicomponent complex. In the present study, we characterized a TFIIH subunit Tfb5 by analysing loss- and gain-of-function mutants to gain a better understanding of the molecular mechanisms underlying stress resistance and pathogenicity in the citrus fungal pathogen Alternaria alternata. Tfb5 deficiency mutants (ΔAatfb5) decreased sporulation and pigmentation, and were impaired in the maintenance of colony surface hydrophobicity and cell wall integrity. ΔAatfb5 increased sensitivity to ultraviolet light, DNA-damaging agents, and oxidants. The expression of Aatfb5 was up-regulated in the wild type upon infection in citrus leaves, implicating the requirement of Aatfb5 in fungal pathogenesis. Biochemical and virulence assays revealed that ΔAatfb5 was defective in toxin production and cellwall-degrading enzymes, and failed to induce necrotic lesions on detached citrus leaves. Aatfb5 fused with green fluorescent protein (GFP) was localized in the cytoplasm and nucleus and physically interacted with another subunit, Tfb2, based on yeast two-hybrid and co-immunoprecipitation analyses. Transcriptome and Antibiotics & Secondary Metabolite Analysis Shell (antiSMASH) analyses revealed the positive and negative roles of Aatfb5 in the production of various secondary metabolites and in the regulation of many metabolic and biosynthetic processes in A. alternata. Aatfb5 may play a negative role in oxidative phosphorylation and a positive role in peroxisome biosynthesis. Two cutinase-coding genes (AaCut2 and AaCut15) required for full virulence were down-regulated in ΔAatfb5. Overall, this study expands our understanding of how A. alternata uses the basal transcription factor to deal with stress and achieve successful infection in the plant host.
... This approach can help The use of color as alternative to size measurements in Fusarium graminearum growth studies and prediction of deoxynivalenol synthesis 20 overcome spatial constraints in fungal studies or applications, such as the limited size or particular shape of a Petri dish or bioreactor, or even predict toxin production through sole analysis of the mold surface color. For instance, mutants lacking in aurofusarin seem to produce an increased amount of ZEA [53] , and histone H3 lysine 4 methylation (H3K4me) is important for the transcription of genes required for the biosynthesis of both DON and aurofusarin [66]. Thus, there is some correlation between the production of major Fusarium mycotoxins and pigments. ...
... Limited but convincing literature is available regarding the relationship between pigmentation and mycotoxins. For instance, the genetic and biosynthetic origins of aurofusarin and both DON and ZEA have been previously described [53,66]. As mentioned earlier, Malz, et al. [53] demonstrated that mutants for the gene pks12, unable to produce aurofusarin, produced an increased quantity of ZEA. ...
Fusarium graminearum Schwabe is a fungal plant parasite responsible for head blight (FHB) in cereals. This mold also synthesizes deoxynivalenol (DON), an emetic mycotoxin responsible for at least eight outbreaks since the beginning of the 20th century in Japan, including Hokkaido. The toxin is also a major problem all over the world. Although size-based variables (e.g. radius, diameter) represent fungal growth in the recently used models, they do not provide important growth information such as metabolic activity or state of maturity, especially during the stationary phase. Changes in the color during fungal growth would be suitable for the purpose, because the changes in color are a product of the mold’s metabolism, changing even when the fungus is no longer expanding in the substrate. The present study aimed to analyze if F. graminearum surface colors are suitable as alternative to size in studies of growth and DON synthesis. The present studies demonstrated that changes in the color of fungal metabolites will be appropriate as an alternative for size variables in F. graminearum growth studies and prediction of synthesis of DON quantity．
... Protein samples were mixed with 5 × loading buffer and boiled for 5 min. The resulting protein samples were separated by 12 % SDS-PAGE gel and transferred onto a PVDF membrane using a Bio-Rad electroblotting apparatus (Liu et al., 2015;Ma et al., 2016). The monoclonal anti-GFP (Sigma-Aldrich, St. Louis, USA) antibodies were used as the primary antibody and goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Vazyme, Nanjing, China) was used the secondary antibody. ...
... The pGBKT 7 -LAM paired with pGADT 7 was used as a negative control and pGBKT 7 -53 paired with pGADT 7 was used as a positive control. Y2H assays were performed as described (Liu et al., 2015). At least three independent experiments were performed to confirm the Y2H assay results. ...
Subtilisin-like serine protease secreted by pathogenic fungi can facilitate the infection and acquisition of nutrients. Functions of subtilisin-like serine proteases in the phytopathogenic fungus Alternaria alternata remains unknown. In the current study, 15 subtilisin-like serine proteases were individually deleted in the citrus fungal pathogen A. alternata. Only one, designated AaPrb1, was found to be required for A. alternata pathogenesis. The AaPrb1 deficiency strain (ΔAaprb1) reduced growth, conidiation, the formation of aerial hyphae, protease production, and virulence on citrus leaves. However, biochemical analyses and bioassays revealed that ΔAaprb1 plays no role in the production of ACT toxin. Through Y2H assays, Aaprb1 was found to interact with Aapep4, a vacuole-localized proteinase A in A. alternata. Furthermore, silencing AaPep4 in A. alternata resulted in phenotypes similar with those of ΔAaprb1. Expression of AaPrb1 was found to be regulated by AaPep4. TEM showed that AaPrb1and AaPep4 were involved in the suppression of the degradation of autophagosomes. Deletion of the autophagy gene AaAtg8 in A. alternata decreased conidiation, the formation of aerial hyphae and pathogenicity similar to ΔAaprb1, implying that some phenotypes of ΔAaprb1 were due to the impairment of autophagy. Overall, this study expands our understanding of how A. alternata utilizes the subtilisin-like serine protease to achieve successful infection in the plant host.
... Studies on Fusarium species revealed several findings such as methylation of fourth lysine residue of H3 acts as a transcriptional activator for BGCs. Moreover, the knockout mutant of histone methyl transferase encoded gene, Set1, led to repress fumonisins and deoxynivalenol (DON) synthesis in Fusarium species (Gu et al. 2017;Liu et al. 2015;Studt et al. 2017). Interestingly, the loss of Set1 increased the bioavailability of fusarins and bikaervin in F. fujikuroi (Studt et al. 2017 (Hueza et al. 2014;Kong et al. 2018). ...
The microbial flora has always been a center of interest for the identification of novel bioactive secondary metabolites (SMs) used against a diversity of infections and ailments. These bioactive compounds are produced by the hosts in response to different stress conditions. Novobiocin naturally synthesized in Streptomyces spheroids, Echinocandin B from Emericella rugulosa, Penicillin from Penicillium chrysogenum, and Gentamycin from Micromonospora purpurea are such examples. In nature, the secondary metabolites genes, either cryptic or microbial bioactive compounds, synthesize in trace amounts under specific environmental conditions, physiological conditions which make their bioavailability scarcer. Different strategies such as induced stress stimuli, cloning the genes under the control of strong promoters, downregulating the expression of negative regulators, epigenetic manipulations, etc. have been tried to increase SM bioavailability. The posttranslational modifications of histones proteins render to regulate the secondary metabolites and have also been implied. Here, in this chapter, we emphasized the epigenetic regulation for enhancement of the production of therapeutically significant bioactive compounds in fungi. DNA wrapped around the histones proteins undergo different posttranslational modifications that facilitate chromatin DNA packaging around the histones proteins either loose (euchromatin) or tight (heterochromatin). Such alterations in the histones proteins epigenetically modulate the expression of genes that are responsible for regulating different metabolic events. Relevance of different epigenetic approaches such as the use of Histone deacetylase inhibitors (HDACs) or DNA methyl transferase inhibitors (DNMTs) concern different fungal bioactive compounds. These modifications could be intrinsically programmed or affected externally. Different studies have provided supportive arguments about the use of epigenetic modifiers for increasing the bioavailability of different fungal bioactive compounds. These epigenetic modifiers either act locally or globally for inducing or repressing the secondary metabolites synthesis genes. Different epigenetic modifiers have been earlier studied for the modulation of secondary metabolite synthesis in fungi. For instance, 5-azacytidine, hydralazine hydrochloride, trichostatin A, trapoxin B, etc. The chapter elaborates on a more developed understanding of histone modifiers for activation/induction of secondary metabolites yield of pharmacologically significant fungal-derived bioactive compounds.
... The preparation of protein samples was performed using previously reported methods [44]. For about 80 µL protein samples, a quarter volume of 5× loading buffer was added, and then, it was boiled for ten minutes. ...
ATP synthase catalyzes the synthesis of ATP by consuming the proton electrochemical gradient, which is essential for maintaining the life activity of organisms. The peripheral stalk belongs to ATP synthase and plays an important supporting role in the structure of ATP synthase, but their regulation in filamentous fungi are not yet known. Here, we characterized the subunits of the peripheral stalk, FfATPh, FfATP5, and FfATPb, and explored their functions on development and pathogenicity of Fusarium Fujikuroi. The FfATPh, FfATP5, and FfATPb deletion mutations (∆FfATPh, ∆FfATP5, and ∆FfATPb) presented deficiencies in vegetative growth, sporulation, and pathogenicity. The sensitivity of ∆FfATPh, ∆FfATP5, and ∆FfATPb to fludioxonil, phenamacril, pyraclostrobine, and fluazinam decreased. In addition, ∆FfATPh exhibited decreased sensitivity to ionic stress and osmotic stress, and ∆FfATPb and ∆FfATP5 were more sensitive to oxidative stress. FfATPh, FfATP5, and FfATPb were located on the mitochondria, and ∆FfATPh, ∆FfATPb, and ∆FfATP5 disrupted mitochondrial location. Furthermore, we demonstrated the interaction among FfATPh, FfATP5, and FfATPb by Bimolecular Fluorescent Complimentary (BiFC) analysis. In conclusion, FfATPh, FfATP5, and FfATPb participated in regulating development, pathogenicity, and sensitivity to fungicides and stress factors in F. fujikuroi.
... Transcriptionally active euchromatin is frequently enriched in histones that are methylated at lysine 4 (H3K4me) (Gates et al. 2017). Deletion of histone methyltransferases in F. graminearum, Colletotrichum fructicola, and M. oryzae led to the loss of H3K4me2/me3 (i.e., fourth lysine with di-or tri-methylation), defects in growth, reduced spore production, decreased metabolism, and virulence (Liu et al. 2015;Zhou et al. 2021;Gao et al. 2022). H3K9me3 is frequently found in transcriptionally silent heterochromatin such as the centromeres and repeat-rich compartments and its disruption is associated with changes in the development and regulation of metabolism in fungal plant pathogens, including L. maculans, Fusarium verticillioides, Fusarium mangiferae, a or V. dahliae Gu et al. 2017;Gates et al. 2017;Atanasoff-Kardjalieff et al. 2021;Kramer et al. 2022). ...
Plant pathogenic fungi have emerged as major concerns for plant health in agriculture and occupy important niches in natural ecosystems. Pathogen species are often highly diverse and respond rapidly to external factors. Our understanding of how this diverse group of fungi interacts with hosts and the environment has been critically advanced by analyses of fungal genomes. Here, we provide an overview of recent research into surveys of genomic diversity within species and review drivers of diversification including transposable elements. Analyses of epigenetic modifications of the genome have revealed important links to pathogenicity factors and genomic defense mechanisms of the genome. We also review population genomics analyses focused on short-term dynamics in plant pathogens such as recombination, genetic exchange, host adaptation, and the genetic basis of phenotypic traits.KeywordsPlant pathogensAgricultureGenome analysesStructural variationTransposable elementsPopulation geneticsPhenotypic traitsGenome-wide association mapping
... Deletion of SET1 and other components caused similar defects regarding invasive hyphal development and pathogenicity, as H3K4me3-marked genes are often involved in spore germination and pathogenesis in M. oryzae [144,175]. In Fusarium graminearum, Set1, together with Bre2, SPP1 and Swd2, mediates H3K4 methylation, which plays critical roles not only in the regulation of fungal growth and secondary metabolism but also in multiple stress responses [176]. The transcription factor AreA regulates putrescine-mediated transcription of TRIs by facilitating the enrichment of H3K4 me2/3 and histone H2B monoubiquitination (H2B ub1) on TRIs, whereas H2B ub1 regulates H3K4 me2/3 via the COMPASS component FgBre2 in F. graminearum [177]. ...
As a sessile organism, plants have evolved a complex and sophisticated immune system to defend against various pathogenic microbes effectively. However, microbes have also developed complicated and delicate strategies to suppress host immunity and successfully colonize the host. Dynamic plant‒pathogen interactions require rapid and fine-tuned regulation of their gene expression. Increasing evidence has revealed that epigenetic regulation plays key roles in plant defense-related transcriptional reprogramming, as well as microbe pathogenicity. In this review, we summarize and highlight the current progress in understanding the roles of epigenetic regulation and factors, including DNA/RNA modification, histone modification, chromatin remodeling and noncoding RNAs, in plant immunity, phytopathogen pathogenicity and their interactions. We also discuss that epigenetic regulation emerges as an efficient strategy for crop breeding and plant disease control.
... KEGG analysis found that lysine-succinylated proteins were enriched primarily on carbon metabolism, glyoxylic acid, and dicarboxylic acid. Many photosynthesis-related succinylation sites and proteins were also detected in many plants, suggesting that lysine succinylation can regulate the activity of photosynthesis-related enzymes [15,[36][37][38][39]. Additionally, succinylated proteins were found in mitochondria, cytoplasm, and chloroplasts, indicating their potential role in energy metabolism. ...
Danshen, belongs to the Lamiaceae family, and its scientific name is Salvia miltiorrhiza Bunge. It is a valuable medicinal plant to prevent and treat cardiovascular and cerebrovascular diseases. Lysine succinylation, a widespread modification found in various organisms, plays a critical role in regulating secondary metabolism in plants. The hairy roots of Salvia miltiorrhiza were subject to proteomic analysis to identify lysine succinylation sites using affinity purification and HPLC-MS/MS in this investigation. Our findings reveal 566 lysine succinylation sites in 348 protein sequences. We observed 110 succinylated proteins related to secondary metabolism, totaling 210 modification sites. Our analysis identified 53 types of enzymes among the succinylated proteins, including phenylalanine ammonia-lyase (PAL) and aldehyde dehydrogenase (ALDH). PAL, a crucial enzyme involved in the biosynthesis of rosmarinic acid and flavonoids, displayed succinylation at two sites. ALDH, which participates in the phenylpropane metabolic pathway, was succinylated at 8 eight sites. These observations suggest that lysine succinylation may play a vital role in regulating the production of secondary metabolites in Salvia miltiorrhiza. Our study may provide valuable insights for further investigation on plant succinylation, specifically as a reference point. SIGNIFICANCE: Salvia miltiorrhiza Bunge is a valuable medicinal plant that prevents and treats cardiovascular and cerebrovascular diseases. Lysine succinylation plays a critical role in regulating secondary metabolism in plants. The hairy roots of Salvia miltiorrhiza were subject to proteomic analysis to identify lysine succinylation sites using affinity purification and HPLC-MS/MS in this investigation. These observations suggest that lysine succinylation may act as a vital role in regulating the production of secondary metabolites in Salvia miltiorrhiza. Our study may provide valuable insights for further investigation on succinylation in plants, specifically as a reference point.
... The inactivation of specific epigenetic regulators, deacetylases, and histone methyltransferases has been reported as an effective approach to uncover new compounds in fungi (Bok et al. 2009;Palmer et al. 2013;Liu et al. 2015b;Mao et al. 2015;Shinohara et al. 2016). Wu et al., (2016) successfully identified 15 new polyketides (135-149) by deleting the histone methyltransferase PfCclA, and a deacetylase PfHdaA in the plant endophytic fungus Pestalotiopsis fici, and the biosynthetic origin of these macrolide skeletons was identified by isotope-labeling experiments. ...
Strains of the fungal genus Pestalotiopsis are reported as large promising sources of structurally varied biologically active metabolites. Many bioactive secondary metabolites with diverse structural features have been derived from Pestalotiopsis. Moreover, some of these compounds can potentially be developed into lead compounds. Herein, we have systematically reviewed the chemical constituents and bioactivities of the fungal genus Pestalotiopsis, covering a period ranging from January 2016 to December 2022. As many as 307 compounds, including terpenoids, coumarins, lactones, polyketides, and alkaloids, were isolated during this period. Furthermore, for the benefit of readers, the biosynthesis and potential medicinal value of these new compounds are also discussed in this review. Finally, the perspectives and directions for future research and the potential applications of the new compounds are summarized in various tables.
... Transcriptionally active euchromatin is frequently enriched in histones that are methylated at lysine 4 (H3K4me) (Gates et al. 2017). Deletion of histone methyltransferases in F. graminearum, Colletotrichum fructicola, and M. oryzae led to the loss of H3K4me2/me3 (i.e., fourth lysine with di-or tri-methylation), defects in growth, reduced spore production, decreased metabolism, and virulence (Liu et al. 2015;Zhou et al. 2021;Gao et al. 2022). H3K9me3 is frequently found in transcriptionally silent heterochromatin such as the centromeres and repeat-rich compartments and its disruption is associated with changes in the development and regulation of metabolism in fungal plant pathogens, including L. maculans, Fusarium verticillioides, Fusarium mangiferae, a or V. dahliae Gu et al. 2017;Gates et al. 2017;Atanasoff-Kardjalieff et al. 2021;Kramer et al. 2022). ...
The activity of transposable elements (TEs) contributes significantly to genome evolution. TEs often destabilize genome integrity but may also confer adaptive variation in phenotypic traits. De-repression of epigenetically silenced TEs often initiates bursts of transposition activity that may be counteracted by purifying selection and genome defenses. However, how these forces interact to determine the expansion routes of TEs within a species remains largely unknown. Here, we analyzed a set of 19 telomere-to-telomere genomes of the fungal wheat pathogen Zymoseptoria tritici . Phylogenetic reconstruction and ancestral state estimates of individual TE families revealed that TEs have undergone distinct activation and repression periods resulting in highly uneven copy numbers between genomes of the same species. Most TEs are clustered in gene poor niches, indicating strong purifying selection against insertions near coding sequences. TE families with high copy numbers have low sequence divergence and strong signatures of defense mechanisms (i.e., RIP). In contrast, small non-autonomous TEs (i.e., MITEs) are less impacted by defense mechanisms and are often located in close proximity to genes. Individual TE families have experienced multiple distinct burst events that generated many nearly identical copies. We found that a Copia element burst was initiated from recent copies inserted substantially closer to genes compared to older insertions. Overall, TE bursts tended to initiate from copies in GC-rich niches that escaped inactivation by genomic defenses. Our work shows how specific genomic environments features provide triggers for TE proliferation.
... In the past 25 years, since the identification of the first histone acetyltransferase enzyme from Tetrahymena thermophila (Brownell and Allis, 1995;Brownell et al., 1996), the field of histone modification has grown quickly. As one of the most important ways of histone modification, histone methylation has been shown to play critical roles in fungal growth, cell development, multi-stress resistance, pathogenicity, and photoperiod regulation (Palmer et al., 2008;Lee et al., 2009;Connolly et al., 2013;Minh et al., 2015;Liu et al., 2016;Zhang et al., 2016;Gu et al., 2017a,b). ...
Histone methylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by histone methyltransferases (HMTs) and histone demethylases (HDMs). This study determined the function of 5 HMTs (AaDot1, AaHMT1, AaHnrnp, AaSet1, and AaSet2) and 1 HDMs (AaGhd2) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. The vegetative growth, conidiation, and pathogenicity of ∆AaSet1 and ∆AaSet2 were severely inhibited indicating that AaSet1 and AaSet2 play critical roles in cell development in A. alternata. Multiple stresses analysis revealed that both AaSet1 and AaSet2 were involved in the adaptation to cell wall interference agents and osmotic stress. Meanwhile, ∆AaSet1 and ∆AaSet2 displayed serious vegetative growth defects in sole carbon source medium, indicating that AaSet1 and AaSet2 play an important role in carbon source utilization. In addition, ∆AaSet2 colony displayed white in color, while the wild-type colony was dark brown, indicating AaSet2 is an essential gene for melanin biosynthesis in A. alternata. AaSet2 was required for the resistance to oxidative stress. On the other hand, all of ∆AaDot1, ∆AaHMT1, and ∆AaGhd2 mutants displayed wild-type phenotype in vegetative growth, multi-stress resistance, pathogenicity, carbon source utilization, and melanin biosynthesis. To explore the regulatory mechanism of AaSet1 and AaSet2, RNA-seq of these mutants and wild-type strain was performed. Phenotypes mentioned above correlated well with the differentially expressed genes in ∆AaSet1 and ∆AaSet2 according to the KEGG and GO enrichment results. Overall, our study provides genetic evidence that defines the central role of HMTs and HDMs in the pathological and biological functions of A. alternata.
... Loss of function of cclA gene, which is involved in histone H3 lysine 4 methylation, activated the secondary metabolite gene cluster expression and produced emodin, monodictyphenone, and its derivatives (Bok et al., 2009). Deletion of Set1 gene, which codes for histone methyltransferase in F. graminearum, leads to the production of aurofusarin and deoxynivalenol (Liu et al., 2015). The deletion of dot1 gene, which codes for H3K79 transferase in A. flavus, decreased aflatoxin production (Liang et al., 2017). ...
... Loss of function of cclA gene, which is involved in histone H3 lysine 4 methylation, activated the secondary metabolite gene cluster expression and produced emodin, monodictyphenone, and its derivatives (Bok et al., 2009). Deletion of Set1 gene, which codes for histone methyltransferase in F. graminearum, leads to the production of aurofusarin and deoxynivalenol (Liu et al., 2015). The deletion of dot1 gene, which codes for H3K79 transferase in A. flavus, decreased aflatoxin production (Liang et al., 2017). ...
Microorganisms are stupendous source of secondary metabolites, having significant pharmaceutical and industrial importance. Genome mining has led to the detection of several cryptic metabolic pathways in the natural producer of secondary metabolites (SMs) such as actinobacteria and fungi. Production of these bioactive compounds in considerable amount is, however, somewhat challenging. This led to the search of using epigenetics as a key mechanism to alter the expression of genes that encode the SMs toward higher production in microorganisms. Epigenetics is defined as any heritable change without involving the changes in the underlying DNA sequences. Epigenetic modifications include chromatin remodeling by histone posttranslational modifications, DNA methylation, and RNA interference. Biosynthetic gene cluster for SMs remains in heterochromatin state in which the transcription of constitutive gene is regulated by epigenetic modification. Therefore, small-molecule epigenetic modifiers, which promote changes in the structure of chromatin, could control the expression of silent genes and may be rationally employed for discovery of novel bioactive compounds. This review article focuses on the types of epigenetic modifications and their impact on gene expression for enhancement of SM production in microorganisms.
... Gene-specific primer pairs for the selected genes were designed using primer design software (Primer Premier 5) and CmEF1α was used as a reference (Supplementary Data Table S6). Each sample was studied in three biological replicates and qRT-PCR assays were performed as previously reported [84]. The relative expression levels of genes were computed using the 2 − CT method [85]. ...
Chrysanthemum, one of the most important commercial ornamental crops, is susceptible to salinity, which limits its cultivation and application in coastal and inland saline areas. Grafting is widely used to improve the salt tolerance of horticultural crops, but the mechanisms of grafted chrysanthemum responses to salt stress remain unclear. In this study, we showed that hetero-grafted chrysanthemum with Artemisia annua as rootstock exhibited increased salt tolerance compared to self-grafted and self-rooted chrysanthemum. Under high salt stress, the roots of hetero-grafted chrysanthemum enrich Na+, resulting in a reduction of the Na+ toxicity in the scion, with only a small amount of Na+ being transported to the leaves. On the other hand, the roots of hetero-grafted chrysanthemum alleviated high Na+ stress via enhanced catalase (CAT) enzyme activity, down-regulated the expression of reactive oxygen species (ROS) accumulation-related genes, massive accumulation of soluble sugars and proline, and up-regulated the expression of heat shock protein (HSP)-related genes to enhance salt tolerance. In addition, the leaves of hetero-grafted chrysanthemum respond to low Na+ stress by increasing peroxidase (POD) enzyme activity and soluble sugar and proline content, to maintain a healthy state. However, self-grafted and self-rooted plants could not integrate ROS, soluble sugars, and proline in response to salt stress, and thus exhibit a salt-sensitive phenotype. Our research reveals the mechanisms underlying the increased salt tolerance of hetero-grafted chrysanthemums and makes it possible to have large-scale cultivation of chrysanthemums in saline areas.
... Interestingly, AurJ is an O-methyltransferase, and its expression may be inhibited by the accumulation of AdoHcy in this study (Figure 3(e)). The lack of AurJ leads to the accumulation of nor-rubrofusarin and thereby negatively regulates the expression of PKS12; it has also been reported that the ΔFgAurJ mutant lacks aurofusarin [37,55]. Additionally, both asexual and sexual reproduction were defective in ΔFgSah1 mutants; the production and germination rate of spores declined and the deletion mutants even produced no perithecia. ...
The S-adenosyl-L-homocysteine hydrolase (Sah1) plays a crucial role in methylation and lipid metabolism in yeast and mammals, yet its function remains elusive in filamentous fungi. In this study, we characterized Sah1 in the phytopathogenic fungus F. graminearum by generating knockout and knockout-complemented strains of FgSAH1. We found that the FgSah1-GFP fusion protein was localized to the cytoplasm, and that deletion of FgSAH1 resulted in defects in vegetative growth, asexual and sexual reproduction, stress responses, virulence, lipid metabolism, and tolerance against fungicides. Moreover, the accumulations of S-adenosyl-L-homocysteine (AdoHcy) and S-adenosyl-L-methionine (AdoMet) (the methyl group donor in most methyl transfer reactions) in ΔFgSah1 were seven- and ninefold higher than those in the wild-type strain, respectively. All of these defective phenotypes in ΔFgSah1 mutants were rescued by target gene complementation. Taken together, these results demonstrate that FgSah1 plays essential roles in methylation metabolism, fungal development, full virulence, multiple stress responses, lipid metabolism, and fungicide sensitivity in F. graminearum. To our knowledge, this is the first report on the systematic functional characterization of Sah1 in F. graminearum.
... To test the pathogenicity, the infection assays of wild-type strain PH-1, ΔFgNsf1 and ΔFgNsf1-C to coleoptiles and flowering heads of susceptible wheat cultivar Zhenmai 5 were conducted by the previously described method (Wu et al. 2005;Liu et al. 2016). A total of 10-μL aliquot of spore suspension (3×10 5 mL -1 ) of each strain harvested from MBL cultures for 5 days was injected to a floret in the central spikelet of single flowering head, or a 2.5-μL aliquot was injected to the tip of coleoptile from the 3-dayold seedlings without the 2-3 mm top, and each strain was inoculated with 20 replicates. ...
Nutrient and stress factor 1 (Nsf1), a transcription factor containing the classical Cys2-His2 (C2H2) zinc finger motif, is expressed under non-fermentable carbon conditions and in response to salt stress in Saccharomyces cerevisiae. However, the role of Nsf1 in filamentous fungi is not well understood. In this study, the orthologue of Nsf1 was investigated in Fusarium graminearum (named FgNsf1), a causal agent of Fusarium head blight (FHB). The functions of FgNsf1 were evaluated by constructing a FgNSF1 deletion mutant, designated as ΔFgNsf1, and its functional complementation mutant ΔFgNsf1-C. Gene deletion experiments showed that the mycelial growth rate, asexual and sexual reproduction of ΔFgNsf1 were significantly reduced, but the pigment production of ΔFgNsf1 was remarkably increased compared with the PH-1 and ΔFgNsf1-C. In addition, the tolerance of ΔFgNsf1 to osmotic pressures, cell wall-damaging agents and oxidative stress increased significantly. Sensitivity tests to different fungicides revealed that ΔFgNsf1 exhibited increased sensitivity to carbendazim (MBC) and tebuconazole, and enhanced tolerance to fludioxonil and iprodione than PH-1 and ΔFgNsf1-C. The virulence of ΔFgNsf1 to wheat coleoptiles and flowering wheat heads were dramatically decreased, which was consistent with the decrease in the yield of deoxynivalenol (DON). All of these defects were restored by target gene complementation. These results indicated that FgNsf1 plays a crucial role in vegetative growth, asexual and sexual reproduction, stress responses, fungicide sensitivity, and full virulence in F. graminearum.
... After homogenization with a vortex shaker, the lysate was centrifuged at 15000 × g for 20 min at 4°C. The resulting proteins were separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P transfer membrane (Millipore, Billerica, MA, USA) 55 . GFP-and RFP-tagged proteins were detected with monoclonal anti-GFP (ab32146, Abcam, Cambridge, UK, 1:10000 dilution) and anti-RFP (ab65856, Abcam, Cambridge, UK, 1:10000 dilution) antibodies, respectively. ...
Precursor messenger RNA (pre-mRNA) splicing is an essential and tightly regulated process in eukaryotic cells; however, the regulatory mechanisms for the splicing are not well understood. Here, we characterize a RNA binding protein named FgRbp1 in Fusarium graminearum , a fungal pathogen of cereal crops worldwide. Deletion of FgRbp1 leads to reduced splicing efficiency in 47% of the F. graminearum intron-containing gene transcripts that are involved in various cellular processes including vegetative growth, development, and virulence. The human ortholog RBM42 is able to fully rescue the growth defects of ΔFgRbp1. FgRbp1 binds to the motif CAAGR in its target mRNAs, and interacts with the splicing factor FgU2AF23, a highly conserved protein involved in 3’ splice site recognition, leading to enhanced recruitment of FgU2AF23 to the target mRNAs. This study demonstrates that FgRbp1 is a splicing regulator and regulates the pre-mRNA splicing in a sequence-dependent manner in F. graminearum .
... These domains are crucial for histone methylation [69,70]. In Fusarium graminearum and F. verticillioides, the SET domain is essential for morphogenesis, secondary metabolism and stress responses [71,72]. The SET domain also helps in growth and development in Neurospora crassa [73]. ...
Aim: To understand the phylogenomics, pathogenic/virulence-associated genes and genomic evolution of pathogenic Sporothrix species. Materials & methods: We performed in silico comparative genome analysis of Sporothrix species using ab initio tools and in-house scripts. We predicted genes and repeats, compared genomes based on synteny, identified orthologous clusters, assessed genes family expansion/contraction, predicted secretory proteins and finally searched for similar sequences from various databases. Results: The phylogenomics revealed that Sporothrix species are closely related to Ophiostoma species. The gene family evolutionary analysis revealed the expansion of genes related to virulence (CFEM domain, iron acquisition genes, lysin motif domain), stress response (Su[var]3-9, Enhancer-of-zeste and Trithorax domain and Domain of unknown function 1996), proteases (aspartic protease, x-pro dipeptidyl-peptidase), cell wall composition associated genes (chitin deacetylase, chitinase) and transporters (major facilitator superfamily transporter, oligo-peptide transporter family) in Sporothrix species. Conclusion: The present study documents the putative pathogenic/virulence-associated genes in the Sporothrix species.
... CmEF1α was used as a reference. Three biological replicates were performed per sample, and the qRT-PCR assays were conducted as previously reported by Liu et al. (2015). The relative expression levels of genes were calculated by using the 2 − ΔΔCT method (Livak and Schmittgen, 2001). ...
Chrysanthemum (Chrysanthemum morifolium), as one of the most commercially important ornamental crops, is threatened by dodder (Cuscuta spp.) during cultivation. When chrysanthemum was parasitized by dodder, growth of chrysanthemum slowed down and the leaves became dry and yellow. In the study, RNA sequencing assays were used for elucidating the immune response of chrysanthemum after dodder invasion and a total of 20,789 genes were differentially expressed. KEGG enrichment analysis revealed that these differentially expressed genes were involved in multiple pathways, “plant hormone signal transduction”, “cutin, suberine and wax biosynthesis”, and “plant-pathogen interaction”, etc. Combined with weighted gene co-expression network analysis (hereafter named WGCNA), we found that when the vascular connections were formed through haustoria, chrysanthemum recognized and fended off pathogens by the two stages of plant immune systems (PTI and ETI). Membrane bound receptors RLKs and the intracellular NLR proteins perceived pathogen-derived proteins and delivered the defense signals to trigger a series of defense responses in chrysanthemum. Reactive oxygen species (ROS), calcium, and MAPK-related pathways were triggered and a series of defense genes were up‐regulated. Besides, in stems of chrysanthemum, ET and SA signal transduction pathways played vital roles and had strong regulatory relationship with each other in response to dodder invasion. Our study provides an understanding of how chrysanthemum responds to dodder’s invasion, which could lay the basis for further revealing the interaction mechanism between chrysanthemum and dodder.
... For example, in F. graminearum, it was shown that the histone deacetylase HDF1 could be involved in the activation of DON production (62). Similarly, H3K4me3 deposited by FgSet1 is required for the active transcription of genes involved in the biosynthesis of both DON and the pigment aurofusarin (63). By contrast, the histone mark H3K27me3 represses 14 % of F. graminearum's genome, including genes involved in secondary metabolism (2). ...
La contamination des aliments par les mycotoxines représente un risque potentiel pour la santé humaine et animale. Des rapports de l'Autorité européenne de sécurité des aliments, l’EFSA, indiquent que près de la moitié des aliments dérivés des céréales et de céréales non transformées collectées entre 2007 et 2012 dans 21 pays européens étaient contaminées par des trichothécènes de type B, ou TCTB, et en particulier du déoxynivalénol, ou DON. Ces mycotoxines sont produites par des champignons phytopathogènes sur les grains en cours de remplissage, avant la récolte. Dans un contexte de changement climatique, cette situation pourrait s’aggraver. Ainsi, maitriser les contaminations en mycotoxines est une tâche urgente qui ne peut être repoussée. En Europe, le DON est principalement produit par Fusarium graminearum. Si les étapes de la voie de biosynthèse des TCTB sont assez bien décrites, les mécanismes moléculaires impliqués dans la régulation de cette voie restent, à ce jour, mal compris.Des études récentes ont mis en évidence que les modifications post-traductionnelles des histones canoniques ainsi que de leurs variantes joue un rôle critique dans les régulations des biosynthèses des mycotoxines et autres métabolites secondaires chez les champignons filamenteux, en modifiant la structure de la chromatine. Chez F. graminearum, il a été prouvé que l'histone désacétylase HDF1 est impliquée dans l'activation de la production de DON. Par contre, la marque histone H3K27me3 réprime 14 % de son génome, y compris des gènes impliqués dans les voies métaboliques secondaires. La variante d'histone H2A.Z, trouvée de façon ubiquitaire chez les eucaryotes, participe à de nombreux processus biologiques dont la stabilité génomique, la réparation de l’ADN, la régulation de transcription et la désactivation des télomères. Toutefois, les mécanismes sous-jacents de ces fonctions restent flous. Chez certaines espèces, la fonction de H2A.Z semble essentielle. À ce jour, la seule étude ciblant directement la fonction de H2A.Z chez les champignons filamenteux a été réalisée chez Neurospora crassa et a identifié son rôle dans la réponse au stress oxydatif.Nous avons ici fait l’hypothèse que H2A.Z est impliquée dans des processus biologiques importants chez F. graminearum, y compris ceux impliqués dans la production de métabolites secondaires dont les mycotoxines. Ce projet vise ainsi à caractériser les rôles joués par H2A.Z dans le contrôle du développement, du métabolisme et de la virulence chez F. graminearum.À l’aide d’une approche de génétique inverse, nous avons créé six mutants ne possédant plus le gène codant H2A.Z dans trois souches différentes de F. graminearum. Tous les mutants présentent un déficit en sporulation, germination, croissance radiale et production de DON. Cependant, l'intensité des effets observés dépend du fond génétique considéré. En outre, le rajout du gène sauvage codant H2A.Z ne restaure pas les phénotypes sauvages. Les séquençages des génomes complets des mutants ont montrés que, bien que H2A.Z ait été totalement éliminé du génome, des mutations compensatoires se produisent à d'autres loci, indépendamment du fond génétique, dans des gènes impliqués dans le remodelage de la chromatine. De manière frappante, une mutation supplémentaire a été détectée dans les mutants délétés pour H2AZ dans lesquels l’allèle sauvage a été réintroduit. Nous avons également construit les mutants surexprimés H2A.Z, mais aucune différence significative de phénotype entre les mutants et la souche sauvage n’a été observée. L’ensemble de ces résultats indique que H2A.Z est essentielle chez F. graminearum, l’occurrence de mutations compensatoires ayant compensé l’effet létal de la délétion infligée. Nous émettons l'hypothèse que les profondes réorganisations des réseaux génétique permettent cette plasticité, avec certaines conséquences en termes d'évolution et d'adaptation.
Secondary metabolites comprise the large repository of biomolecules, which are biosynthesized by bacteria, plants, and microorganisms. The metabolites necessary to perform day-to-day routine activities are termed primary metabolites and are the outcome of primary metabolism. Secondary metabolism forms more diverse and complex biomolecules termed as secondary metabolites; these are the end product of secondary metabolism. Depending upon the diverse functional group or basic carbon skeleton, secondary metabolites are categorized as terpenes, phenolics, and alkaloids. All the secondary metabolites are biosynthesized in either one of the shikimic acid, malonic acid, mevalonic acid, and methylerythritol phosphate pathways. These secondary metabolites enable the plant’s survival in different habitats and fluctuating environment conditions. The secondary metabolites are more economical and have lesser side effects as compared to chemical drugs, therefore an indispensable part of the traditional healthcare system. They are also useful in food, aroma, spices, and perfume industry. Owing to their diverse and multiple uses, there exists a huge gap between their production and demand. Due to the uniqueness and complexity in the chemical structures of secondary metabolites, often complete plants/organisms are used for harvesting secondary metabolites. The production of secondary metabolite in their native systems has the problems, such as low yield, tissue- and organ-specific compartmentalization, and accumulation in response to specific growth or environmental and geographical conditions. Moreover, harvesting secondary metabolites from the wild or native stage is often not a sustainable way, as this might result in the overharvesting of concerned plant as well as to deterioration of biodiversity. Apart from this, the pharmaceutical industry demands homogeneous samples having uniform compositions of the bioactive principles that is difficult to be achieved when harvesting, or collection is done randomly from the wild. The practices, such as cultivation, culturing, and domestication of the source organism, might be a valuable alternative, providing more uniform conditions and delivering homogenous composition of desired valuable secondary metabolites. But in most of the cases, the feasibility of this approach is limited because of various reasons. To overcome these hurdles concerning to the low synthesis, heterogeneity in composition, and accumulation in response to specific cues or specific stage of secondary metabolites, genetic manipulation of host organism seems to be a viable option. The research related to secondary metabolism through genetic manipulation is expanding at a fast pace and is challenging in molecular biology and biotechnology, holding unlimited opportunities. New advents in molecular biology, functional genomics, metabolomics, and proteomics are expanding our understanding of the pathways, networks, genes, and enzymes involved in the synthesis of secondary metabolites. These inputs from different dimensions of genetic manipulations are contributing determinant role in developing efficient strategies for targeted biosynthesis of valuable secondary metabolites. With the ever-increasing demand for novel drugs related to recently identified molecular targets, genetic manipulation will likely become more and more relevant. The lucrative economic aspects of commercial and industrial production of secondary metabolite related to pharmaceuticals, food, nutraceutical, aromatic, and perfume industries could magnetize investments and interest and build up new opportunities in this promising research field. This chapter discusses the various approaches and strategies used for the genetic manipulation of secondary metabolites and manipulation of the biosynthetic pathway of secondary metabolite products, leading to an improved quantity of secondary metabolites or more valuable and desired biomolecules. The various examples concerned with each approach have been also mentioned.
Light, magnetic field and methylation affected the growth and secondary metabolism of fungi. The regulation effect of the three factors on the growth and Monascus pigments (MPs) synthesis of M. purpureus was investigated in this study. 5-azacytidine (5-AzaC), DNA methylation inhibitor, was used to treat M. purpureus (wild-type, WT). 20 μM 5-AzaC significantly promoted the growth, development and MPs yield. Moreover, 250 lux red light and red light coupled magnetic field (RLCMF) significantly promoted the biomass. For WT, red light and RLCMF significantly promoted MPs yield. But compared with red light treatment, only 0.2 mT RLCMF promoted the alcohol-soluble MPs yield. For histone H3K4 methyltransferase complex subunit Ash2 gene knockout strain (ΔAsh2), only 0.2 mT RLCMF significantly promoted water-soluble MPs yield. Yet red light, 1.0 mT and 0.2 mT RLCMF significantly promoted alcohol-soluble MPs yield. This indicated that methylation affected the MPs biosynthesis. Red light and weaker MF had a synergistic effect on growth and MPs synthesis of ΔAsh2. This result was further confirmed by the expression of related genes. Therefore, histone H3K4 methyltransferase was involved in the regulation of the growth, development and MPs synthesis of M. purpureus by the RLCMF.
Magnaporthe oryzae ( M. oryzae ) is a devastating pathogenic fungal disease that has a serious threat to the global rice security. Recently, an in‐depth study of the pathogenic mechanism and developmental process of M. oryzae became a primary model in the study of host–fungal pathogen interactions. Here, we have identified a gene MoJMJD3 ( MGG_02032 , accession XM_003708733) encoding the JumonjiC (JmjC) domain through bioinformatics analysis in M. oryzae. The Δ Mojmjd3 deletion mutants showed defects in early appressorium formation and fungal virulence during pathogenesis. At 4 h after inoculation, the early appressorium formation of Δ Mojmjd3 was decreased when compared with that of the wild‐type P131. Analysis of intracellular localization of MoJMJD3 with RFP fluorescence observation showed that MoJMJD3 was mainly localized in the cytoplasm of the conidia, appressoria, hyphae, and infective hyphae. Furthermore, by spray inoculation analysis, the Δ Mojmjd3 deletion mutants reduced virulence on the leaves of rice. These data suggest that MoJMJD3 plays important roles in fungal virulence during pathogenesis in M. oryzae .
Fusarium graminearum est un champignon filamenteux phytopathogène contaminant de nombreuses céréales, telles le blé et le maïs, entraînant des pertes économiques majeures à travers le monde. De plus, cette espèce produit des mycotoxines, métabolites secondaires extrêmement stables qui sont toxiques pour l’humain et l’animal après ingestion. A l’heure actuelle, il n’existe pas de moyen de lutte efficace et durable contre les infections par ce pathogène et l’accumulation de mycotoxines dans les grains. Parmi les mécanismes impliqués dans les régulations du développement et de la production de mycotoxines par ce champignon, les dynamiques de modifications de la structure chromatinienne sont d’importance majeure. Un des exemples de modulateurs de la conformation chromatinienne est la variante histone H2A.Z, identifiée comme essentielle chez F. graminearum. Considérant l’importance de la marque, ces travaux de thèse se sont intéressés aux régulations médiées par H2A.Z chez F. graminearum. Ces travaux ont, dans un premier temps, nécessité de mettre au point diverses méthodes adaptées aux études de la structure nucléaire et chromatinienne chez F. graminearum. A l’aide de ces nouveaux outils, nous avons étudié le lien entre H2A.Z et la régulation de l’expression de gènes. Pour ce faire, nous avons tout d’abord utilisé une approche transcriptomique afin d’identifier les réseaux de régulation au sein desquels H2A.Z est impliqué. Ensuite, nous nous sommes focalisés sur la localisation génomique de H2A.Z au sein du génome et mis cette distribution en perspective grâce à nos données d’expression de gènes. Ces premières approches nous ont permis de dessiner la fonction de H2A.Z sur la régulation de l’expression des gènes au sein de son réseau. Ensuite, la distribution relative de H2A.Z par rapport à deux modifications post-traductionnelles (PTMs) d’histone H3K4me3 et H3K27me3 identifiées comme respectivement associées à une activation ou à une répression de l’expression de gènes, nous a permis de confirmer nos hypothèses quant au rôle de H2A.Z dans cette régulation. Les résultats préalablement obtenus ont également été comparés à l’organisation tridimensionnelle du génome nucléaire en compartiments associés à de la chromatine active et inactive, respectivement. Ces premières approches ont ensuite été complétées par une analyse fonctionnelle des points précédemment cités sur des mutants délétés et surexprimés pour H2A.Z, ceci permettant de mettre en exergue de potentiels remaniements. Enfin, une approche exploratoire sans a priori d’identification du répertoire de PTMs d’histones chez F. graminearum a été menée, ainsi qu’une analyse comparative chez des souches de F. graminearum modifiées génétiquement pour ne plus posséder ou surexprimer H2A.Z. L’ensemble de ces données fournit des clefs indispensables pour la compréhension de la fonction de H2A.Z chez F. graminearum. De plus cette analyse globale permet également de mettre en évidence le rôle majeur de la composante épigénétique dans le développement et la production de métabolites secondaire chez F. graminearum.
Fusarium head blight (FHB), mainly caused by Fusarium graminearum , is one of the most devastating diseases in wheat and barley worldwide. In addition to causing severe yield losses, F. graminearum produces deoxynivalenol (DON), a trichothecene mycotoxin which is harmful to human health and serves as an important virulence factor. Currently, changes in global climate and tillage systems have made FHB epidemics more frequent and severe. During the past decade, considerable efforts have been deployed to reveal the pathogenic mechanisms of F. graminearum , identify resistance genes in wheat, and breed FHB-resistant varieties. In this review, we highlight recent advances in FHB pathogenesis, F . graminearum -wheat interaction, and wheat defense mechanisms. This review contains four main sections: (1) signal sensing and transduction associated with the pathogenesis of F . graminearum ; (2) regulation and functions of DON during wheat infection; (3) roles of F . graminearum -secreted enzymes and effectors in facilitating pathogen infection of wheat; (4) wheat components involved in interactions with F. graminearum . A comprehensive understanding of the molecular interactions between F . graminearum and wheat will contribute to the development of novel and efficient strategies for controlling FHB.
Fungi are an understudied resource possessing huge potential for developing products that can greatly improve human well-being. In the current paper, we highlight some important discoveries and developments in applied mycology and interdisciplinary Life Science research. These examples concern recently introduced drugs for the treatment of infections and neurological diseases; application of –OMICS techniques and genetic tools in medical mycology and the regulation of mycotoxin production; as well as some highlights of mushroom cultivaton in Asia. Examples for new diagnostic tools
in medical mycology and the exploitation of new candidates for therapeutic drugs, are also given. In addition, two entries illustrating the latest developments in the use of fungi for biodegradation and fungal biomaterial production are provided.
Some other areas where there have been and/or will be significant developments are also included. It is our hope that this paper will help realise the importance of fungi as a potential industrial resource and see the next two decades bring forward
many new fungal and fungus-derived products.
Filamentous fungal pathogens have evolved diverse strategies to infect a variety of hosts including plants and insects. The dynamic infection process requires rapid and fine-tuning regulation of fungal gene expression programs in response to the changing host environment and defenses. Therefore, transcriptional reprogramming of fungal pathogens is critical for fungal development and pathogenicity. Histone post-translational modification, one of the main mechanisms of epigenetic regulation, has been shown to play an important role in the regulation of gene expressions, and is involved in, e.g., fungal development, infection-related morphogenesis, environmental stress responses, biosynthesis of secondary metabolites, and pathogenicity. This review highlights recent findings and insights into regulatory mechanisms of histone methylation and acetylation in fungal development and pathogenicity, as well as their roles in modulating pathogenic fungi–host interactions.
Epigenetic modification is important for cellular functions. Trimethylation of histone H3 lysine 4 (H3K4me3), which associates with transcriptional activation, is one of the important epigenetic modifications. In this study, the biological functions of UvKmt2-mediated H3K4me3 modification were characterized in Ustilaginoidea virens, which is the causal agent of the false smut disease, one of the most destructive diseases in rice. Phenotypic analyses of the ΔUvkmt2 mutant revealed that UvKMT2 is necessary for growth, conidiation, secondary spore formation, and virulence in U. virens. Immunoblotting and chromatin immunoprecipitation assay followed by sequencing (ChIP-seq) showed that UvKMT2 is required for the establishment of H3K4me3, which covers 1729 genes of the genome in U. virens. Further RNA-seq analysis demonstrated that UvKmt2-mediated H3K4me3 acts as an important role in transcriptional activation. In particular, H3K4me3 modification involves in the transcriptional regulation of conidiation-related and pathogenic genes, including two important mitogen-activated protein kinases UvHOG1 and UvPMK1. The down-regulation of UvHOG1 and UvPMK1 genes may be one of the main reasons for the reduced pathogenicity and stresses adaptability of the ∆Uvkmt2 mutant. Overall, H3K4me3, established by histone methyltransferase UvKMT2, contributes to fungal development, secondary spore formation, virulence, and various stress responses through transcriptional regulation in U. virens.
The strategies of genetic dereplication and manipulation of epigenetic regulators to activate the cryptic gene clusters are effective to discover natural products with novel structure in filamentous fungi. In this study, a combination of genetic dereplication (deletion of pesthetic acid biosynthetic gene, PfptaA) and manipulation of epigenetic regulators (deletion of histone methyltransferase gene PfcclA and histone deacetylase gene PfhdaA) was developed in plant endophytic fungus Pestalotiopsis fici. The deletion of PfptaA with PfcclA and/or PfhdaA led to isolation of 1 novel compound, pestaloficiol X (1), as well as another 11 known compounds with obvious yield changes. The proposed biosynthesis pathway of pestaloficiol X was speculated using comparative analysis of homologous biosynthetic gene clusters. Moreover, phenotypic effects on the conidial development and response to oxidative stressors in the mutants were explored. Our results revealed that the new strain with deletion of PfcclA or PfhdaA in ΔPfptaA background host can neutralise the hyperformation of conidia in the PfptaA mutant, and that the ΔPfptaA ΔPfhdaA mutant was generally not sensitive to oxidative stressors as much as the ΔPfptaA ΔcclA mutant in comparison with the single mutant ΔPfptaA or the parental strains. This combinatorial approach can be applied to discover new natural products in filamentous fungi.
The transcriptional expression pattern of lignocellulolytic enzyme‐encoding genes in white‐rot fungi differs depending on the culture conditions. Recently, it was shown that 13 putative cellulolytic enzyme‐encoding genes were significantly upregulated in most Pleurotus ostreatus ligninolysis‐deficient mutant strains on beech wood sawdust medium. However, the mechanisms by which this transcriptional shift is triggered remain unknown. In this study, we identified one mechanism. Our previous study implied that histone H3 N‐dimethylation at lysine 4 level possibly affects the shift; therefore, we analysed the expression pattern in the disruptants of P. ostreatus ccl1, which encodes a putative component of the COMPASS complex mediating the methylation. The results showed upregulation of five of the 13 cellulolytic enzyme‐encoding genes. We also found that rho1b, encoding a putative GTPase regulating signal transduction pathways, was upregulated in the ccl1 disruptants and ligninolysis‐deficient strains. Upregulation of at least three of the five the cellulolytic enzyme‐encoding genes was observed in rho1b‐overexpressing strains, but not in ccl1/rho1b double gene disruptants, during the 20‐day culture period. These results suggest that Rho1b may be involved in the upregulation of cellulolytic enzyme‐encoding genes observed in the ccl1 disruptants. Furthermore, we suggest that Mpk1b, a putative Agaricomycetes‐specific mitogen‐activated protein kinase, functions downstream of Rho1b. This article is protected by copyright. All rights reserved.
Fusarium graminearum produces the mycotoxin deoxynivalenol (DON) which promotes its expansion during infection on its plant host wheat. Conditional expression of DON production during infection is poorly characterized.
Wheat produces the defense compound putrescine, which induces hypertranscription of DON biosynthetic genes ( FgTRI s) and subsequently leads to DON accumulation during infection. Further, the regulatory mechanisms of FgTRI s hypertranscription upon putrescine treatment were investigated.
The transcription factor FgAreA regulates putrescine‐mediated transcription of FgTRI s by facilitating the enrichment of histone H2B monoubiquitination (H2B ub1) and histone 3 lysine 4 di‐ and trimethylations (H3K4 me2/3) on FgTRIs . Importantly, a DNA‐binding domain (bZIP) specifically within the Fusarium H2B ub1 E3 ligase Bre1 othologs is identified, and the binding of this bZIP domain to FgTRIs depends on FgAreA‐mediated chromatin rearrangement. Interestingly, H2B ub1 regulates H3K4 me2/3 via the methyltransferase complex COMPASS component FgBre2, which is different from Saccharomyces cerevisiae .
Taken together, our findings reveal the molecular mechanisms by which host‐generated putrescine induces DON production during F. graminearum infection. Our results also provide a novel insight into the role of putrescine during phytopathogen–host interactions and broaden our knowledge of H2B ub1 biogenesis and crosstalk between H2B ub1 and H3K4 me2/3 in eukaryotes.
Fusarium head blight(FHB)caused by Fusarium graminearum species complex (FGSC) is one of the most important diseases around the world. Deoxynivalenol (DON) is a type of mycotoxin produced by FGSC when infecting cereal crops. It is a serious threat to the health of both humans and livestock. Trehalose-6-phosphate phosphatase (TPP), a conserved metabolic enzyme found in many plants and pathogens, catalyzes the formation of trehalose. N-(phenylthio) phthalimide (NPP) has been reported to inhibit the normal growth of nematodes by inhibiting the activity of TPP, but this inhibitor of nematodes has not previously been tested against F. graminearum. In this study, we found that TPP in F. graminearum (FgTPP) had similar secondary structures and conserved cysteine (Cys356) to nematodes by means of bioinformatics. At the same time, the sensitivity of F. graminearum strains to NPP was determined. NPP exhibited a better inhibitory effect on conidia germination than mycelial growth. In addition, the effects of NPP on DON biosynthesis and trehalose biosynthesis pathway in PH-1 were also determined. We found that NPP decreased DON production, trehalose content, glucose content and TPP enzyme activity but increased trehalose-6-phosphate content and trehalose-6-phosphate synthase (TPS) enzyme activity. Moreover, the expression of TRl1, TRl4, TRl5, TRl6, and TPP genes were downregulated, on the contrary, the TPS gene was upregulated. Finally, in order to further determine the control ability of NPP on DON production in the field, we conducted a series of field experiments, and found that NPP could effectively reduce the DON content in wheat grain and had a general control effect on FHB. In conclusion, the research in this study will provide important theoretical basis for controlling FHB caused by F. graminearum and reducing DON production in the field.
Fusarium fujikuroi and Fusarium graminearum are agronomically important plant pathogens, both infecting important staple food plants and thus leading to huge economic losses worldwide. F.fujikuroi belongs to the Fusarium fujikuroi species complex (FFSC) and causes bakanae disease on rice, whereas F.graminearum, a member of the Fusarium graminearum species complex (FGSC), is the causal agent of Fusarium Head Blight (FHB) disease on wheat, barley and maize. In recent years, the importance of chromatin regulation became evident in the plant-pathogen interaction. Several processes, including posttranslational modifications of histones, have been described as regulators of virulence and the biosynthesis of secondary metabolites. In this study, we have functionally characterised methylation of lysine 20 histone 4 (H4K20me) in both Fusarium species. We identified the respective genes solely responsible for H4K20 mono-, di- and trimethylation in F.fujikuroi (FfKMT5) and F.graminearum (FgKMT5). We show that loss of Kmt5 affects colony growth in F.graminearum while this is not the case for F.fujikuroi. Similarly, FgKmt5 is required for full virulence in F.graminearum as Δfgkmt5 is hypovirulent on wheat, whereas the F.fujikuroi Δffkmt5 strain did not deviate from the wild type during rice infection. Lack of Kmt5 had distinct effects on the secondary metabolism in both plant pathogens with the most pronounced effects on fusarin biosynthesis in F.fujikuroi and zearalenone biosynthesis in F.graminearum. Next to this, loss of Kmt5 resulted in an increased tolerance towards oxidative and osmotic stress in both species.
Posttranslational modifications (PTMs) play crucial roles in regulating protein function and thereby control many cellular processes and biological phenotypes in both eukaryotes and prokaryotes. Several recent studies illustrate how plant fungal and bacterial pathogens use these PTMs to facilitate development, stress response, and host infection. In this review, we discuss PTMs that have key roles in the biological and infection processes of plant-pathogenic fungi and bacteria. The emerging roles of PTMs during pathogen–plant interactions are highlighted. We also summarize traditional tools and emerging proteomics approaches for PTM research. These discoveries open new avenues for investigating the fundamental infection mechanisms of plant pathogens and the discovery of novel strategies for plant disease control.
Expected final online publication date for the Annual Review of Phytopathology, Volume 59 is August 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
The soil‐borne ascomycete Verticillium dahliae causes wilt disease in more than two hundred dicotyledonous plants including the economically important crop cotton, and results in a severe reduction in cotton fiber yield and quality. During infection, V. dahliae secretes numerous secondary metabolites, which act as toxic factors to promote the infection process. However, the mechanism underlying how V. dahliae secondary metabolites regulate cotton infection remains largely unexplored. In this study, we report that VdBre1, an ubiquitin ligase (E3) enzyme to modify H2B, regulates radial growth and conidia production of V. dahliae. The VdBre1 deletion strains show nonpathogenic symptoms on cotton, and microscopic inspection and penetration assay indicated that penetration ability of the ∆VdBre1 strain was dramatically reduced. RNA‐seq revealed that a total of 1643 differentially expressed genes between the ∆VdBre1 strain and the wild type strain V592, among which genes related to lipid metabolism were significantly overrepresented. Remarkably, the volume of lipid droplets in the ∆VdBre1 conidia was shown to be smaller than that of wild‐type strains. Further metabolomics analysis revealed that the pathways of lipid metabolism and secondary metabolites, such as steroid biosynthesis and metabolism of terpenoids and polyketides, have dramatically changed in the ∆VdBre1 metabolome. Taken together, these results indicate that VdBre1 plays crucial roles in cotton infection and pathogenecity, by globally regulating lipid metabolism and secondary metabolism of V. dahliae.
Fusarium head blight is a destructive disease of grains resulting in reduced yields and contamination of grains with mycotoxins worldwide; Fusarium graminearum is its major causal agent. Chromatin structure changes play key roles in regulating mycotoxin biosynthesis in filamentous fungi. Using a split-marker approach in three F. graminearum strains INRA156, INRA349 and INRA812 (PH-1), we knocked out the gene encoding H2A.Z, a ubiquitous histone variant reported to be involved in a diverse range of biological processes in yeast, plants and animals, but rarely studied in filamentous fungi. All ΔH2A.Z mutants exhibit defects in development including radial growth, sporulation, germination and sexual reproduction, but with varying degrees of severity between them. Heterogeneity of osmotic and oxidative stress response as well as mycotoxin production was observed in ΔH2A.Z strains. Adding-back wild-type H2A.Z in INRA349ΔH2A.Z could not rescue the phenotypes. Whole genome sequencing revealed that, although H2A.Z has been removed from the genome and the deletion cassette is inserted at H2A.Z locus only, mutations occur at other loci in each mutant regardless of the genetic background. Genes affected by these mutations encode proteins involved in chromatin remodeling, such as the helicase Swr1p or an essential subunit of the histone deacetylase Rpd3S, and one protein of unknown function. These observations suggest that H2A.Z and the genes affected by such mutations are part or the same genetic interaction network. Our results underline the genetic plasticity of F. graminearum facing detrimental gene perturbation. These findings suggest that intergenic suppressions rescue deleterious phenotypes in ΔH2A.Z strains, and that H2A.Z may be essential in F. graminearum. This assumption is further supported by the fact that H2A.Z deletion failed in another Fusarium spp., i.e., the rice pathogen Fusarium fujikuroi.
The histone deacetylases (HDACs) are involved in growth, development and stress responses in many plants. However, the functions of HDACs in tea plant (Camellia sinensis L. O. Kuntze) and other woody plants remain unclear. Here, 18 CsHDAC genes were identified by genome-wide analysis in tea plant. The phylogenetic analysis demonstrated that the CsHDAC proteins were divided into three subfamilies, namely，the RPD3/HDA1 subfamily (8 members), the SIR2 subfamily (4 members) and the plant specific HD2 subfamily (6 members). The expression patterns showed that most members of CsHDACs family were regulated by different abiotic stress. High correlation was found between the expression of the CsHDACs and the accumulation of theanine, catechin, EGCG and other metabolites in tea plant. Most of the CsHDAC proteins were negative regulators. We further studied a specific gene CsHD2C (NCBI-ID: KY364373) in tea plant, which is the homolog of AtHD2C, encoded a protein of 306 aa. CsHD2C was highly expressed in leaves, young buds and stems. The transcription of CsHD2C was inhibited by ABA, NaCl and low temperature. It was found localized in the nucleus when fused with a YFP reporter gene. Overexpression of CsHD2C can rescue the phenotype related to different abiotic stresses in the mutant of AtHD2C in Arabidopsis. The stress-responsive genes RD29A, RD29B, ABI1 and ABI2 were also investigated to understand the regulating role of CsHD2C under abiotic stresses. We also found that CsHD2C could renew the change of acetylation level for histone H4 and the RNAP-II occupancy accumulation in the promoter of abiotic stress responses gene in the hd2c Arabidopsis mutant. Together, our results suggested that CsHD2C may act as a positive regulator in abiotic stress responses in tea plant.
The mitogen‐activated protein kinase ( MAPK ) signaling pathways have been characterized in Fusarium graminearum . Currently, the upstream sensors of these pathways are unknown.
Biological functions of a transmembrane protein FgSho1 were investigated using a target gene deletion strategy. The relationship between FgSho1 and the MAPK cassette FgSte50‐Ste11‐Ste7 was analyzed in depth.
The transmembrane protein FgSho1 is required for conidiation, full virulence, and deoxynivalenol ( DON ) biosynthesis in F. graminearum . Furthermore, FgSho1 and FgSln1 have an additive effect on virulence of F. graminearum . The yeast two‐hybrid, coimmunoprecipitation, colocalization and affinity capture‐mass spectrometry analyses strongly indicated that FgSho1 physically interacts with the MAPK module FgSte50‐Ste11‐Ste7. Similar to the FgSho1 mutant, the mutants of FgSte50, FgSte11, and FgSte7 were defective in conidiation, pathogenicity, and DON biosynthesis. In addition, FgSho1 plays a minor role in the response to osmotic stress but it is involved in the cell wall integrity pathway, which is independent of the module FgSte50‐Ste11‐Ste7 in F. graminearum .
Collectively, results of this study strongly indicate that FgSho1 regulates fungal development and pathogenicity via the MAPK module FgSte50‐Ste11‐Ste7 in F. graminearum , which is different from what is known in the budding yeast Saccharomyces cerevisiae .
The target of rapamycin ( TOR ) signaling pathway plays critical roles in controlling cell growth in a variety of eukaryotes. However, the contribution of this pathway in regulating virulence of plant pathogenic fungi is unknown.
We identified and characterized nine genes encoding components of the TOR pathway in Fusarium graminearum . Biological, genetic and biochemical functions of each component were investigated.
The FgFkbp12‐rapamycin complex binds to the FgTor kinase. The type 2A phosphatases FgPp2A, FgSit4 and FgPpg1 were found to interact with FgTap42, a downstream component of FgTor. Among these, we determined that FgPp2A is likely to be essential for F. graminearum survival, and FgSit4 and FgPpg1 play important roles in cell wall integrity by positively regulating the phosphorylation of FgMgv1, a key MAP kinase in the cell wall integrity pathway. In addition, the FgPpg1 interacting protein, FgTip41, is involved in regulating mycelial growth and virulence. Notably, FgTip41 does not interact with FgTap42 but with FgPpg1, suggesting the existence of FgTap42:FgPpg1:FgTip41 heterotrimer in F. graminearum , a complex not observed in the yeast model.
Collectively, we defined a genetic regulatory framework that elucidates how the TOR pathway regulates virulence and vegetative development in F. graminearum .
Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth, and pathogenicity, and in responses to the environment. In this study, we characterized a MAP kinase kinase FgMkk1 in Fusarium graminearum, the causal agent of wheat head blight. Phenotypic analyses of the FgMKK1 mutant (ΔFgMKK1) showed that FgMkk1 is involved in the regulation of hyphal growth, pigmentation, conidiation, deoxynivalenol biosynthesis, and virulence of F. graminearum. ΔFgMKK1 also showed increased sensitivity to cell wall-damaging agents, and to osmotic and oxidative stresses, but exhibited decreased sensitivity to the fungicides iprodione and fludioxonil. In addition, the mutant revealed increased sensitivity to a biocontrol agent, Trichoderma atroviride. Western blot assays revealed that FgMkk1 positively regulates phosphorylation of the MAK kinases Mgv1 and FgOs-2, the key component in the cell wall integrity (CWI) and high-osmolarity glycerol (HOG) signaling pathway, respectively. Yeast two-hybrid assay indicated that Mgv1 interacts with a transcription factor FgRlm1. The FgRLM1 mutant (ΔFgRLM1) showed increased sensitivity to cell wall-damaging agents, and exhibited decreased virulence. Taken together, our data indicated that FgMkk1 is an upstream component of Mgv1 and FgOs-2, and regulates vegetative differentiation, multiple stress response and virulence via the CWI and HOG signaling pathways. FgRlm1 may be a downstream component of Mgv1 in the CWI pathway in F. graminearum.
The cereal pathogen Fusarium graminearum produces secondary metabolites toxic to humans and animals, yet coordinated transcriptional regulation of gene clusters remains largely a mystery. By chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing. H3K27me3 was found predominantly in regions that lack synteny with other Fusarium species, generally subtelomeric regions. Di- or trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found in mutually exclusive regions of the genome. To find functions for H3K27me3, we deleted the gene for the putative H3K27 methyltransferase, KMT6, a homolog of Drosophila Enhancer of zeste, E(z). The kmt6 mutant lacks H3K27me3, as shown by western blot and ChIP-seq, displays growth defects, is sterile, and constitutively expresses genes for mycotoxins, pigments and other secondary metabolites. Transcriptome analyses showed that 75% of 4,449 silent genes are enriched for H3K27me3. A subset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in kmt6. A largely overlapping set of genes showed increased expression in kmt6. Almost 95% of the remaining 2,720 annotated silent genes showed no enrichment for either H3K27me3 or H3K4me2/3 in kmt6. In these cases mere absence of H3K27me3 was insufficient for expression, which suggests that additional changes are required to activate genes. Taken together, we show that absence of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity factors. Results from this study provide the framework for novel targeted strategies to control the "cryptic genome", specifically secondary metabolite expression.
Fusarium graminearum, the causal agent of Fusarium head blight in cereal crops, produces mycotoxins such as trichothecenes and zearalenone in infected plants. Here, we focused on the function of FgLaeA in F. graminearum, a homolog of Aspergillus nidulans LaeA encoding the global regulator for both secondary metabolism and sexual development. Prior to gene analysis, we constructed a novel luciferase reporter system consisting of a transgenic F. graminearum strain expressing a firefly luciferase gene under control of the promoter for either TRI6 or ZEB2 controlling the biosynthesis of these mycotoxins. Targeted deletion of FgLaeA led to a dramatic reduction of luminescence in reporter strains, indicating that FgLaeA controls the expression of these transcription factors in F. graminearum; reduced toxin accumulation was further confirmed by GC-MS analysis. Overexpression of FgLaeA caused the increased production of trichothecenes and additional metabolites. RNA seq-analysis revealed that gene member(s) belonging to ∼70% of total tentative gene clusters, which were previously proposed, were differentially expressed in the ΔFgLaeA strain. In addition, ΔFgLaeA strains exhibited an earlier induction of sexual fruiting body (perithecia) formation and drastically reduced disease symptoms in wheat, indicating that FgLaeA seems to negatively control perithecial induction, but positively control virulence toward the host plant. FgLaeA was constitutively expressed under both mycotoxin production and sexual development conditions. Overexpression of a GFP-FgLaeA fusion construct in the ΔFgLaeA strain restored all phenotypic changes to wild-type levels and led to constitutive expression of GFP in both nuclei and cytoplasm at different developmental stages. A split luciferase assay demonstrated that FgLaeA was able to interact with FgVeA, a homolog of A. nidulans veA. Taken together, these results demonstrate that FgLaeA, a member of putative FgVeA complex, controls secondary metabolism, sexual development, and virulence in F. graminearum, although the specific regulation pattern differs from that of LaeA in A. nidulans.
Secondary metabolite (SM) production in filamentous fungi is mechanistically associated with chromatin remodeling of specific SM clusters. One locus recently shown to be involved in SM suppression in Aspergillus nidulans was CclA, a member of the histone 3 lysine 4 methylating COMPASS complex. Here we examine loss of CclA and a putative H3K4 demethylase, HdmA, in the human pathogen Aspergillus fumigatus. Although deletion of hdmA showed no phenotype under the conditions tested, the cclA deletant was deficient in tri- and di-methylation of H3K4 and yielded a slowly growing strain that was rich in the production of several SMs, including gliotoxin. Similar to deletion of other chromatin modifying enzymes, ΔcclA was sensitive to 6-azauracil indicating a defect in transcriptional elongation. Despite the poor growth, the ΔcclA mutant had wild-type pathogenicity in a murine model and the Toll-deficient Drosophila model of invasive aspergillosis. These data indicate that tri- and di-methylation of H3K4 is involved in the regulation of several secondary metabolites in A. fumigatus, however does not contribute to pathogenicity under the conditions tested.
Set1 is a conserved histone H3 lysine 4 (H3K4) methyltransferase that exists as a multisubunit complex. Although H3K4 methylation is located on many actively transcribed genes, few studies have established a direct connection showing that loss of Set1 and H3K4 methylation results in a phenotype caused by disruption of gene expression. In this study, we determined that cells lacking Set1 or Set1 complex members that disrupt H3K4 methylation have a growth defect when grown in the presence of the antifungal drug Brefeldin A (BFA), indicating that H3K4 methylation is needed for BFA resistance. To determine the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate-limiting enzyme HMG-CoA reductase. Consequently, deletion of SET1 leads to a reduction in HMG-CoA reductase protein and total cellular ergosterol. In addition, the lack of Set1 results in an increase in the expression of DAN1 and PDR11, two genes involved in ergosterol uptake. The increase in expression of uptake genes in set1Δ cells allows sterols such as cholesterol and ergosterol to be actively taken up under aerobic conditions. Interestingly, when grown in the presence of ergosterol set1Δ cells become resistant to BFA, indicating that proper ergosterol levels are needed for antifungal drug resistance. These data show that H3K4 methylation impacts gene expression and output of a biologically and medically relevant pathway and determines why cells lacking H3K4 methylation have antifungal drug sensitivity.
The ergosterol biosynthesis pathway is well understood in Saccharomyces cerevisiae, but currently little is known about the pathway in plant-pathogenic fungi. In this study, we characterized the Fusarium graminearum FgERG4 gene encoding sterol C-24 reductase, which catalyses the conversion of ergosta-5,7,22,24-tetraenol to ergosterol in the final step of ergosterol biosynthesis. The FgERG4 deletion mutant ΔFgErg4-2 failed to synthesize ergosterol. The mutant exhibited a significant decrease in mycelial growth and conidiation, and produced abnormal conidia. In addition, the mutant showed increased sensitivity to metal cations and to various cell stresses. Surprisingly, mycelia of ΔFgErg4-2 revealed increased resistance to cell wall-degrading enzymes. Fungicide sensitivity tests revealed that ΔFgErg4-2 showed increased resistance to various sterol biosynthesis inhibitors (SBIs), which is consistent with the over-expression of SBI target genes in the mutant. ΔFgErg4-2 was impaired dramatically in virulence, although it was able to successfully colonize flowering wheat head and tomato, which is in agreement with the observation that the mutant produces a significantly lower level of trichothecene mycotoxins than does the wild-type progenitor. All of these phenotypic defects of ΔFgErg4-2 were complemented by the reintroduction of a full-length FgERG4 gene. In addition, FgERG4 partially rescued the defect of ergosterol biosynthesis in the Saccharomyces cerevisiae ERG4 deletion mutant. Taken together, the results of this study indicate that FgERG4 plays a crucial role in ergosterol biosynthesis, vegetative differentiation and virulence in the filamentous fungus F. graminearum.
The velvet genes are conserved in ascomycetous fungi and function as global regulators of differentiation and secondary metabolism. Here, we characterized one of the velvet genes, designated FgVelB, in the plant-pathogenic fungus Fusarium graminearum, which causes fusarium head blight in cereals and produces mycotoxins within plants. FgVelB-deleted (ΔFgVelB) strains produced fewer aerial mycelia with less pigmentation than those of the wild-type (WT) during vegetative growth. Under sexual development conditions, the ΔFgVelB strains produced no fruiting bodies but retained male fertility, and conidiation was threefold higher compared with the WT strain. Production of trichothecene and zearalenone was dramatically reduced compared with the WT strain. In addition, the ΔFgVelB strains were incapable of colonizing host plant tissues. Transcript analyses revealed that FgVelB was highly expressed during the sexual development stage, and may be regulated by a mitogen-activated protein kinase cascade. Microarray analysis showed that FgVelB affects regulatory pathways mediated by the mating-type loci and a G-protein alpha subunit, as well as primary and secondary metabolism. These results suggest that FgVelB has diverse biological functions, probably by acting as a member of a possible velvet protein complex, although identification of the FgVelB-FgVeA complex and the determination of its roles require further investigation.
The Set1 complex (also known as complex associated with Set1 or COMPASS) methylates histone H3 on lysine 4, with different levels of methylation affecting transcription by recruiting various factors to distinct regions of active genes. Neither Set1 nor its associated proteins are essential for viability with the notable exception of Swd2, a WD repeat protein that is also a subunit of the essential transcription termination factor APT (associated with Pta1). Cells lacking Set1 lose COMPASS recruitment but show increased promoter cross-linking of TFIIE large subunit and the serine 5 phosphorylated form of the Rpb1 C-terminal domain. Although Swd2 is normally required for bringing APT to genes, deletion of SET1 restores both viability and APT recruitment to a strain lacking Swd2. We propose a model in which Swd2 is required for APT to overcome antagonism by COMPASS.
As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study.
The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed.
The velvet protein, VeA, is involved in the regulation of diverse cellular processes. In this study, we explored functions of FgVeA in the wheat head blight pathogen, Fusarium graminearum,using a gene replacement strategy. The FgVEA deletion mutant exhibited a reduction in aerial hyphae formation, hydrophobicity, and deoxynivalenol (DON) biosynthesis. Deletion of FgVEA gene led to an increase in conidial production, but a delay in conidial germination. Pathogencity assays showed that the mutant was impaired in virulence on flowering wheat head. Sensitivity tests to various stresses exhibited that the FgVEA deletion mutant showed increased resistance to osmotic stress and cell wall-damaging agents, but increased sensitivity to iprodione and fludioxonil fungicides. Ultrastructural and histochemical analyses revealed that conidia of FgVeA deletion mutant contained an unusually high number of large lipid droplets, which is in agreement with the observation that the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Serial analysis of gene expression (SAGE) in the FgVEA mutant confirmed that FgVeA was involved in various cellular processes. Additionally, six proteins interacting with FgVeA were identified by yeast two hybrid assays in current study. These results indicate that FgVeA plays a critical role in a variety of cellular processes in F. graminearum.
In F. graminearum, the transcriptional regulator Tri6 is encoded within the trichothecene gene cluster and regulates genes involved in the biosynthesis of the secondary metabolite deoxynivalenol (DON). The Tri6 protein with its Cys₂His₂ zinc-finger may also conform to the class of global transcription regulators. This class of global transcriptional regulators mediate various environmental cues and generally responds to the demands of cellular metabolism. To address this issue directly, we sought to find gene targets of Tri6 in F. graminearum grown in optimal nutrient conditions. Chromatin immunoprecipitation followed by Illumina sequencing (ChIP-Seq) revealed that in addition to identifying six genes within the trichothecene gene cluster, Tri1, Tri3, Tri6, Tri7, Tri12 and Tri14, the ChIP-Seq also identified 192 additional targets potentially regulated by Tri6. Functional classification revealed that, among the annotated genes, ∼40% are associated with cellular metabolism and transport and the rest of the target genes fall into the category of signal transduction and gene expression regulation. ChIP-Seq data also revealed Tri6 has the highest affinity toward its own promoter, suggesting that this gene could be subject to self-regulation. Electro mobility shift assays (EMSA) performed on the promoter of Tri6 with purified Tri6 protein identified a minimum binding motif of GTGA repeats as a consensus sequence. Finally, expression profiling of F. graminearum grown under nitrogen-limiting conditions revealed that 49 out of 198 target genes are differentially regulated by Tri6. The identification of potential new targets together with deciphering novel binding sites for Tri6, casts new light into the role of this transcriptional regulator in the overall growth and development of F. graminearum.
Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila melanogaster Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2),
and Trr (human MLL3/4). Here, we report the biochemical purification and molecular characterization of the Drosophila COMPASS family. We observed a one-to-one similarity in subunit composition with their mammalian counterparts, with the exception
of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple
PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. Our study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di- and trimethylase in
Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their
role in the regulation of histone H3K4 methylation throughout development in Drosophila.
In Saccharomyces cerevisiae, the Nrd1-Nab3-Sen1 pathway mediates the termination of snoRNAs and cryptic unstable transcripts (CUTs). Both Nrd1 and the
Set1 histone H3K4 methyltransferase complex interact with RNA polymerase II (Pol II) during early elongation, leading us to
test whether these two processes are functionally linked. The deletion of SET1 exacerbates the growth rate and termination defects of nrd1 mutants. Set1 is important for the appropriate recruitment of Nrd1. Additionally, Set1 modulates histone acetylation levels
in the promoter-proximal region via the Rpd3L deacetylase and NuA3 acetyltransferase complexes, both of which contain PHD
finger proteins that bind methylated H3K4. Increased levels of histone acetylation reduce the efficiency of Nrd1-dependent
termination. We speculate that Set1 promotes proper early termination by the Nrd1-Nab3-Sen1 complex by affecting the kinetics
of Pol II transcription in early elongation.
Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. Here, we report that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. Our data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation.
Trichothecenes are toxic secondary metabolites produced by filamentous fungi mainly belonging to the Fusarium genus. Production of these mycotoxins occurs during infection of crops and is a threat to human and animal health. Although the pathway for biosynthesis of trichothecenes is well established, the regulation of the Tri genes implicated in the pathway remains poorly understood. Most of the Tri genes are gathered in a cluster which contains two transcriptional regulators controlling the expression of the other Tri genes. The regulation of secondary metabolites biosynthesis in most fungal genera has been recently shown to be controlled by various regulatory systems in response to external environment. The control of the "Tri cluster" by non-cluster regulators in Fusarium was not clearly demonstrated until recently. This review covers the recent advances concerning the regulation of trichothecene biosynthesis in Fusarium and highlights the potential implication of various general regulatory circuits. Further studies on the role of these regulatory systems in the control of trichothecene biosynthesis might be useful in designing new strategies to reduce mycotoxin accumulation.
Chromatin, composed of DNA wrapped around an octamer of histones, is the relevant substrate for all genetic processes in eukaryotic nuclei. Changes in chromatin structure are associated with the activation and silencing of gene transcription and reversible post-translational modifications of histones are now known to direct chromatin structure transitions. Recent studies in several fungal species have identified a chromatin-based regulation of secondary metabolism (SM) gene clusters representing an upper-hierarchical level for the coordinated control of large chromosomal elements. Regulation by chromatin transition processes provides a mechanistic model to explain how different SM clusters located at dispersed genomic regions can be simultaneously silenced during primary metabolism. Activation of SM clusters has been shown to be associated with increased acetylation of histones H3 and H4 and, consequently, inhibition of histone de-acetylase activities also leads to increased production of secondary metabolites. New findings suggest that SM clusters are silenced by heterochromatic histone marks and that the "closed" heterochromatic structures are reversed during SM activation. This process is mediated by the conserved activator of SM, LaeA. Despite the increase in knowledge about these processes, much remains to be learned from chromatin-level regulation of SM. For example, which proteins "position" the chromatin restructuring signal onto SM clusters or how exactly LaeA works to mediate the low level of heterochromatic marks inside different clusters remain open questions. Answers to these and other chromatin-related questions would certainly complete our understanding of SM gene regulation and signaling and, because for many predicted SM clusters corresponding products have not been identified so far, anti-silencing strategies would open new ways for the identification of novel bioactive substances.
Fungal secondary metabolites are important bioactive compounds but the conditions leading to expression of most of the putative secondary metabolism (SM) genes predicted by fungal genomics are unknown. Here we describe a novel mechanism involved in SM-gene regulation based on the finding that, in Aspergillus nidulans, mutants lacking components involved in heterochromatin formation show de-repression of genes involved in biosynthesis of sterigmatocystin (ST), penicillin and terrequinone A. During the active growth phase, the silent ST gene cluster is marked by histone H3 lysine 9 trimethylation and contains high levels of the heterochromatin protein-1 (HepA). Upon growth arrest and activation of SM, HepA and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3. SM-specific chromatin modifications are restricted to genes located inside the ST cluster, and constitutive heterochromatic marks persist at loci immediately outside the cluster. LaeA, a global activator of SM clusters in fungi, counteracts the establishment of heterochromatic marks. Thus, one level of regulation of the A. nidulans ST cluster employs epigenetic control by H3K9 methylation and HepA binding to establish a repressive chromatin structure and LaeA is involved in reversal of this heterochromatic signature inside the cluster, but not in that of flanking genes.
Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.
Post-translational modifications of histones play important roles in maintaining normal transcription patterns by directly or indirectly affecting the structural properties of the chromatin. In plants, methylation of histone H3 lysine 4 (H3K4me) is associated with genes and required for normal plant development.
We have characterized the genome-wide distribution patterns of mono-, di- and trimethylation of H3K4 (H3K4me1, H3K4me2 and H3K4me3, respectively) in Arabidopsis thaliana seedlings using chromatin immunoprecipitation and high-resolution whole-genome tiling microarrays (ChIP-chip). All three types of H3K4me are found to be almost exclusively genic, and two-thirds of Arabidopsis genes contain at least one type of H3K4me. H3K4me2 and H3K4me3 accumulate predominantly in promoters and 5' genic regions, whereas H3K4me1 is distributed within transcribed regions. In addition, H3K4me3-containing genes are highly expressed with low levels of tissue specificity, but H3K4me1 or H3K4me2 may not be directly involved in transcriptional activation. Furthermore, the preferential co-localization of H3K4me3 and H3K27me3 found in mammals does not appear to occur in plants at a genome-wide level, but H3K4me2 and H3K27me3 co-localize at a higher-than-expected frequency. Finally, we found that H3K4me2/3 and DNA methylation appear to be mutually exclusive, but surprisingly, H3K4me1 is highly correlated with CG DNA methylation in the transcribed regions of genes.
H3K4me plays widespread roles in regulating gene expression in plants. Although many aspects of the mechanisms and functions of H3K4me appear to be conserved among all three kingdoms, we observed significant differences in the relationship between H3K4me and transcription or other epigenetic pathways in plants and mammals.
Fungi display astounding diversity in their patho- genicity.Wecategorizethemasbiotrophsornecrotrophs and as obligate or facultative pathogens, but these categories do not reflect their polymorphic nature. Our approach to controlling disease would be better served by improving our understanding of the complete life cycle of the fungal pathogen, from infection and colo- nization to overwintering, using all of the tools at our disposal. A handful of fungal plant pathogens are now being studied at this level, including Fusarium grami- nearum(sexualstage,Gibberellazeae),thecausalagentof head blight of wheat (Triticum aestivum), oat (Avena sativa), and barley (Hordeum vulgare) and ear and stalk rot of maize (Zea mays). Although F. graminearum is arguably one of the best studied fungal plant patho- gens,thegeneticbasesofitslifecycleandpathogenicity are poorly defined. This article focuses on the contri- butions that genomics and postgenomic studies are making to our understanding of the entire life cycle of this important pathogen. The genome of this fungal pathogen cannot be viewed in isolation, just as the fungus does not exist in isolation. Genomics can be used to elucidate F. graminearum's interaction with its hosts, leading toaclearerpictureof itsecologicalniche. Fusarium head blight is a global problem. F. grami- nearum is the most prominent causal agent of head blight in the United States, Canada, and Europe (McMullen et al., 1997). A study of a worldwide col- lection of F. graminearum isolates has resulted in the proposeddivisionoftheoriginalspeciesinto11distinct species based on phylogenetic analyses of DNA se- quences(O'Donnelletal.,2004;Starkeyetal.,2007).The name F. graminearum was retained for the proposed species predominantly causing head blight in the United States and Europe. The proposed division of F. graminearum has not been accepted by all Fusarium researchers (Leslie and Bowden, 2008). Other Fusarium species, including F. culmorum, F. poae, F. pseudogrami- nearum, and F. avenaceum, may also be associated with kernel blight and related diseases throughout the world. Interestingly, individual species may predomi- nate in specific grain hosts, regions of the world, or
A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.
The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases specific for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated with Set1 and Set1C and found that it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new specificity and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, we screened the databases and found other Set1 homologues. We propose that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues.
The trithorax (Trx) family of proteins is required for maintaining a specific pattern of gene expression in some organisms.
Recently we reported the isolation and characterization of COMPASS, a multiprotein complex that includes the Trx-related protein
Set1 of the yeast Saccharomyces cerevisiae. Here we report that COMPASS catalyzes methylation of the fourth lysine of histone H3in vitro. Set1 and several other components of COMPASS are also required for histone H3 methylation in vivo and for transcriptional silencing of a gene located near a chromosome telomere.
Understanding variation in pathogen virulence and cultivar resistance is important for development of effective strategies for breeding wheat cultivars resistant to scab. Six isolates of Fusarium graminearum from China and the United States were compared for variation in cultural characteristics and virulence on nine wheat cultivars with different degrees of resistance to scab. The isolates varied in their cultural characteristics and ability to cause scab, but there was no consistent specificity of cultivar resistance or pathogen virulence. Therefore, a mixture of local isolates is an appropriate inoculum to screen for scab resistance. Subculturing the fungus on potato dextrose agar for eight generations did not reduce virulence. In the greenhouse, eight cultivars were tested five times over 3 years by inoculating one central floret in a spike with an Indiana isolate of the fungus. Cultivars Ning 7840, Sumai 49, Fu 5114, and Sumai 3 were consistently resistant. The fungus spread from the inoculated spikelet to noninoculated spikelets of resistant cultivars in less than 20% of the plants, and spread was not evident until 12 days after inoculation. All plants of susceptible cultivar Clark showed spread of infection, and symptoms appeared on noninoculated spikelets by 8 days after inoculation. Sudden blight on the top part of the spike may be an important characteristic of highly susceptible cultivars. Measurement of spread of scab within a spike is a stable and reliable estimate of cultivar resistance.
Epichloë festucae is a filamentous fungus that forms a mutually beneficial symbiotic association with Lolium perenne. This endophyte synthesizes bioprotective lolitrems (ltm) and ergot alkaloids (eas) in planta but the mechanisms regulating expression of the corresponding sub-telomeric gene clusters are not known. We show here that the status of histone H3 lysine 9 and lysine 27 trimethylation (H3K9me3/H3K27me3) at these alkaloid gene loci are critical determinants of transcriptional activity. Using ChIP-qPCR we found that levels of H3K9me3 and H3K27me3 were reduced at these loci in plant infected tissue compared to axenic culture. Deletion of E. festucae genes encoding the H3K9- (ClrD) or H3K27- (EzhB) methyltransferases led to derepression of ltm and eas gene expression under non-symbiotic culture conditions and a further enhancement of expression in the double deletion mutant. These changes in gene expression were matched by corresponding reductions in H3K9me3 and H3K27me3 marks. Both methyltransferases are also important for the symbiotic interaction between E. festucae and L. perenne. Our results show that the state of H3K9 and H3K27 trimethylation of E. festucae chromatin is an important regulatory layer controlling symbiosis-specific expression of alkaloid bioprotective metabolites and the ability of this symbiont to form a mutualistic interaction with its host.
The vitamin folate is required for methionine homeostasis in all organisms. In addition to its role in protein synthesis, methionine is the precursor to S-adenosyl-methionine (SAM), which is used in myriad cellular methylation reactions, including all histone methylation reactions. Here, we demonstrate that folate and methionine deficiency led to reduced methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. The effect of nutritional deficiency on H3K79 methylation was less pronounced, but was exacerbated in S. cerevisiae carrying a hypomorphic allele of Dot1, the enzyme responsible for H3K79 methylation. This result suggested a hierarchy of epigenetic modifications in terms of their susceptibility to nutritional limitations. Folate deficiency caused changes in gene transcription that mirrored the effect of complete loss of H3K4 methylation. Histone methylation was also found to respond to nutritional deficiency in the fission yeast Schizosaccharomyces pombe, and in human cells in culture.
Recent epidemics of Fusarium head blight (FHB) severely damaged the hard red spring wheat and barley crops in Minnesota. Samples of commercial grain were analyzed in 1993 and 1994 to determine the effects of FHB on several quality parameters. Wheat test weight (TW) averaged 832 kg m-3 (55.4 lb/bu), thousand kernel weight (TKW) averaged 27.4 g, and the proportion of visually scabby kernels (VSK) averaged 11.0%. Deoxynivalenol (DON) was detected in 493 of 500 samples (98.6%). The mean concentration was 8.3 μg/g (range = 0.0 to 44.7 μg/g). Scab in wheat could rapidly be estimated using easy-to-prepare visual comparison standards. Scores of percent VSK were correlated with DON concentration at r = 0.897 and 0.908 in 1993 and 1994, respectively. TW and TKW were less effective estimators of DON (r = -0.622 and -0.550, respectively). DON was detected in 100 of 100 six-row harley samples collected during the survey and averaged 10.4 μg/g (range = 0.5 to 39.7 μg/g). DON concentration in barley could not be effectively estimated with grading parameters including TW, TKW, percent plump kernels, or a visual index of kernel discoloration. In 28 samples of oats, DON averaged 1.4 μg/g (range = 0.0 to 6.4 μg/g). Nivalenol was not detected in any of the 628 samples analyzed during the two-year study.
Trichothecenes and fumonisins are mycotoxins produced by Fusarium, a filamentous fungus that can cause disease in barley, maize, rice, wheat, and some other crop plants. Research on the genetics and biochemistry of trichothecene and fumonisin biosynthesis has provided important insights into the genetic and biochemical pathways in Fusarium that lead to formation of these mycotoxins. In Fusarium, trichothecene biosynthetic enzymes are encoded by genes at three loci: the single-gene TRI101 locus, the two-gene TRI1-TRI16 locus, and the 12-gene core TRI cluster. In contrast, fumonisin biosynthetic enzymes identified to date are all located at one locus, the 17-gene FUM cluster. The FUM and core TRI clusters also encode proteins that regulate expression of the cluster genes and proteins that are involved in mycotoxin transport across the cell membrane. Biosynthetic pathways for both mycotoxins have been proposed based on a combination of biochemical and genetic evidence, including toxin production phenotypes of Fusarium mutants in which individual TRI or FUM genes have been inactivated. Some TRI and FUM gene mutants have also been employed to examine the role of mycotoxin production in plant pathogenesis. The studies indicate that trichothecene production can contribute to the ability of F. graminearum to cause wheat head blight, one of the most important wheat diseases in the world. Thus, studies into the genetic basis of mycotoxin production have identified a potential target to enhance resistance of wheat to a major plant disease and mycotoxin contamination problem.
This paper describes a method for the analysis of DON and its derivatives in wheat and barley using combination gas chromatography/mass spectrometry (GC/MS). The method has been designed for the analysis of large batch (100 g) samples and single kernels. The sensitivity of the method is 50 ppb (ng/g), and the precision in terms of percent standard deviation lies between 0.0 and 11.1. The percent recovery for 1, 5, and 10 μg/g (ppm) recovered from wheat is 97.2, 88.4 and 87.9% respectively. A comparison of methods was made between two laboratories using GC/EC and our method. There was no significant difference between the results of the two methods. The method is also applicable to 15-acetyldeoxynivalenol (15-ADON) as well as nivalenol (NIV). Standard curves constructed for DON, 15-ADON, and NIV show a linear relationship between 0.025 ng (limit of sensitivity) and 8 ng. Keywords: Deoxynivalenol; nivalenol; analytical method
The velvet complex containing VeA, VelB and LaeA has been showed to play critical roles in the regulation of secondary metabolism and diverse cellular processes in Aspergillus spp. In this study, we identified FgVelB, a homolog of Aspergillus nidulans VelB, from Fusarium graminearum using the BLASTP program. Disruption of FgVELB gene led to several phenotypic defects, including suppression of aerial hyphae formation, reduced hyphal hydrophobicity and highly increased conidiation. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents, which may be related to a high level of glycerol accumulation in the mutant. Additionally, the mutant exhibited increased sensitivity to the phenylpyrrole fungicide fludioxonil. Ultrastructural and histochemical analyses revealed that conidia of FgVELB deletion mutant contained numerous lipid droplets. Pathogenicity assays showed FgVELB deletion mutant was impaired in virulence on flowering wheat head, which is consistent with the observation that FgVelB is involved in the regulation of deoxynivalenol biosynthesis in F. graminearum. All of the defects were restored by genetic complementation of the mutant with wild-type FgVELB gene. Yeast two hybrid assays showed that FgVelB does not interact with FgVeA. Taken together, results of this study indicated that FgVelB plays a critical role in the regulation of various cellular processes in F. graminearum.
Growth and toxigenesis by Fusarium graminearum R6576, were compared in four liquid media. Parameters monitored during the fermentation were deoxynivalenol (DON) and 15-acetyl deoxynivalenol (15-ADON) production, fungal mass, carbohydrate utilization, and pH. Factors which were varied included basal medium composition, corn steep liquor (CSL) concentration, sucrose concentration and ammonium tartrate concentration. Growth in modified Fries medium resulted in only low levels of DON (0.25 mg/ L) and 15-ADON (0.25 mg/ L) after 20 days. Addition of 4% CSL to modified Fries medium raised the 20 day DON yield to 16.5 mg/ l. Increasing the sucrose concentration in modified Fries medium amended with 4% CSL resulted in increased mycelial dry weight but decreased levels of DON. Concentrations of ammonium tartrate greater than 1% in modified Fries amended with 4% CSL greatly reduced DON yield. Use of glucose-yeast extract-peptone (GYEP) for toxin production resulted in higher yields of 15-ADON (14.0 mg/ L) than DON (5.5 mg/ L) after 20 days. However, supplementation of GYEP with 4% CSL resulted primarily in DON production (4.5 mg/ L) after 20 days. In general, qualitative and quantitative production of DON and 15-ADON by Fusarium graminearum R6576 were dependent on the composition of the complex liquid medium.
The Saccharomyces cerevisiae Set1/COMPASS was the first histone H3 lysine 4 (H3K4) methylase identified over 10 years ago. Since then, it has been demonstrated that Set1/COMPASS and its enzymatic product, H3K4 methylation, is highly conserved across the evolutionary tree. Although there is only one COMPASS in yeast, Drosophila possesses three and humans bear six COMPASS family members, each capable of methylating H3K4 with nonredundant functions. In yeast, the histone H2B monoubiquitinase Rad6/Bre1 is required for proper H3K4 and H3K79 trimethylations. The machineries involved in this process are also highly conserved from yeast to human. In this review, the process of histone H2B monoubiquitination-dependent and -independent histone H3K4 methylation as a mark of active transcription, enhancer signatures, and developmentally poised genes is discussed. The misregulation of histone H2B monoubiquitination and H3K4 methylation result in the pathogenesis of human diseases, including cancer. Recent findings in this regard are also examined.
The high-osmolarity glycerol signal pathway plays an important role in the response of fungi to various environmental stresses. In this study, we characterized a mitogen-activated protein kinase kinase kinase gene BcOS4 in Botrytis cinerea, which is homologous to Saccharomyces cerevisiae SSK2/SSK22. The BcOS4 deletion mutant was significantly impaired in vegetative growth and conidial formation. The mutant exhibited increased sensitivity to the osmotic, oxidative stresses and to the fungicides iprodione and fludioxonil. Western blot analysis showed that BcSak1, a putative downstream component of BcOs4, was not phosphorylated in the mutant. In addition, the BcOS4 mutant was unable to infect leaves of rapeseed and cucumber, and grape fruits, although it can cause disease on apple fruits. All the defects were restored by genetic complementation of the BcOS4 deletion mutant with the wild-type BcOS4 gene. The data of this study indicate that BcOS4 is involved in vegetative differentiation, virulence, adaption to hyperosmotic and oxidative stresses, and to fungicides in B. cinerea.
Fusarium graminearum (Fg) can cause head blight and crown rot (CR) diseases of wheat but fungal colonisation and mycotoxin production by Fg during CR are little understood. Studies of an Australian strain of Fg demonstrated that expression of the Tri5 gene of Fg and deoxynivalenol (DON) production were induced during infection of the stem base and to levels equivalent to those observed in inoculated heads. To study fungal colonisation and DON production in CR disease, we inoculated stem bases, 14 days after sowing, with macroconidia of Australian and USA strains of Fg and of an Australian strain of Fusarium pseudograminearum, a related pathogen frequently associated with CR. At maturity, the fungal pathogen was subsequently detected by isolation on growth media in high percentages of plants at the stem base and the lowest node adjacent to the inoculation site (50–100%), in the non-inoculated flag-leaf node (FLN) (35–95%) and in asymptomatic non-inoculated mature heads (50–60%) and kernels (20%). Microscopic analysis suggested that colonisation of upper nodes occurred primarily via the pith parenchyma and lumen. Significant concentrations of DON (up to 35 ppm) were detected in the FLN and the head/rachis following inoculation of the stem base. To test the role of DON in CR a transgenic strain of Fg from the USA with the Tri5 gene deleted was compared with its original wild-type and these produced similar levels of CR lesion development and necrosis indicating that DON production was not necessary to cause CR disease symptoms. However, the DON-minus mutant was less frequently recovered from the FLN than the wild type suggesting that DON has a role in stem colonisation by the fungus.
Posttranslational modifications of histones are coupled in the regulation of the cellular processes involving chromatin, such as transcription, replication, repair, and genome stability. Recent biochemical and genetic studies have clearly demonstrated that many aspects of chromatin, in addition to posttranslational modifications of histones, provide surfaces that can interact with effectors and the modifying machineries in a context-dependent manner, all as a part of the "chromatin signaling pathway." Here, we have reviewed recent findings on the molecular basis for the recruitment of the chromatin-modifying machineries and their diverse and varied biological outcomes.
Heritable changes to the transcriptome that are independent to changes in the genome are defined as epigenetics. DNA methylation and posttranslational modifications of histones, such as acetylation/deacetylation and methylation/demethylation of lysine residues, underlie these epigenetic phenomena, which impact on many physiological processes. This perspective focuses on the emerging biology of histone methylation and demethylation, highlighting how these reactions depend on metabolic coenzymes like S-adenosylmethionine, flavin adenine dinucleotide, and α-ketoglutarate. Furthermore, we illustrate that methyltranferases and demethylases affect many metabolic pathways. Despite the preliminary evidence that methyltranferases and demethylases could link metabolic signals to chromatin and alter transcription, further research is indispensable to consolidate these enticing observations.
Filamentous fungi produce a vast array of small molecules called secondary metabolites, which include toxins as well as antibiotics. Coregulated gene clusters are the hallmark of fungal secondary metabolism, and there is a growing body of evidence that suggests regulation is at least, in part, epigenetic. Chromatin-level control is involved in several silencing phenomena observed in fungi including mating type switching, telomere position effect (TPE), silencing of ribosomal DNA, regulation of genes involved in nutrient acquisition, and as presented here, secondary metabolite cluster expression. These phenomena are tied together by the underlying theme of chromosomal location, often near centromeres and telomeres, where facultative heterochromatin plays a role in transcription. Secondary metabolite gene clusters are often located subtelomerically and recently it has been shown that proteins involved in chromatin remodeling, such as LaeA, ClrD, CclA, and HepA mediate cluster regulation.
We have characterized two post-translational histone modifications in Caenorhabditis elegans on a genomic scale. Micrococcal nuclease digestion and immunoprecipitation were used to obtain distinct populations of single nucleosome cores, which were analyzed using massively parallel DNA sequencing to obtain positional and coverage maps. Two methylated histone H3 populations were chosen for comparison: H3K4 histone methylation (associated with active chromosomal regions) and H3K9 histone methylation (associated with inactivity). From analysis of the sequence data, we found nucleosome cores with these modifications to be enriched in two distinct partitions of the genome; H3K4 methylation was particularly prevalent in promoter regions of widely expressed genes, while H3K9 methylation was enriched on specific chromosomal arms. For each of the six chromosomes, the highest level of H3K9 methylation corresponds to the pairing center responsible for chromosome alignment during meiosis. Enrichment of H3K9 methylation at pairing centers appears to be an early mark in meiotic chromosome sorting, occurring in the absence of components required for proper pairing of homologous chromosomes. H3K9 methylation shows an intricate pattern within the chromosome arms with a particular anticorrelation to regions that display a strong approximately 10.5 bp periodicity of AA/TT dinucleotides that is known to associate with germline transcription. By contrast to the global features observed with H3K9 methylation, H3K4 methylation profiles were most striking in their local characteristics around promoters, providing a unique promoter-central landmark for 3,903 C. elegans genes and allowing a precise analysis of nucleosome positioning in the context of transcriptional initiation.
Covalent modification of histones on chromatin is a dynamic mechanism by which various nuclear processes are regulated. Methylation of histone H3 on lysine 4 (H3K4) implemented by the macromolecular complex COMPASS and its related complexes is associated with transcriptionally active regions of chromatin. Enzymes that catalyze H3K4 methylation were initially characterized genetically as regulators of Hox loci, long before their catalytic functions were recognized. Since their discovery, genetic and biochemical studies of H3K4 methylases and demethylases have provided important mechanistic insight into the role of H3K4 methylation in HOX gene regulation during development.
FPPI database provides information of protein−protein interactions of F. graminearum predicted based on both interologs and domain−domain interactions experimentally determined based on protein structures. FPPI contains 223 166 interactions among 7406 proteins for F. graminearum, and a core PPI data set that consists of 27 102 interactions and 3745 proteins. FPPI also contains other functional information for F. graminearum genes. Public access to the FPPI database is available at http://csb.shu.edu.cn/fppi.
Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression.
Technological limitations have so far prevented a global analysis of lysine acetylation’s cellular roles. We used high-resolution
mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response
to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large
macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear
transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast
cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable
with that of other major posttranslational modifications.
Cotranscriptional histone methylations by Set1 and Set2 have been shown to affect histone acetylation at promoters and 3' regions of genes, respectively. While histone H3K4 trimethylation (H3K4me3) is thought to promote nucleosome acetylation and remodeling near promoters, we show here that H3K4 dimethylation (H3K4me2) by Set1 leads to reduced histone acetylation levels near 5' ends of genes. H3K4me2 recruits the Set3 complex via the Set3 PHD finger, localizing the Hos2 and Hst1 subunits to deacetylate histones in 5' transcribed regions. Cells lacking the Set1-Set3 complex pathway are sensitive to mycophenolic acid and have reduced polymerase levels at a Set3 target gene, suggesting a positive role in transcription. We propose that Set1 establishes two distinct chromatin zones on genes: H3K4me3 leads to high levels of acetylation and low nucleosome density at promoters, while H3K4me2 just downstream recruits the Set3 complex to suppress nucleosome acetylation and remodeling.
Trichothecenes are isoprenoid mycotoxins produced in wheat infected with the filamentous fungus Fusarium graminearum. Some fungal genes for trichothecene biosynthesis (Tri genes) are known to be under control of transcription factors encoded by Tri6 and Tri10. Tri6 and Tri10 deletion mutants were constructed in order to discover additional genes regulated by these factors in planta. Both mutants were greatly reduced in pathogenicity and toxin production and these phenotypes were largely restored by genetic complementation with the wild-type gene. Transcript levels for over 200 genes were altered > or = twofold for Deltatri6 or Deltatri10 mutants including nearly all known Tri genes. Also reduced were transcript levels for enzymes in the isoprenoid biosynthetic pathway leading to farnesyl pyrophosphate, the immediate molecular precursor of trichothecenes. DNA sequences 5' to isoprenoid biosynthetic genes were enriched for the Tri6p DNA binding motif, YNAGGCC, in F. graminearum but not in related species that do not produce trichothecenes. To determine the effect of trichothecene metabolites on gene expression, cultures were treated with trichodiene, the first metabolic intermediate specific to the trichothecene biosynthetic pathway. A total of 153 genes were upregulated by added trichodiene and were significantly enriched for genes likely involved in cellular transport. Differentially regulated genes will be targeted for functional analysis to discover additional factors involved in toxin biosynthesis, toxin resistance and pathogenesis.
S-adenosyl-L-methionine (AdoMet) is synthesized by transfer of the adenosyl moiety of ATP to the sulfur atom of methionine. This reaction is catalysed by AdoMet synthetase. In all eukaryotic organisms studied so far, multiple forms of AdoMet synthetases have been reported and from their recent study, it appears that AdoMet synthetase is an exceptionally well conserved enzyme through evolution. In Saccharomyces cerevisiae, we have demonstrated the existence of two AdoMet synthetases encoded by genes SAM1 and SAM2. Yeast, which is able to concentrate exogenously added AdoMet, is thus a particularly useful biological system to understand the role and the physiological significance of the preservation of two almost identical AdoMet synthetases. The analysis of the expression of the two SAM genes in different genetic backgrounds during growth under different conditions shows that the expression of SAM1 and SAM2 is regulated differently. The regulation of SAM1 expression is identical to that of other genes implicated in AdoMet metabolism, whereas SAM2 shows a specific pattern of regulation. A careful analysis of the expression of the two genes and of the variations in the methionine and AdoMet intracellular pools during the growth of different strains lead us to postulate the existence of two different AdoMet pools, each one supplied by a different AdoMet synthetase but in equilibrium with each other. This could be a means of storing AdoMet whenever this metabolite is overproduced, thus avoiding the degradation of a metabolite the synthesis of which is energetically expensive.
The production of trichothecene mycotoxins by some plant pathogenic species of Fusarium is thought to contribute to their virulence. Gibberella zeae (F. graminearum) is an important cereal pathogen that produces the trichothecene deoxynivalenol. To determine if trichothecene production contributes to the virulence of G. zeae, we generated trichothecene-deficient mutants of the fungus by gene disruption. The disrupted gene, Tri5, encodes the enzyme trichodiene synthase, which catalyzes the first step in trichothecene biosynthesis. To disrupt Tri5, G. zeae was transformed with a plasmid carrying a doubly truncated copy of the Tri5 coding region interrupted by a hygromycin B resistance gene. Tri5- transformants were selected by screening for the inability to produce trichothecenes and by Southern blot analysis. Tri5- strains exhibited reduced virulence on seedlings of Wheaton wheat and common winter rye, but wild-type virulence on seedlings of Golden Bantam maize. On Caldwell and Marshall wheat and Porter oat seedlings, Tri5- strains were inconsistent in causing less disease than their wild-type progenitor strain. Head blight developed more slowly on Wheaton when inoculated with Tri5- mutants than when inoculated with wild-type strains. These results suggest that trichothecene production contributes to the virulence of G. zeae on some hosts.
The trithorax gene family contains members implicated in the control of transcription, development, chromosome structure, and human leukemia. A feature shared by some family members, and by other proteins that function in chromatin-mediated transcriptional regulation, is the presence of a 130- to 140-amino acid motif dubbed the SET or Tromo domain. Here we present analysis of SET1, a yeast member of the trithorax gene family that was identified by sequence inspection to encode a 1080-amino acid protein with a C-terminal SET domain. In addition to its SET domain, which is 40-50% identical to those previously characterized, SET1 also shares dispersed but significant similarity to Drosophila and human trithorax homologues. To understand SET1 function(s), we created a null mutant. Mutant strains, although viable, are defective in transcriptional silencing of the silent mating-type loci and telomeres. The telomeric silencing defect is rescued not only by full-length episomal SET1 but also by the conserved SET domain of SET1. set1 mutant strains display other phenotypes including morphological abnormalities, stationary phase defects, and growth and sporulation defects. Candidate genes that may interact with SET1 include those with functions in transcription, growth, and cell cycle control. These data suggest that yeast SET1, like its SET domain counterparts in other organisms, functions in diverse biological processes including transcription and chromatin structure.