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The root invading pathogen Fusarium oxysporum targets pattern‐triggered immunity using both cytoplasmic and apoplastic effectors
Abstract
Plant pathogens use effector proteins to promote host colonization. The mode of action of effectors from root invading pathogens, such as Fusarium oxysporum (Fo), is poorly understood. Here, we investigated whether Fo effectors suppress pattern‐triggered immunity (PTI), and whether they enter host cells during infection. ‐ Eight candidate effectors of an Arabidopsis‐infecting Fo strain were expressed with and without signal peptide in Nicotiana benthamiana and their effect on flg22‐ and chitin‐triggered ROS burst was monitored. To detect uptake, effector biotinylation by an intracellular Arabidopsis‐produced biotin ligase was examined following root infection. ‐ Four effectors suppressed PTI signaling; two act intracellularly and two apoplastically. Heterologous expression of a PTI‐suppressing effector in Arabidopsis enhanced bacterial susceptibility. Consistent with an intracellular activity, host cell uptake of five effectors, but not of the apoplastically acting ones, was detected in Fo infected Arabidopsis roots. ‐ Multiple Fo effectors target PTI signaling, uncovering a surprising overlap in infection strategies between foliar‐ and root pathogens. Extracellular targeting of flg22 signaling by a microbial effector provides a new mechanism on how plant pathogens manipulate their host. Effector translocation appears independent of protein size, charge, presence of conserved motifs or the promoter driving its expression.
... During the infection process, effector proteins are secreted into the apoplastic spaces of the root cortex and into the xylem sap; fourteen of these Fol-secreted proteins have been identified as Six (Secreted In Xylem) proteins (Houterman et al., 2007;Schmidt et al., 2013;Redkar et al., 2022a). Some effectors function inside the apoplast, while others can be taken up by the host cell and exert their function intercellularly (e.g., Avr2, Six6, Six8) (Gawehns et al., 2014;Tintor et al., 2020). Six1, Avr2, Six5, and Six6 are required for full Fol pathogenicity, defining them as genuine effectors (Rep et al., 2004;Houterman et al., 2009;Gawehns et al., 2014;Ma et al., 2015). ...
... PTI involves a series of defense outputs, ranging from early responses, such as changes in ion fluxes, ROS production, and activation of mitogen-activated protein kinases (MAPK), to late responses, such as callose deposition and growth inhibition (Couto and Zipfel, 2016;Saijo et al., 2018). Avr2 suppresses PTI responses, including ROS accumulation, MAPK activation, callose deposition, and growth inhibition, upon Flg22, chitin, chitosan, or nlp24 application (Di et al., 2017;Tintor et al., 2020;Coleman et al., 2021;de Lamo et al., 2021). Besides its PTI-suppressing activity, Avr2 also acts as an avirulence factor upon its recognition in the plant nucleus by the resistance protein I-2 (Houterman et al., 2009;Ma et al., 2015) inducing effector-triggered immunity (ETI). ...
... To test whether SIX5 expression affected the disease susceptibility of Arabidopsis, disease assays were performed using the Arabidopsis-infecting F. oxysporum strain Fo5167 (Thatcher et al., 2009). Since Six5 has been reported to function in conjunction with Avr2 (Cao et al., 2018), a transgenic AVR2expressing Fo5167 was included in the assays (Figure 1 and Supplementary Figure 2). ...
Pathogens produce effector proteins to manipulate their hosts. While most effectors act autonomously, some fungal effectors act in pairs and rely on each other for function. During the colonization of the plant vasculature, the root-infecting fungus Fusarium oxysporum (Fo) produces 14 so-called Secreted in Xylem (SIX) effectors. Two of these effector genes, Avr2 (Six3) and Six5 , form a gene pair on the pathogenicity chromosome of the tomato-infecting Fo strain. Avr2 has been shown to suppress plant defense responses and is required for full pathogenicity. Although Six5 and Avr2 together manipulate the size exclusion limit of plasmodesmata to facilitate cell-to-cell movement of Avr2, it is unclear whether Six5 has additional functions as well. To investigate the role of Six5, we generated transgenic Arabidopsis lines expressing Six5 . Notably, increased susceptibility during the early stages of infection was observed in these Six5 lines, but only to Fo strains expressing Avr2 and not to wild-type Arabidopsis-infecting Fo strains lacking this effector gene. Furthermore, neither PAMP-triggered defense responses, such as ROS accumulation and callose deposition upon treatment with Flg22, necrosis and ethylene-inducing peptide 1-like protein (NLP), or chitosan, nor susceptibility to other plant pathogens, such as the bacterium Pseudomonas syringae or the fungus Verticilium dahlia , were affected by Six5 expression. Further investigation of the ability of the Avr2/Six5 effector pair to manipulate plasmodesmata (PD) revealed that it not only permits cell-to-cell movement of Avr2, but also facilitates the movement of two additional effectors, Six6 and Six8. Moreover, although Avr2/Six5 expands the size exclusion limit of plasmodesmata (i.e., gating) to permit the movement of a 2xFP fusion protein (53 kDa), a larger variant, 3xFP protein (80 kDa), did not move to the neighboring cells. The PD manipulation mechanism employed by Avr2/Six5 did not involve alteration of callose homeostasis in these structures. In conclusion, the primary function of Six5 appears to function together with Avr2 to increase the size exclusion limit of plasmodesmata by an unknown mechanism to facilitate cell-to-cell movement of Fo effectors.
... In order to identify and characterize additional Avr effectors of Fo5176, Tintor et al. (2020) further analyzed the Fo5176 genome sequence and, in addition to the four previously described Six genes, identified four novel effector candidates designated FoaEffector1-4 (Foa1-Foa4). When expressed transiently in N. benthamiana leaves, Foa2 and Foa3 suppressed the flg22-and chitin-induced ROS burst, while Six1 and Foa1 suppressed the flg22-induced burst, but not the chitin-induced burst. ...
... When expressed transiently in N. benthamiana leaves, Foa2 and Foa3 suppressed the flg22-and chitin-induced ROS burst, while Six1 and Foa1 suppressed the flg22-induced burst, but not the chitin-induced burst. Additionally, Six1 and Foa1 had to be targeted to the apoplast to suppress the immune response, while Foa2 and 3 acted both in the apoplast and intracellularly (Tintor et al., 2020). This mode of action was confirmed in A. thaliana for Foa2, where flg22-and chitin-induced ROS burst and MPK3/6 phosphorylation were dampened in the presence of the Foa2 transgene. ...
... This mode of action was confirmed in A. thaliana for Foa2, where flg22-and chitin-induced ROS burst and MPK3/6 phosphorylation were dampened in the presence of the Foa2 transgene. Using an elegant in vivo effector labeling approach, the authors furthermore confirmed that Foa2 and 3 are injected into the cell by Fo5176, while Six1 and Foa1 could only be detected in traces intracellularly, thereby confirming their action in the apoplast (Tintor et al., 2020). The function of Six4 could not be clearly determined in this study, but the authors could not confirm its earlier described pattern-triggered immunity (PTI) suppression function (Thatcher et al., 2012a). ...
Fusarium oxysporum is a soil-borne fungal pathogen of several major food crops. Research on understanding the molecular details of fungal infection and the plant’s defense mechanisms against this pathogen has long focused mainly on the tomato-infecting F. oxysporum strains and their specific host plant. However, in recent years, the Arabidopsis thaliana - Fusarium oxysporum strain 5176 pathosystem has additionally been established to study this plant-pathogen interaction with all the molecular biology, genetic and genomic tools available for the A. thaliana model system. Work on this system has since produced several new insights, especially in regards to the role of phytohormones involved in the plant’s defense response, and the receptor proteins and peptide ligands involved in pathogen-detection. Furthermore, work with the pathogenic strain Fo5176 and the related endophytic strain Fo47 has demonstrated the suitability of this system for comparative studies of the plant’s specific responses to general microbe- or pathogen-associated molecular patterns. In this review, we highlight the advantages of this specific pathosystem, summarize the advances made in studying the molecular details of this plant-fungus interaction, and point out open questions that remain to be answered.
... Individual strains carry partially overlapping effector sets, potentially determining their host range (Van Dam et al., 2016). Several Fo effectors were shown to act intracellularly as (a)virulence factors (Houterman et al., 2009;Di et al., 2017;Tintor et al., 2020). However, it remains unknown how they enter plant cells, since they do not share obvious motifs, or have common physio-chemical properties such as size or charge. ...
... However, it remains unknown how they enter plant cells, since they do not share obvious motifs, or have common physio-chemical properties such as size or charge. Here we focus on Foa3, an effector of the Arabidopsis-infecting strain Fo5176 that inhibits both the flg22-and chitin-triggered ROS burst when expressed in planta without its endogenous signal peptide, indicating that it acts via an intracellular mechanism (Tintor et al., 2020). However, expression of full length Foa3 (e.g. ...
... To generate SPpr1-NS24-Foa3, the backbone of pBIN-SPpr1-NS24-YFP was amplified by PCR using primers FP8847/FP8848. The Foa3 coding sequence (genbank ID: FOXB_16928) lacking the signal peptide sequence (dsp for deleted sp) was amplified from pBIN-dspFoa3-HB (Tintor et al., 2020) with primers FP8849/FP8850, thereby introducing overlaps with NS24 and the vector backbone. Finally, these two fragments were fused using the In-Fusion technology, following the manufacturer's instructions (New England Biolabs). ...
Plant pathogens employ secreted proteins, among which are effectors, to manipulate and colonize their hosts. A large fraction of effectors is translocated into host cells, where they can suppress defense signaling. Bacterial pathogens directly inject effectors into host cells via the type three secretion system, but it is little understood how eukaryotic pathogens, such as fungi, accomplish this critical process and how their secreted effectors enter host cells. The root-infecting fungus Fusarium oxysporum ( Fo ) secrets numerous effectors into the extracellular space. Some of these, such as Foa3, function inside the plant cell to suppress host defenses. Here, we show that Foa3 suppresses pattern-triggered defense responses to the same extent when it is produced in planta irrespective of whether the protein carries the PR1 secretory signal peptide or not. When a GFP-tagged Foa3 was targeted for secretion it localized, among other locations, to mobile subcellular structures of unknown identity. Furthermore, like the well-known cell penetrating peptide Arginine 9, Foa3 was found to deliver an orthotospovirus avirulence protein-derived peptide into the cytosol, resulting in the activation of the matching resistance protein. Finally, we show that infiltrating Foa3 into the apoplast results in strong suppression of the pattern-triggered immune responses, potentially indicating its uptake by the host cells in absence of a pathogen.
... Interaction of Foc with the host entails coordinated expression of several genes in both pathogen and host (Di Pietro et al. 2003). During the invasion, the pathogen secretes small effector proteins, majority of which are cysteine rich, which enable the pathogen to successfully invade the host plant (Tintor et al. 2020). The effector proteins can alter the structure and function of host cells to promote pathogenicity. ...
Vascular wilt disease caused by Fusarium oxysporum f. sp. carthami (Foc) is one of the biggest constraints for safflower production in India. Understanding the basis of pathogenicity and molecular dissection of its complex processes is of immense economic importance for the effective management of the wilt disease in safflower. In this study, a forward genetic approach was employed as an unbiased tool to identify the candidate pathogenicity-related genes. Agrobacterium mediated random T-DNA mutagenesis in Foc resulted in the generation of 178 Foc transformants. A hydroponics-based pathogenicity screening of generated mutants led to the identification of 12 avirulent mutants. Genome walking with two of the single insertion mutants revealed T-DNA insertion in the intergenic region of one mutant, while in the other mutant T-DNA was inserted in the coding region of a transcription factor. The genes identified in the present study can be targeted by host-delivered RNAi to generate transgenic safflower lines resistant to Foc.
... For instance, the SIX proteins, the first set of effectors described in F. oxysporum (Rep et al. 2004), are encoded by genes localized on Chromosome 14, an AC (Ma et al. 2010;Schmidt et al. 2013). Among them, SIX1, SIX3, SIX5, and SIX6 confer full virulence to Fol4287 (Rep et al. 2004;Ma et al. 2013;Gawehns et al. 2014), and SIX1 suppresses plant immunity (Tintor et al. 2020). Another important group of effectors in the FOSC are enzymes involved in the degradation of plant compounds (such as carbohydrate-active enzymes, i.e., CAZymes) (Ma lab, unpublished). ...
The genome of Fusarium oxysporum, an ascomycete fungus, can be divided into two compartments: core chromosomes (CCs) and accessory chromosomes (ACs). CCs are conserved, vertically transmitted from parent to offspring, and involved in essential housekeeping functions, whereas lineage- or strain-specific ACs are horizontally transmitted and associated with specialized functions. These two genomic compartments differ in terms of gene density, transposable element distribution, and epigenetic markers. Although commonly observed in eukaryotes, the functional importance of ACs stands out among phytopathogenic fungi, especially in relation to their pathogenicity and adaptability to hosts and other environmental conditions. Recent studies confirmed that these structural and functional variations observed at the genomic level contribute to the colonization of both plant and human hosts by different F. oxysporum strains, most likely through coordination and crosstalk between these two compartments. In this review, we focus on the cross-kingdom fungal pathogenicity of F. oxysporum, providing a summary of the genome dynamics of F. oxysporum and describing how these dynamics shape the host niche through molecular dialogues.KeywordsAccessory chromosomeCross-kingdom fungal pathogenicityFungal genome compartmentalization
Fusarium oxysporum
Genome crosstalkHost niche adaptation
... effector Pst18363 has been shown to target and stabilize the negative defense regulator TaNUDX23 (Nudex hydrolase), which inhibits ROS accumulation to promote pathogen infection [303]. The F. oxysporum apoplasts (SIX1 and Foa1) and cytoplasmic effectors (Avr2, Foa2 and Foa3) promote host colonization by inhibiting Flg22-or chitin-induced ROS [304]. Recent studies have shown that AVR-Pita, an effector of M. oryzae, interacts with the cytochrome c oxidase (COX) assembly protein OsCOX11, a key regulator of reactive oxygen metabolism in rice mitochondria [305]. ...
Plant pathogens are one of the main factors hindering the breeding of cash crops. Pathogens, including oomycetes, fungus, and bacteria, secrete effectors as invasion weapons to successfully invade and propagate in host plants. Here, we review recent advances made in the field of plant-pathogen interaction models and the action mechanisms of phytopathogenic effectors. The review illustrates how effectors from different species use similar and distinct strategies to infect host plants. We classify the main action mechanisms of effectors in plant-pathogen interactions according to the infestation process: targeting physical barriers for disruption, creating conditions conducive to infestation, protecting or masking themselves, interfering with host cell physiological activity, and manipulating plant downstream immune responses. The investigation of the functioning of plant pathogen effectors contributes to improved understanding of the molecular mechanisms of plant-pathogen interactions. This understanding has important theoretical value and is of practical significance in plant pathology and disease resistance genetics and breeding.
... Recently, SIX1 and three other effectors (Foa1,Foa2,and Foa3) from Fo f. sp. conglutinans were found to suppress the pattern-triggered immunity (PTI)-associated oxidative burst in Arabidopsis thaliana (Tintor et al., 2020). Moreover, the SIX5-Avr2 effector pair alters plasmodesmatal exclusion to promote cell-to cell movement of Avr2 (Cao et al., 2018). ...
Fungal interactions with plant roots, either beneficial or detrimental, have a crucial impact on agriculture and ecosystems. The cosmopolitan plant pathogen Fusarium oxysporum (Fo) provokes vascular wilts in more than a hundred different crops. Isolates of this fungus exhibit host-specific pathogenicity, which is conferred by lineage-specific Secreted In Xylem (SIX) effectors encoded on accessory genomic regions. However, such isolates also can colonize the roots of other plants asymptomatically as endophytes or even protect them against pathogenic strains. The molecular determinants of endophytic multi-host compatibility are largely unknown. Here we characterized a set of Fo candidate effectors from tomato (Solanum lycopersicum) root apoplastic fluid; these Early Root Colonization effectors (ERCs) are secreted during early biotrophic growth on main and alternative plant hosts. In contrast to SIX effectors, ERCs have homologs across the entire Fo species complex as well as in other plant-interacting fungi, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence and rapid activation of plant immune responses, while ERC deletion in a non-pathogenic isolate led to impaired root colonization and biocontrol ability. Strikingly, some ERCs contribute to Fo infection on the non-vascular land plant Marchantia polymorpha, revealing an evolutionarily conserved mechanism for multi-host colonization by root infecting fungi.
Fusarium oxysporum f. sp. niveum (Fon), a soilborne phytopathogenic fungus, causes watermelon Fusarium wilt, resulting in serious yield losses worldwide. However, the underlying molecular mechanism of Fon virulence is largely unknown. The present study investigated the biological functions of six FonPUFs, encoding RNA binding Pumilio proteins, and especially explored the molecular mechanism of FonPUF1 in Fon virulence. A series of phenotypic analyses indicated that FonPUFs have distinct but diverse functions in vegetative growth, asexual reproduction, macroconidia morphology, spore germination, cell wall, or abiotic stress response of Fon. Notably, the deletion of FonPUF1 attenuates Fon virulence by impairing the invasive growth and colonization ability inside the watermelon plants. FonPUF1 possesses RNA binding activity, and its biochemical activity and virulence function depend on the RNA recognition motif or Pumilio domains. FonPUF1 associates with the actin-related protein 2/3 (ARP2/3) complex by interacting with FonARC18, which is also required for Fon virulence and plays an important role in regulating mitochondrial functions, such as ATP generation and reactive oxygen species production. Transcriptomic profiling of ΔFonPUF1 identified a set of putative FonPUF1-dependent virulence-related genes in Fon, possessing a novel A-rich binding motif in the 3' untranslated region (UTR), indicating that FonPUF1 participates in additional mechanisms critical for Fon virulence. These findings highlight the functions and molecular mechanism of FonPUFs in Fon virulence. IMPORTANCE Fusarium oxysporum is a devastating plant-pathogenic fungus that causes vascular wilt disease in many economically important crops, including watermelon, worldwide. F. oxysporum f. sp. nievum (Fon) causes serious yield loss in watermelon production. However, the molecular mechanism of Fusarium wilt development by Fon remains largely unknown. Here, we demonstrate that six putative Pumilio proteins-encoding genes (FonPUFs) differentially operate diverse basic biological processes, including stress response, and that FonPUF1 is required for Fon virulence. Notably, FonPUF1 possesses RNA binding activity and associates with the actin-related protein 2/3 complex to control mitochondrial functions. Furthermore, FonPUF1 coordinates the expression of a set of putative virulence-related genes in Fon by binding to a novel A-rich motif present in the 3' UTR of a diverse set of target mRNAs. Our study disentangles the previously unexplored molecular mechanism involved in regulating Fon virulence, providing a possibility for the development of novel strategies for disease management.
Host apoplastic nutrients might influence the proliferating biotroph, therefore, we analysed the effect of host apoplastic nutrients on the virulence strategy of E. orontii (a biotroph model). E. orontii was initially treated with the analogs of commonly found apoplastic nutrients in a culture media. The analogs included Cyanoalanine (CAL), Trehalose, H2O2, acidic water (0.3% H3PO4), NaNO3, and MgSO4. After pre-treatment with the analogs of apoplastic nutrients, E. orontii was then inoculated to the host plants. After 5 and 8 days of post-infection (dpi), the virulence of E. orontii was determined through the expression of 20 virulence factor and 7 host defence genes. The expression of AVRk1, Ekal1, Ekal2, Ekal3, Ekal4, Ekal5, Ekal6, Ekal7, Ekal8, Epul5, Epul6, Epul7, Epul8, Epul9, Epul10, Epul11, Epul12, Epul5, Epul17 was determined as a virulence factor and expression of PR1, ERF1, PDF1.2a, PMRs, DMRs, IOS1, MYB3R4 was determined as host defence genes. The results revealed that the tested analogs vigorously affected the phenotypic and genotypic properties of E. orontii and thus its biotrophic virulence strategy. Certainly, the exogenous application of fungal growth-promoting apoplastic nutrients (CAL or TRE) severely reduced the virulence of E. orontii. Contrarily, the stress-inducing apoplastic analogs (H2O2 or acidic water) surprisingly increased the virulence of E. orontii. Moreover, the application of NaNO3 or MgSO4 decoyed the fungal growth and thus decreasing the E. orontii virulence.
Apple replant disease (ARD) is a complex syndrome caused by various biotic and abiotic stresses contained in replanted soil, leading to reduced plant growth and fruit yields and causing serious economic loss. Breeding disease-resistant varieties is an effective and practical method to control ARD. Effective plant defense depends in part on the plant immune responses induced by the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). BAK1 participates in the regulation of plant immunity as an important PRR-binding protein. In this study, MdBAK1 overexpression activated indeterminate immune responses in tissue-cultured apple plants. MdBAK1-overexpressing rooted apple plants exhibited enhanced resistance to ARD, as the inhibition of plant growth was significantly alleviated during the replanted soil treatment. In addition, MdBAK1-overexpressing apple plants showed abolished growth inhibition, wilting and root rot induced by Fusarium oxysporum, which is the main pathogen that causes ARD in China. MdBAK1 overexpression changed the microbial community structure in the rhizosphere soil, as reflected by the increase in bacterial content and the decrease in fungal content, and the root exudates of MdBAK1-overexpressing plants inhibited F. oxysporum spore germination compared with that of wild-type plants. Furthermore, the constitutive immunity and cell necrosis induced by the upregulation of MdBAK1 expression were involved in the inhibition of colonization and expansion of F. oxysporum in host plants. In short, MdBAK1 plays an important role in the regulation of apple resistance to ARD, suggesting that MdBAK1 may be a valuable gene for molecular breeding of ARD resistance.
Recognition of microbe-associated molecular patterns (MAMPs) is crucial for the plant's immune response. How this sophisticated perception system can be usefully deployed in roots, continuously exposed to microbes, remains a mystery. By analyzing MAMP receptor expression and response at cellular resolution in Arabidopsis, we observed that differentiated outer cell layers show low expression of pattern-recognition receptors (PRRs) and lack MAMP responsiveness. Yet, these cells can be gated to become responsive by neighbor cell damage. Laser ablation of small cell clusters strongly upregulates PRR expression in their vicinity, and elevated receptor expression is sufficient to induce responsiveness in non-responsive cells. Finally, localized damage also leads to immune responses to otherwise non-immunogenic, beneficial bacteria. Damage-gating is overridden by receptor overexpression, which antagonizes colonization. Our findings that cellular damage can "switch on" local immune responses helps to conceptualize how MAMP perception can be used despite the presence of microbial patterns in the soil.
Environmental adaptation of organisms relies on fast perception and response to external signals, which lead to developmental changes. Plant cell growth is strongly dependent on cell wall remodeling. However, little is known about cell wall-related sensing of biotic stimuli and the downstream mechanisms that coordinate growth and defense responses. We generated genetically encoded pH sensors to determine absolute pH changes across the plasma membrane in response to biotic stress. A rapid apoplastic acidification by phosphorylation-based proton pump activation in response to the fungus Fusarium oxysporum immediately reduced cellulose synthesis and cell growth and, furthermore, had a direct influence on the pathogenicity of the fungus. In addition, pH seems to influence cellulose structure. All these effects were dependent on the COMPANION OF CELLULOSE SYNTHASE proteins that are thus at the nexus of plant growth and defense. Hence, our discoveries show a remarkable connection between plant biomass production, immunity, and pH control, and advance our ability to investigate the plant growth-defense balance.
Significance
Multiple effectors of bacterial pathogens target immune kinases such as BAK1 and BIK1, but it is unclear whether this strategy is employed by fungal pathogens. We reveal here that a fungal effector named NIS1 is broadly conserved in filamentous fungi in the Ascomycota and Basidiomycota, thus being regarded as a core effector, and has the ability to suppress PAMP-triggered immunity. Importantly, NIS1 targets BAK1 and BIK1, interfering with their essential functions for immune activation upon pathogen recognition. Multifaceted analyses including the knockout of NIS1 revealed that it plays a critical role in fungal infection. These findings demonstrate that to infect host plants, filamentous fungi deploy a core effector that attacks conserved immune kinases critical for the ancestral defense system.
The potato blight pathogen Phytophthora infestans secretes effector proteins that are delivered inside (cytoplasmic) or can act outside (apoplastic) plant cells to neutralize host immunity. Little is known about how and where effectors are secreted during infection, yet such knowledge is essential to understand and combat crop disease.
We used transient Agrobacterium tumefaciens‐mediated in planta expression, transformation of P. infestans with fluorescent protein fusions and confocal microscopy to investigate delivery of effectors to plant cells during infection.
The cytoplasmic effector Pi04314, expressed as a monomeric red fluorescent protein (mRFP) fusion protein with a signal peptide to secrete it from plant cells, did not passively re‐enter the cells upon secretion. However, Pi04314‐mRFP expressed in P. infestans was translocated from haustoria, which form intimate interactions with plant cells, to accumulate at its sites of action in the host nucleus. The well‐characterized apoplastic effector EPIC1, a cysteine protease inhibitor, was also secreted from haustoria. EPIC1 secretion was inhibited by brefeldin A (BFA), demonstrating that it is delivered by conventional Golgi‐mediated secretion. By contrast, Pi04314 secretion was insensitive to BFA treatment, indicating that the cytoplasmic effector follows an alternative route for delivery into plant cells.
Phytophthora infestans haustoria are thus sites for delivery of both apoplastic and cytoplasmic effectors during infection, following distinct secretion pathways.
The Fusarium oxysporum species complex includes both plant pathogenic and nonpathogenic strains, which are commonly found in soils. F. oxysporum has received considerable attention from plant pathologists for more than a century owing to its broad host range and the economic losses it causes. The narrow host specificity of pathogenic strains has led to the concept of formae speciales, each forma specialis grouping strains with the same host range. Initially restricted to one plant species, this host range was later found to be broader for many formae speciales. In addition, races were identified in some formae speciales, generally with cultivar-level specialization. In 1981, Armstrong and Armstrong listed 79 F. oxysporum formae speciales and mentioned races in 16 of them. Since then, the known host range of F. oxysporum has considerably increased, and many new formae speciales and races have been identified. We carried out a comprehensive search of the literature to propose this review of F. oxysporum formae speciales and races. We recorded 106 well-characterized formae speciales, together with 37 insufficiently documented ones, and updated knowledge on races and host ranges. We also recorded 58 plant species/genera susceptible to F. oxysporum but for which a forma specialis has not been characterized yet. This review raises issues regarding the nomenclature and the description of F. oxysporum formae speciales and races.
Pathogens use effector proteins to manipulate their hosts. During infection of tomato the fungus Fusarium oxysporum secretes the effectors Avr2 and Six5. Whereas Avr2 suffices to trigger I-2-mediated cell death in heterologous systems, both effectors are required for I-2-mediated disease resistance in tomato. How Six5 participates in triggering resistance is unknown. Using BiFC assays we found that Avr2 and Six5 interact at plasmodesmata. Single-cell transformation revealed that a 2xRFP marker protein and Avr2-GFP only move to neighboring cells in the presence of Six5. Six5 alone does not alter plasmodesmatal transductivity as 2xRFP was only translocated in the presence of both effectors. In SIX5-expressing transgenic plants the distribution of virally expressed Avr2-GFP, and subsequent onset of I-2-mediated cell death, differed from that in wild type tomato. Together our data shows that in the presence of Six5 Avr2 moves from cell-to-cell, which in susceptible plants contributes to virulence, but in I-2 containing plants induces resistance.
Plant pathogens employ effector proteins to manipulate their hosts. Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, produces effector protein Avr2. Besides being a virulence factor, Avr2 triggers immunity in I‐2 carrying tomato (Solanum lycopersicum). Fol strains that evade I‐2 recognition carry point mutations in Avr2 (e.g. Avr2R45H), but retain full virulence.
Here we investigate the virulence function of Avr2 and determine its crystal structure. Transgenic tomato and Arabidopsis expressing either wild‐type ΔspAvr2 (deleted signal‐peptide) or the ΔspAvr2R45H variant become hypersusceptible to fungal, and even bacterial infections, suggesting that Avr2 targets a conserved defense mechanism. Indeed, Avr2 transgenic plants are attenuated in immunity‐related readouts, including flg22‐induced growth inhibition, ROS production and callose deposition.
The crystal structure of Avr2 reveals that the protein shares intriguing structural similarity to ToxA from the wheat pathogen Pyrenophora tritici‐repentis and to TRAF proteins. The I‐2 resistance‐breaking Avr2V41M, Avr2R45H and Avr2R46P variants cluster on a surface‐presented loop. Structure‐guided mutagenesis enabled uncoupling of virulence from I‐2‐mediated recognition.
We conclude that I‐2‐mediated recognition is not based on monitoring Avr2 virulence activity, which includes suppression of immune responses via an evolutionarily conserved effector target, but by recognition of a distinct epitope.
In both plants and animals, defense against pathogens relies on a complex surveillance system for signs of danger. Danger signals may originate from the infectious agent or from the host itself. Immunogenic plant host factors can be roughly divided into two categories: molecules which are passively released upon cell damage ('classical' damage-associated molecular patterns, DAMPs), and peptides which are processed and/or secreted upon infection to modulate the immune response (phytocytokines). We highlight the ongoing challenge to understand how plants sense various danger signals and integrate this information to produce an appropriate immune response to diverse challenges.
The innate immune system of plants recognizes microbial pathogens and terminates their growth. However, recent findings suggest that at least one layer of this system is also engaged in cooperative plant-microbe interactions and influences host colonization by beneficial microbial communities. This immune layer involves sensing of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) that initiate quantitative immune responses to control host-microbial load, whereas diversification of MAMPs and PRRs emerges as a mechanism that locally sculpts microbial assemblages in plant populations. This suggests a more complex microbial management role of the innate immune system for controlled accommodation of beneficial microbes and in pathogen elimination. The finding that similar molecular strategies are deployed by symbionts and pathogens to dampen immune responses is consistent with this hypothesis but implies different selective pressures on the immune system due to contrasting outcomes on plant fitness. The reciprocal interplay between microbiota and the immune system likely plays a critical role in shaping beneficial plant-microbiota combinations and maintaining microbial homeostasis. Expected final online publication date for the Annual Review of Phytopathology Volume 55 is August 4, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.