Plant infection by oomycete pathogens is a complex process. It requires precise expression of a plethora of genes in the pathogen that contribute to a successful interaction with the host. Whereas much effort has been made to uncover the molecular systems underlying this infection process, mechanisms of transcriptional regulation of the genes involved remain largely unknown. We performed the first systematic de-novo DNA motif discovery analysis inPhytophthora. To this end, we utilized the genome sequence of the late blight pathogenPhytophthora infestans and two related Phytophthora species (P. ramorum and P. sojae), as well as genome-wide in planta gene expression data to systematically predict 19 conserved DNA motifs. This catalog describes common eukaryotic promoter elements whose functionality is supported by the presence of orthologs of known general transcription factors. Together with strong functional enrichment of the common promoter elements towards effector genes involved in pathogenicity, we obtained a new and expanded picture of the promoter structure in P. infestans. More intriguingly, we identified specific DNA motifs that are either highly abundant or whose presence is significantly correlated with gene expression levels during infection. Several of these motifs are observed upstream of genes encoding transporters, RXLR effectors, but also transcriptional regulators. Motifs that are observed upstream of known pathogenicity-related genes are potentially important binding sites for transcription factors. Our analyses add substantial knowledge to the as of yet virtually unexplored question regarding general and specific gene regulation in this important class of pathogens. We propose hypotheses on the effects of cis-regulatory motifs on the gene regulation of pathogenicity-related genes and pinpoint motifs that are prime targets for further experimental validation.
Phytophthora infestans is the oomycete pathogen responsible for the devastating late blight disease on potato and tomato. There is presently an intense research focus on the role(s) of effectors in promoting late blight disease development. However, little is known about how they are regulated, or how diversity in their expression may be generated among different isolates. Here we present data from investigation of RNA silencing processes, characterized by non-coding small RNA molecules (sRNA) of 19–40 nt. From deep sequencing of sRNAs we have identified sRNAs matching numerous RxLR and Crinkler (CRN) effector protein genes in two isolates differing in pathogenicity. Effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Small RNAs in P. infestans grouped into three clear size classes of 21, 25/26 and 32 nt. Small RNAs from all size classes mapped to RxLR effector genes, but notably 21 nt sRNAs were the predominant size class mapping to CRN effector genes. Some effector genes, such as PiAvr3a, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. The P. infestans genome is rich in transposable elements, and the majority of sRNAs of all size classes mapped to these sequences, predominantly to long terminal repeat (LTR) retrotransposons. RNA silencing of Dicer and Argonaute genes provided evidence that generation of 21 nt sRNAs is Dicer-dependent, while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, we identified six microRNA (miRNA) candidates from our sequencing data, their precursor sequences from the genome sequence, and target mRNAs. These miRNA candidates have features characteristic of both plant and metazoan miRNAs.
Host specificity of plant pathogens can be dictated by genes that enable pathogens to circumvent host defenses. Upon recognition of a pathogen, plants initiate defense responses that can include the production of antimicrobial compounds such as phytoalexins. The pea pathogen Nectria haematococca mating population VI (MPVI) is a filamentous ascomycete that contains a cluster of genes known as the pea pathogenicity (PEP) cluster in which the pisatin demethylase (PDA) gene resides. The PDA gene product is responsible for the detoxification of the phytoalexin pisatin, which is produced by the pea plant (Pisum sativum L.). This detoxification activity allows the pathogen to evade the phytoalexin defense mechanism. It has been proposed that the evolution of PDA and the PEP cluster reflects horizontal gene transfer (HGT). Previous observations consistent with this hypothesis include the location of the PEP cluster and PDA gene on a dispensable portion of the genome (a supernumerary chromosome), a phylogenetically discontinuous distribution of the cluster among closely related species, and a bias in G + C content and codon usage compared to other regions of the genome. In this study we compared the phylogenetic history of PDA, beta-tubulin, and translation elongation factor 1-alpha in three closely related fungi (Nectria haematococca, Fusarium oxysporum, and Neocosmospora species) to formally evaluate hypotheses regarding the origin and evolution of PDA. Our results, coupled with previous work, robustly demonstrate discordance between the gene genealogy of PDA and the organismal phylogeny of these species, and illustrate how HGT of pathogenicity genes can contribute to the expansion of host specificity in plant-pathogenic fungi.
The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.
In 2007, Comparative Fungal Genomics Platform (CFGP; http://cfgp.snu.ac.kr/) was publicly open with 65 genomes corresponding to 58 fungal and Oomycete species. The CFGP provided six bioinformatics tools, including a novel tool entitled BLASTMatrix that enables search homologous genes to queries in multiple species simultaneously. CFGP also introduced Favorite, a personalized virtual space for data storage and analysis with these six tools. Since 2007, CFGP has grown to archive 283 genomes corresponding to 152 fungal and Oomycete species as well as 201 genomes that correspond to seven bacteria, 39 plants and 105 animals. In addition, the number of tools in Favorite increased to 27. The Taxonomy Browser of CFGP 2.0 allows users to interactively navigate through a large number of genomes according to their taxonomic positions. The user interface of BLASTMatrix was also improved to facilitate subsequent analyses of retrieved data. A newly developed genome browser, Seoul National University Genome Browser (SNUGB), was integrated into CFGP 2.0 to support graphical presentation of diverse genomic contexts. Based on the standardized genome warehouse of CFGP 2.0, several systematic platforms designed to support studies on selected gene families have been developed. Most of them are connected through Favorite to allow of sharing data across the platforms.
Modern agriculture favours the selection and spread of novel plant diseases. Furthermore, crop genetic resistance against pathogens is often rendered ineffective within a few years of its commercial deployment. Leptosphaeria maculans, the cause of phoma stem canker of oilseed rape, develops gene-for-gene interactions with its host plant, and has a high evolutionary potential to render ineffective novel sources of resistance in crops. Here, we established a four-year field experiment to monitor the evolution of populations confronted with the newly released Rlm7 resistance and to investigate the nature of the mutations responsible for virulence against Rlm7. A total of 2551 fungal isolates were collected from experimental crops of a Rlm7 cultivar or a cultivar without Rlm7. All isolates were phenotyped for virulence and a subset was genotyped with neutral genetic markers. Virulent isolates were investigated for molecular events at the AvrLm4-7 locus. Whilst virulent isolates were not found in neighbouring crops, their frequency had reached 36% in the experimental field after four years. An extreme diversity of independent molecular events leading to virulence was identified in populations, with large-scale Repeat Induced Point mutations or complete deletion of AvrLm4-7 being the most frequent. Our data suggest that increased mutability of fungal genes involved in the interactions with plants is directly related to their genomic environment and reproductive system. Thus, rapid allelic diversification of avirulence genes can be generated in L. maculans populations in a single field provided that large population sizes and sexual reproduction are favoured by agricultural practices.
Genome sequencing has been carried out on a small selection of major fungal ascomycete pathogens. These studies show that simple models whereby pathogens evolved from phylogenetically related saprobes by the acquisition or modification of a small number of key genes cannot be sustained.The genomes show that pathogens cannot be divided into three clearly delineated classes (biotrophs, hemibiotrophs and necrotrophs) but rather into a complex matrix of categories each with subtly different properties. It is clear that the evolution of pathogenicity is ancient, rapid and ongoing. Fungal pathogens have undergone substantial genomic rearrangements that can be appropriately described as ‘genomic tillage’. Genomic tillage underpins the evolution and expression of large families of genes – known as effectors – that manipulate and exploit metabolic and defence processes of plants so as to allow the proliferation of pathogens.
Pest and pathogen losses jeopardise global food security and ever since the 19th century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.
Comparative analyses of pathogen genomes provide new insights into how pathogens have evolved common and divergent virulence strategies to invade related plant species. Fusarium crown and root rots are important diseases of wheat and barley world-wide. In Australia, these diseases are primarily caused by the fungal pathogen Fusarium pseudograminearum. Comparative genomic analyses showed that the F. pseudograminearum genome encodes proteins that are present in other fungal pathogens of cereals but absent in non-cereal pathogens. In some cases, these cereal pathogen specific genes were also found in bacteria associated with plants. Phylogenetic analysis of selected F. pseudograminearum genes supported the hypothesis of horizontal gene transfer into diverse cereal pathogens. Two horizontally acquired genes with no previously known role in fungal pathogenesis were studied functionally via gene knockout methods and shown to significantly affect virulence of F. pseudograminearum on the cereal hosts wheat and barley. Our results indicate using comparative genomics to identify genes specific to pathogens of related hosts reveals novel virulence genes and illustrates the importance of horizontal gene transfer in the evolution of plant infecting fungal pathogens.
The eukaryotic microbes known as oomycetes are common inhabitants of terrestrial and aquatic environments, and include saprophytes and pathogens. Lifestyles of the pathogens extend from biotrophy to necrotrophy, obligate to facultative pathogenesis, and narrow to broad host ranges on plants or animals. Sequencing of several pathogens has revealed striking variation in genome size and content, a plastic set of genes related to pathogenesis, and adaptations associated with obligate biotrophy. Features of genome evolution include repeat-driven expansions, deletions, gene fusions, and horizontal gene transfer, in a landscape organized into gene-dense and gene-sparse sectors and influenced by transposable elements. Gene expression profiles are also highly dynamic throughout oomycete life cycles, with transcriptional polymorphisms as well as differences in protein sequence contributing to variation. The genome projects have set the foundation for functional studies and should spur the sequencing of additional species, including more diverse pathogens and nonpathogens.
Compatible/incompatible interactions between the tomato wilt fungus Fusarium oxysporum f. sp. lycopersici (FOL) and tomato Solanum lycopersicum are controlled by three avirulence genes (AVR1–3) in FOL and the corresponding resistance genes (I–I3) in tomato. The three known races (1, 2 and 3) of FOL carry AVR genes in different combinations. The current model to explain the proposed order of mutations in AVR genes is: i) FOL race 2 emerged from race 1 by losing the AVR1 and thus avoiding host resistance mediated by I (the resistance gene corresponding to AVR1), and ii) race 3 emerged when race 2 sustained a point mutation in AVR2, allowing it to evade I2-mediated resistance of the host. Here, an alternative mechanism of mutation of AVR genes was determined by analyses of a race 3 isolate, KoChi-1, that we recovered from a Japanese tomato field in 2008. Although KoChi-1 is race 3, it has an AVR1 gene that is truncated by the transposon Hormin, which belongs to the hAT family. This provides evidence that mobile genetic elements may be one of the driving forces underlying race evolution. KoChi-1 transformants carrying a wild type AVR1 gene from race 1 lost pathogenicity to cultivars carrying I, showing that the truncated KoChi-1 avr1 is not functional. These results imply that KoChi-1 is a new race 3 biotype and propose an additional path for emergence of FOL races: Race 2 emerged from race 1 by transposon-insertion into AVR1, not by deletion of the AVR1 locus; then a point mutation in race 2 AVR2 resulted in emergence of race 3.
Hemibiotrophic fungal plant pathogens represent a group of agronomically significant disease-causing agents that grow first on living tissue and then cause host death in later, necrotrophic growth. Among these, Colletotrichum spp. are devastating pathogens of many crops. Identifying expanded classes of genes in the genomes of phytopathogenic Colletotrichum, especially those associated with specific stages of hemibiotrophy, can provide insights on how these pathogens infect a large number of hosts.The genomes of Colletotrichum orbiculare, which infects cucurbits and Nicotiana benthamiana, and C. gloeosporioides, which infects a wide range of crops, were sequenced and analyzed, focusing on features with potential roles in pathogenicity. Regulation ofC. orbiculare gene expression was investigated during infection of N. benthamiana using a custom microarray.Genes expanded in both genomes compared to other fungi included sequences encoding small, secreted proteins (SSPs), secondary metabolite synthesis genes, proteases and carbohydrate-degrading enzymes. Many SSP and secondary metabolite synthesis genes were upregulated during initial stages of host colonization, whereas the necrotrophic stage of growth is characterized by upregulation of sequences encoding degradative enzymes.Hemibiotrophy in C. orbiculare is characterized by distinct stage-specific gene expression profiles of expanded classes of potential pathogenicity genes.
For a comprehensive survey of the structure and dynamics of the Dutch Phytophthora infestans population, 652 P. infestans isolates were collected from commercial potato fields in the Netherlands during the 10-year period 2000–2009. Genotyping was performed using 12 highly informative microsatellite markers and mitochondrial haplotypes. In addition, for each isolate, the mating type was determined. STRUCTURE analysis grouped the 322 identified genotypes in three clusters. Cluster 1 consists of a single clonal lineage NL-001, known as “Blue_13”; all isolates in this cluster have the A2 mating type and the Ia mitochondrial haplotype. Clusters 2 and 3 display a more elaborate substructure containing many unique genotypes. In Cluster 3, several distinct clonal lineages were also identified. This survey witnesses that the Dutch population underwent dramatic changes in the 10 years under study. The most notable change was the emergence and spread of A2 mating type strain NL-001 (or “Blue_13”). The results emphasize the importance of the sexual cycle in generating genetic diversity and the importance of the asexual cycle as the propagation and dispersal mechanism for successful genotypes. Isolates were also screened for absence of the Avrblb1/ipiO class I gene, which is indicative for virulence on Rpi-blb1. This is also the first report of Rpi-blb1 breakers in the Netherlands. Superimposing the virulence screening on the SSR genetic backbone indicates that lack the Avrblb1/ipiO class I gene only occurred in sexual progeny. So far, the asexual spread of the virulent isolates identified has been limited.
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We sequenced and compared the genomes of the Dothideomycete fungal plant pathogens Cladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu >61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation.
Horizontal gene transfer has been postulated to occur between crops to co-occurring parasitic plants, but empirical evidence has been lacking. We present evidence that an HGT event moved a nuclear monocot gene into the genome of the eudicot parasite witchweed (Striga hermonthica), which infects many grass species in Africa. Analysis of expressed sequence tags revealed that the genome of S. hermonthica contains a nuclear gene that is widely conserved among grass species but is not found in other eudicots. Phylogenetically, this gene clusters with sorghum genes, the monocot host of the parasitic weed, suggesting that nuclear genes can be captured by parasitic weeds in nature.
Background - Oomycetes are fungal-like microorganisms evolutionary distinct from true fungi, belonging to the Stramenopile lineage and comprising major plant pathogens. Both oomycetes and fungi express proteins able to interact with cellulose, a major component of plant and oomycete cell walls, through the presence of carbohydrate-binding module belonging to the family 1 (CBM1). Fungal CBM1-containing proteins were implicated in cellulose degradation whereas in oomycetes, the Cellulose Binding Elicitor Lectin (CBEL), a well-characterized CBM1-protein from Phytophthora parasitica, was implicated in cell wall integrity, adhesion to cellulosic substrates and induction of plant immunity.
Results - To extend our knowledge on CBM1-containing proteins in oomycetes, we have conducted a comprehensive analysis on 60 fungi and 7 oomycetes genomes leading to the identification of 518 CBM1-containing proteins. In plant-interacting microorganisms, the larger number of CBM1-protein coding genes is expressed by necrotroph and hemibiotrophic pathogens, whereas a strong reduction of these genes is observed in symbionts and biotrophs. In fungi, more than 70% of CBM1-containing proteins correspond to enzymatic proteins in which CBM1 is associated with a catalytic unit involved in cellulose degradation. In oomycetes more than 90% of proteins are similar to CBEL in which CBM1 is associated with a non-catalytic PAN/Apple domain, known to interact with specific carbohydrates or proteins. Distinct Stramenopile genomes like diatoms and brown algae are devoid of CBM1 coding genes. A CBM1-PAN/Apple association 3D structural modeling was built allowing the identification of amino acid residues interacting with cellulose and suggesting the putative interaction of the PAN/Apple domain with another type of glucan. By Surface Plasmon Resonance experiments, we showed that CBEL binds to glycoproteins through galactose or N-acetyl-galactosamine motifs.
Conclusions - This study provides insight into the evolution and biological roles of CBM1-containing proteins from oomycetes. We show that while CBM1s from fungi and oomycetes are similar, they team up with different protein domains, either in proteins implicated in the degradation of plant cell wall components in the case of fungi or in proteins involved in adhesion to polysaccharidic substrates in the case of oomycetes. This work highlighted the unique role and evolution of CBM1 proteins in oomycete among the Stramenopile lineage.
The button mushroom occupies a prominent place in our diet and in the grocery store where it boasts a tasty multibillion-dollar niche, while in nature, Agaricus bisporus is known to decay leaf matter on the forest floor. Now, owing to an international collaboration of two-dozen institutions led by the French National Institute for Agricultural Research (INRA) and the U.S. Department of Energy Joint Genome Institute (DOE JGI), the full repertoire of A. bisporus genes has been determined. In particular, new work shows how its genes are actually deployed not only in leaf decay but also wood decay and in the development of fruiting bodies (the above ground part of the mushroom harvested for food). The work also suggests how such processes have major implications for forest carbon management. The analysis of the inner workings of the world’s most cultivated mushroom was published online the week of October 8 in the journal, the Proceedings of the National Academy of Sciences (PNAS).
Plants and pathogens evolve in response to each other. This co-evolutionary arms race is fueled by genetic variation underlying the recognition of pathogen proteins by the host and the defeat of host defenses by the pathogen. Together with new mutations, genetic diversity in populations of both the host and pathogen represent a pool of possible variants to maintain adaptation via natural selection.Drastic changes in genetic diversity in crop species have occurred as a consequence of domestication. Whether changes in the genetic composition of these host populations also have affected genetic diversity in pathogen species is, so far, poorly understood. Advances in comparative genomics and population genomic approaches open new avenues to study adaptive processes in plant pathogens and to infer the impact of agro-ecosystems on the evolution of pathogen populations. Here we summarize new insights gained from comparative genome studies and population genomics in host-pathogen systems.
Biotroph pathogens establish intimate interactions with their hosts that are conditioned by the successful secretion of effectors in infected tissues and subsequent manipulation of host physiology. The identification of early-expressed pathogen effectors and early-modulated host functions is currently a major goal to understand the molecular basis of biotrophy. Here, we report the 454-pyrosequencing transcriptome analysis of early stages of poplar leaf colonization by the rust fungus Melampsora larici-populina. Among the 841,301 reads considered for analysis, 616,879 and 649 were successfully mapped to Populus trichocarpa and M. larici-populina genome sequences, respectively. From a methodological aspect, these results indicate that this single approach is not appropriate to saturate poplar transcriptome and to follow transcript accumulation of the pathogen. We identified 19 pathogen transcripts encoding early-expressed small-secreted proteins representing candidate effectors of interest for forthcoming studies. Poplar RNA-Seq data were validated by oligoarrays and quantitatively analysed, which revealed a highly stable transcriptome with a single transcript encoding a sulfate transporter (herein named PtSultr3;5, POPTR_0006s16150) showing a dramatic increase upon colonization by either virulent or avirulent M. larici-populina strains. Perspectives connecting host sulfate transport and biotrophic lifestyle are discussed.