[Show abstract][Hide abstract] ABSTRACT: Background:
Fusarium crown rot (FCR) is a major cereal disease in semi-arid areas worldwide. Of the various QTL reported, the one on chromosome arm 3BL (Qcrs.cpi-3B) has the largest effect that can be consistently detected in different genetic backgrounds. Nine sets of near isogenic lines (NILs) for this locus were made available in a previous study. To identify markers that could be reliably used in tagging the Qcrs.cpi-3B locus, a NIL-derived population consisting of 774 F10 lines were generated and exploited to assess markers selected from the existing linkage map and generated from sequences of the 3B pseudomolecule.
This is the first report on fine mapping a QTL conferring FCR resistance in wheat. By three rounds of linkage mapping using the NILs and the NIL-derived population, the Qcrs.cpi-3B locus was mapped to an interval of 0.7 cM covering a physical distance of about 1.5 Mb. Seven markers co-segregating with the locus were developed. This interval contains a total of 63 gene-coding sequences based on the 3B pseudomolecule, and six of them were known to encode disease resistance proteins. Several of the genes in this interval were among those responsive to FCR infection detected in an earlier study.
The accurate localization of the Qcrs.cpi-3B locus and the development of the markers co-segregating with it should facilitate the incorporation of this large-effect QTL conferring FCR resistance into breeding programs as well as the cloning of the gene(s) underlying the QTL.
[Show abstract][Hide abstract] ABSTRACT: Sugarcane (Saccharum spp. hybrids) accumulates high concentrations of sucrose in its mature stalk and a considerable portion of carbohydrate metabolism is also devoted to cell wall synthesis and fibre production. We examined tissue-specific expression patterns to explore the spatial deployment of pathways responsible for sucrose accumulation and fibre synthesis within the stalk. We performed expression profiling of storage parenchyma, vascular bundles and rind dissected from a maturing stalk internode of sugarcane, identifying ten cellulose synthase subunit genes and examining significant differences in the expression of their corresponding transcripts and those of several sugar transporters. These were correlated with differential expression patterns for transcripts of genes encoding COBRA-like proteins and other cell wall metabolism-related proteins. The sugar transporters genes ShPST2a, ShPST2b and ShSUT4 were significantly up-regulated in storage parenchyma while ShSUT1 was up-regulated in vascular bundles. Two co-ordinately expressed groups of cell wall related transcripts were also identified. One group, associated with primary cell wall synthesis (ShCesA1, ShCesA7, ShCesA9 and Shbk2l3), was up-regulated in parenchyma. The other group, associated with secondary cell wall synthesis (ShCesA10, ShCesA11, ShCesA12 and Shbk-2), was up-regulated in rind. In transformed sugarcane plants, the ShCesA7 promoter conferred stable expression of green fluorescent protein preferentially in the storage parenchyma of the maturing stalk internode. Our results indicate that there is spatial separation for elevated expression of these important targets in both sucrose accumulation and cell wall synthesis, allowing for increased clarity in our understanding of sucrose transport and fibre synthesis in sugarcane.
[Show abstract][Hide abstract] ABSTRACT: Brachypodium distachyon has been widely adopted as a pathosystem for study of cereal pathogens. Fusarium crown rot (FCR), primarily caused by the necrotrophic fungal pathogen Fusarium pseudograminearum, constitutes a major disease risk for the Australian wheat industry. The aim of this work was to assess whether species in the genus Brachypodium will be a useful model for characterizing the molecular basis for resistance to F. pseudograminearum. Two Brachypodium species were found to be readily infected by F. pseudograminearum producing similar symptoms and disease progression to that observed in wheat and barley. A large number of Turkish natural accessions were screened for FCR susceptibility and several ecotypes were found to differ significantly in disease severity. Brachypodium hybridium accessions exhibited significantly greater quantitative resistance compared with B. distachyon accessions. RNAseq was utilized to observe the host molecular response during FCR infection in wheat and B. distachyon. Comparing induction of homologs in wheat and Brachypodium revealed a high degree of similarity in response between crop and model. However, significant differences for induction of defence related phytohormones and other defence related metabolites were observed using liquid chromatography – mass spectrometry (LC-MS). B. distachyon transformants constitutively expressing a salicylate hydroxylase (nahG) from Pseudomonas putida were developed (Bd21-3 background) to assess the importance of salicylic acid mediated defence responses in promoting resistance against necrotrophic pathogens. The potential utility and limitations for Brachypodium as a molecular model for studying cereal-pathogen interactions are discussed in light of observed similarities and differences.
International Brachypodium Conference 2015; 06/2015
[Show abstract][Hide abstract] ABSTRACT: Plants respond to pathogens either by investing more resources into immunity which is costly to development, or by accelerating reproductive processes such as flowering time to ensure reproduction occurs before the plant succumbs to disease. In this study we explored the link between flowering time and pathogen defense using the interaction between Arabi-dopsis thaliana and the root infecting fungal pathogen Fusarium oxysporum. We report that F. oxysporum infection accelerates flowering time and regulates transcription of a number of floral integrator genes, including FLOWERING LOCUS C (FLC), FLOWERING LOCUS T (FT) and GIGANTEA (GI). Furthermore, we observed a positive correlation between late flowering and resistance to F. oxysporum in A. thaliana natural ecotypes. Late-flowering gi and autonomous pathway mutants also exhibited enhanced resistance to F. oxysporum, supporting the association between flowering time and defense. However, epistasis analysis showed that accelerating flowering time by deletion of FLC in fve-3 or fpa-7 mutants did not alter disease resistance, suggesting that the effect of autonomous pathway on disease resistance occurs independently from flowering time. Indeed, RNA-seq analyses suggest that fve-3 mediated resistance to F. oxysporum is most likely a result of altered defense-associated gene transcription. Together, our results indicate that the association between flowering time and pathogen defense is complex and can involve both pleiotropic and direct effects.
PLoS ONE 06/2015; 10(6). DOI:10.1371/journal.pone.0127699 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Some of the most devastating agricultural diseases are caused by root-infecting pathogens, yet the majority of studies on these interactions to date have focused on the host responses of aerial tissues rather than those belowground. Fusarium oxysporum is a root-infecting pathogen that causes wilt disease on several plant species including Arabidopsis thaliana. To investigate and compare transcriptional changes triggered by F. oxysporum in different Arabidopsis tissues, we infected soil-grown plants with F. oxysporum and subjected root and leaf tissue harvested at early and late timepoints to RNA-seq analyses. At least half of the genes induced or repressed by F. oxysporum showed tissue-specific regulation. Regulators of auxin and ABA signalling, mannose binding lectins and peroxidases showed strong differential expression in root tissue. We demonstrate that ARF2 and PRX33, two genes regulated in the roots, promote susceptibility to F. oxysporum. In the leaves, defensins and genes associated with the response to auxin, cold and senescence were strongly regulated while jasmonate biosynthesis and signalling genes were induced throughout the plant.
PLoS ONE 04/2015; 10(4-4):e0121902. DOI:10.1371/journal.pone.0121902 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed.
PLoS ONE 02/2015; 10(2):e0117369. DOI:10.1371/journal.pone.0117369 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Wheat, maize, rye, and some other agriculturally important species in the Poaceae family produce the benzoxazolinone class of phytoalexins upon pest and pathogen attack. Benzoxazolinones can inhibit the growth of pathogens. However, certain fungi can actively detoxify these compounds. Despite this, a clear link between the ability to detoxify benzoxazolinones and pathogen virulence has not been shown. Here, through comparative genome analysis of several Fusarium species, we have identified a conserved genomic region around the FDB2 gene encoding a N-malonyltransferase enzyme known to be involved in benzoxazolinone degradation in the maize pathogen Fusarium verticillioides. Expression analyses demonstrated that a cluster of nine genes were responsive to exogenous benzoxazolinone in the important wheat pathogen Fusarium pseudograminearum. Analysis of independent F. pseudograminearum FDB2 knockouts and complementation of the knockout with FDB2 homologs from F. graminearum and F. verticillioides confirmed that the N-malonyltransferase enzyme encoded by this gene is central to the detoxification of benzoxazolinones, and that Fdb2 contributes quantitatively to virulence towards wheat in head blight inoculation assays. This contrasts with previous observations in F. verticillioides, where no effect of FDB2 mutations on pathogen virulence towards maize was observed. Overall, our results demonstrate detoxification of benzoxazolinones is a strategy wheat infecting F. pseudograminearum has adopted to overcome host-derived chemical defences.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Fusarium pathogens cause two major diseases in cereals, Fusarium crown rot (FCR) and head blight (FHB). A large-effect locus conferring resistance to FCR disease was previously located to chromosome arm 3BL (designated as Qcrs-3B) and several independent sets of near isogenic lines (NILs) have been developed for this locus. In this study, five sets of the NILs were used to examine transcriptional changes associated with the Qcrs-3B locus and to identify genes linked to the resistance locus as a step towards the isolation of the causative gene(s). Of the differentially expressed genes (DEGs) detected between the NILs, 12.7% was located on the single chromosome 3B. Of the expressed genes containing SNP (SNP-EGs) detected, 23.5% was mapped to this chromosome. Several of the DEGs and SNP-EGs are known to be involved in host-pathogen interactions, and a large number of the DEGs were among those detected for FHB in previous studies. Of the DEGs detected, 22 were mapped in the Qcrs-3B interval and they included eight which were detected in the resistant isolines only. The enrichment of DEG, and not necessarily those containing SNPs between the resistant and susceptible isolines, around the Qcrs-3B locus is suggestive of local regulation of this region by the resistance allele. Functions for 13 of these DEGs are known. Of the SNP-EGs, 28 were mapped in the Qcrs-3B interval and biological functions for 16 of them are known. These results provide insights into responses regulated by the 3BL locus and identify a tractable number of target genes for fine mapping and functional testing to identify the causative gene(s) at this QTL.
PLoS ONE 11/2014; 9(11):e113309. DOI:10.1371/journal.pone.0113309 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defense proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialized gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control.
[Show abstract][Hide abstract] ABSTRACT: Studies in Arabidopsis show that DELLA genes may differentially affect responses to biotrophic and necrophic pathogens. A recent report based on the study of DELLA-producing reduced height (Rht) genes in wheat and barley also hypothesized that DELLA genes likely increased susceptibility to necrotrophs but increased resistance to biotrophs.
Effects of uzu, a non-GA (gibberellic acid)-responsive semi-dwarfing gene, on Fusarium crown rot (FCR) resistance in barley were investigated. Fifteen pairs of near isogenic lines for this gene were generated and assessed under two different temperature regimes. Similar to its impacts on plant height, the semi-dwarfing gene uzu also showed larger effects on FCR severity in the high temperature regime when compared with that in the low temperature regime.
Results from this study add to the growing evidence showing that the effects of plant height on Fusarium resistances are unlikely related to DELLA genes but due to direct or indirect effects of height difference per se. The interaction between these two characteristics highlights the importance of understanding relationships between resistance and other traits of agronomic importance as the value of a resistance gene could be compromised if it dramatically affects plant development and morphology.
[Show abstract][Hide abstract] ABSTRACT: In comparison to dicot-infecting bacteria, only limited numbers of genome sequences are available for monocot-infecting and in particular cereal-infecting bacteria. Herein we report the characterisation and genome sequence of Xanthomonas translucens isolate DAR61454 pathogenic on wheat and barley. Based on phylogenetic analysis of the ATP synthase beta subunit (atpD) gene, DAR61454 is most closely related to other X. translucens strains and the sugarcane- and banana- infecting Xanthomonas strains, but shares a type III secretion system (T3SS) with X. translucens pv. graminis and more distantly related xanthomonads. Assays with an adenylate cyclase reporter protein demonstrate that DAR61454's T3SS is functional in delivering proteins to wheat cells. X. translucens DAR61454 also encodes two type VI secretion systems with one most closely related to those found in some strains of the rice infecting strain X. oryzae pv. oryzae but not other xanthomonads. Comparative analysis of 18 different Xanthomonas isolates revealed 84 proteins unique to cereal (i.e. rice) infecting isolates and the wheat/barley infecting DAR61454. Genes encoding 60 of these proteins are found in gene clusters in the X. translucens DAR61454 genome, suggesting cereal-specific pathogenicity islands. However, none of the cereal pathogen specific proteins were homologous to known Xanthomonas spp. effectors. Comparative analysis outside of the bacterial kingdom revealed a nucleoside triphosphate pyrophosphohydrolase encoding gene in DAR61454 also present in other bacteria as well as a number of pathogenic Fusarium species, suggesting that this gene may have been transmitted horizontally from bacteria to the Fusarium lineage of pathogenic fungi. This example further highlights the importance of horizontal gene acquisition from bacteria in the evolution of fungi.
PLoS ONE 01/2014; 9(1):e84995. DOI:10.1371/journal.pone.0084995 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fungal pathogens cause devastating losses in economically important cereal crops by utilising pathogen proteins to infect host plants. Secreted pathogen proteins are referred to as effectors and have thus far been identified by selecting small, cysteine-rich peptides from the secretome despite increasing evidence that not all effectors share these attributes.
We take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov Model analyses followed by unsupervised protein clustering. Our method returns experimentally validated fungal effectors in Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen. Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens. Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing.
Our pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms.
[Show abstract][Hide abstract] ABSTRACT: Fusarium is a genus of filamentous fungi that contains many agronomically important plant pathogens, mycotoxin producers, and opportunistic human pathogens. Comparative analyses have revealed that the Fusarium genome is compartmentalized into regions responsible for primary metabolism and reproduction (core genome), and pathogen virulence, host specialization, and possibly other functions (adaptive genome). Genes involved in virulence and host specialization are located on pathogenicity chromosomes within strains pathogenic to tomato (Fusarium oxysporum f. sp. lycopersici) and pea (Fusarium 'solani' f. sp. pisi). The experimental transfer of pathogenicity chromosomes from F. oxysporum f. sp. lycopersici into a nonpathogen transformed the latter into a tomato pathogen. Thus, horizontal transfer may explain the polyphyletic origins of host specificity within the genus. Additional genome-scale comparative and functional studies are needed to elucidate the evolution and diversity of pathogenicity mechanisms, which may help inform novel disease management strategies against fusarial pathogens.
[Show abstract][Hide abstract] ABSTRACT: The constant interaction between plants and their pathogens has resulted in the evolution of a diverse array of microbial infection strategies. It is increasingly evident that horizontal acquisition of new virulence functions in fungi is one of the evolutionary processes that maintain pathogens' competitive edge over host plants. Genome analyses of fungi are pointing towards this phenomenon being particularly prevalent in the subphylum Pezizomycota. While the extent of cross-kingdom gene transfer can be determined with existing genomic tools and databases, so far very few horizontally transmitted genes have been functionally characterised, and an understanding of their physiological roles in virulence has been determined for even fewer genes. Understanding the evolutionary selection pressures that drive the retention of acquired genes in particular fungal lineages is important, as it will undoubtedly reveal new insights into both fungal virulence mechanisms and corresponding plant defence processes in the future.
[Show abstract][Hide abstract] ABSTRACT: Fusarium pathogens represent a major constraint to wheat and barley production worldwide. To facilitate future comparative studies of Fusarium species that are pathogenic to wheat, the genome sequences of four Fusarium pseudograminearum isolates, a single Fusarium acuminatum isolate, and an organism from the Fusarium incarnatum-F. equiseti species complex are reported.
[Show abstract][Hide abstract] ABSTRACT: FgABC1 (FGSG_04580) is predicted to encode a pleiotropic drug resistance-class ABC transporter in Fusarium graminearum, a globally important pathogen of wheat. Deletion mutants of FgABC1 showed reduced virulence towards wheat in crown and root infection assays but were unaltered in infectivity on barley. Expression of FgABC1 during head blight and crown rot disease increases during the necrotrophic phases of infection suggestive of a role for FgABC1 in late infection stages in different tissue types. Deletion of FgABC1 also led to increased sensitivity of the fungus to the antifungal compound benalaxyl in culture but the response to known cereal defence compounds, gramine, 2-benzoxazalinone and tryptamine, was un-altered. FgABC1 appears to have a role in protecting the fungus from antifungal compounds and is likely to help combat as yet unidentified wheat defence compounds during disease development. This article is protected by copyright. All rights reserved.