Fungi of the Pucciniales order cause rust diseases, which altogether affect thousands of plant species worldwide and pose major threat to several crops. How rust effectors - virulence proteins delivered into infected tissues to modulate host functions - contribute to pathogen virulence remains poorly understood. Melampsora larici-populina is a devastating and widespread rust pathogen of poplars and its genome encodes 1,184 identified small secreted proteins that could potentially act as effectors. Here, following specific criteria we selected 16 candidate effector proteins and characterized their virulence activities and subcellular localizations in the leaf cells of Arabidopsis thaliana. Infection assays using bacterial (Pseudomonas syringae) and oomycete (Hyaloperonospora arabidopsidis) pathogens revealed subsets of candidate effectors that enhanced or decreased pathogen leaf colonization. Confocal imaging of GFP-tagged candidate effectors constitutively expressed in stable transgenic plants revealed that some protein fusions specifically accumulate in nuclei, chloroplasts, plasmodesmata and punctate cytosolic structures. Altogether, our analysis suggests that rust fungal candidate effectors target distinct cellular components in host cells to promote parasitic growth.
Herein provided is the full-genome sequence of Pseudomonas fluorescens LBUM636. This strain is a plant growth-promoting rhizobacterium (PGPR) which produces phenazine-1-carboxylic acid, an antibiotic involved in the biocontrol of numerous plant pathogens, including late blight of potato caused by the plant pathogen Phytophthora infestans.
Pseudomonas brassicacearum LBUM300, a plant rhizosphere-inhabiting bacterium, produces 2,4-diacetylphloroglucinol and hydrogen cyanide and has shown antagonistic activity against the plant pathogens Verticillium dahliae, Phytophthora cactorum, and Clavibacter michiganensis subsp. michiganensis. Here, we report the complete genome sequence of P. brassicacearum LBUM300.
Pseudomonas fluorescens LBUM223 is a plant growth-promoting rhizobacterium (PGPR) with biocontrol activity against various plant pathogens. It produces the antimicrobial metabolite phenazine-1-carboxylic acid, which is involved in the biocontrol of Streptomyces scabies, the causal agent of common scab of potato. Here, we report the complete genome sequence of P. fluorescens LBUM223.
Pseudozyma flocculosa is related to the model plant pathogen Ustilago maydisyet is not a phytopathogen but rather a biocontrol agent of powdery mildews; this relationship makes it unique for the study of the evolution of plant pathogenicity factors. The P. flocculosa genome of ∼23 Mb includes 6877 predicted protein coding genes. Genome features, including hallmarks of pathogenicity, are very similar in P. flocculosa and U. maydis, Sporisorium reilianum, and Ustilago hordei. Furthermore, P. flocculosa, a strict anamorph, revealed conserved and seemingly intact mating-type and meiosis loci typical of Ustilaginales. By contrast, we observed the loss of a specific subset of candidate secreted effector proteins reported to influence virulence in U. maydis as the singular divergence that could explain its nonpathogenic nature. These results suggest that P. flocculosa could have once been a virulent smut fungus that lost the specific effectors necessary for host compatibility. Interestingly, the biocontrol agent appears to have acquired genes encoding secreted proteins not found in the compared Ustilaginales, including necrosis-inducing-Phytophthora-protein- and Lysin-motif- containing proteins believed to have direct relevance to its lifestyle. The genome sequence should contribute to new insights into the subtle genetic differences that can lead to drastic changes in fungal pathogen lifestyles.
The obligate biotrophic rust fungus Melampsora larici-populina is the most devastating and widespread pathogen of poplars. Studies over recent years have identified various small secreted proteins (SSP) from plant biotrophic filamentous pathogens and have highlighted their role as effectors in host–pathogen interactions. The recent analysis of theM. larici-populina genome sequence has revealed the presence of 1,184 SSP-encoding genes in this rust fungus. In the present study, the expression and evolutionary dynamics of these SSP were investigated to pinpoint the arsenal of putative effectors that could be involved in the interaction between the rust fungus and poplar. Similarity with effectors previously described in Melampsora spp., richness in cysteines, and organization in large families were extensively detailed and discussed. Positive selection analyses conducted over clusters of paralogous genes revealed fast-evolving candidate effectors. Transcript profiling of selected M. laricipopulina SSP showed a timely coordinated expression during leaf infection, and the accumulation of four candidate effectors in distinct rust infection structures was demonstrated by immunolocalization. This integrated and multifaceted approach helps to prioritize candidate effector genes for functional studies.
Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.
Poplar leaf rusts belonging to the genus Melampsora are considered as the world’s most important disease of poplars. During infection, Melampsora species secrete a diverse set of effector proteins that aim to reprogram the host into a susceptible state. However, recognition of particular effector proteins by the plant immune system, so-called avirulence proteins, leads to induction of the hypersensitve response, a form of programmed cell death. Based on prevalent models of plant–pathogen coevolution, some of these effectors, notably those with avirulence functions, are predicted to exhibit molecular signatures of accelerated evolution. In this thesis, features described above were used to identify the pathogenicity determinants of poplar leaf rusts with the specific aim of identifying candidate effector proteins. The first part of this thesis includes the development of a multifaceted approach to take advantage of available EST libraries, and included computational prediction of secreted proteins, intra- and interspecific comparative genomics, and testing for the presence of positive selection. Accelerated evolution was found to act more importantly on secreted proteins, and most of those proteins under positive selection were shown to harbour a high number of Cys residues and to share no homology in international databases. The second part of this thesis presents the results of a collaborative effort to characterize the entire secretome of M. larici-populina using expert annotation, screening for positive selection, and transcript profiling approaches. A significant part of the secretome was found to be under positive selection, and commonalities described before were observed again (Cys-rich proteins with no homology in international databases). Most of those with transcript evidence were found to be specifically expressed in planta, and positively selected sites were concentrated in the C-terminal region, consistent with an effector function. As a first step toward a functional characterization, immunolocalization of selected candidates has revealed specific labeling of some proteins in the haustoria periphery.
Obligate biotrophs such as rust fungi are believed to establish long-term relationships by modulating plant defenses through a plethora of effector proteins, whose most recognizable feature is the presence of a signal peptide for secretion. Since the phenotypes of these effectors extend to host cells, their genes are expected to be under accelerated evolution stimulated by host-pathogen coevolutionary arms races. Recently, whole genome sequence data has allowed the prediction of secretomes, facilitating the identification of putative effectors.
We generated cDNA libraries from four poplar leaf rust pathogens (Melampsora spp.) and used computational approaches to identify and annotate putative secreted proteins with the aim of uncovering new knowledge about the nature and evolution of the rust secretome. While more than half of the predicted secretome members encoded lineage-specific proteins, similarities with experimentally characterized fungal effectors were also identified. A SAGE analysis indicated a strong stage-specific regulation of transcripts encoding secreted proteins. The average sequence identity of putative secreted proteins to their closest orthologs in the wheat stem rust Puccinia graminis f. sp. tritici was dramatically reduced compared with non-secreted ones. A comparative genomics approach based on homologous gene groups unravelled positive selection in putative members of the secretome.
We uncovered robust evidence that different evolutionary constraints are acting on the rust secretome when compared to the rest of the genome. These results are consistent with the view that these genes are more likely to exhibit an effector activity and be involved in coevolutionary arms races with host factors.
With the availability of the entire genome of the model tree Populus trichocarpa Torr. & A. Gray and the current genome sequencing project of its rust pathogen Melampsora larici-populinaKleb., rust–poplar interaction research has entered the genomic era. Recent genomics research on poplars has attempted to connect the genetic localizations of loci for qualitative and quantitative disease resistance with putative genes encoding resistance or signalling proteins. The interactions between these putative resistance genes and rust effectors remain unknown. Genomic resources developed for Melampsora spp. promise to contribute to our understanding of the molecular basis of pathogenicity by facilitating the isolation of pathogenicity genes. A multifaceted approach for the identification of such genes that relies largely on trimming and sequence data analysis has been developed. The strategy takes advantage of the resources available and combines EST libraries, bioinformatics data mining for extracellularly expressed secreted proteins, intra- and inter-specific comparative genomics, and testing for the presence of positive selection. It has resulted in the discovery of several putative candidate genes. In silico evidence for candidate genes will be further validated by robust experimental evidence through functional analyses.
In May 2003, a survey was conducted in southwestern Alberta, east of the Rocky Mountains, to determine the extent of the spread and genetic diversity of white pine blister rust, which is caused by Cronartium ribicola J.C. Fisch. Aeciospores were sampled from white pine blister rust cankers in three infected limber pine (Pinus flexilis James) stands separated from one another by 100 to 215 km. DNA genotypes were determined for 12 codominant PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) loci representing genes derived from an EST library. At each site sampled, some aecia displayed DNA genotypes that were heterozygous at all loci and possessed novel alleles (GenBank Accession Nos. DQ009533-DQ009611). At Waterton Lakes, Kananaskis County, and Porcupine Hills, 29%, 11%, and 3% of sampled aecia and 38%, 33%, and 10% of sampled trees, respectively, possessed these unusual profiles. In May 2004, similar genetic profiles were found at two of these sites, Waterton Lakes and Kananaskis County, at 17 and 25% of sampled aecia (25% of sampled trees). In each of these aecia, genotyping and sequence analysis revealed this pattern was due to the presence of oneC. ribicola and one C. comandrae Peck. allele at each of the 12 loci. Scanning electron microscopy (SEM) revealed aeciospore morphology that was intermediate between C. ribicola and C. comandrae. Aeciospores were longer (16 to 20 × 25 to 40 μm) than the expected range for C. ribicola (18 to 20 × 22 to 31 μm). They were also fusiform, obovoid or short-to-long ellipsoid, but not pyriform-acuminate as in C. comandrae, and without a true conspicuous smooth spot as inC. ribicola. This provides evidence for interspecific hybridization between C. ribicola and C. comandrae, the causal agent of comandra blister rust. We hypothesize that the presence of nearby C. comandrae-infected lodgepole pine (P. contorta Dougl.) could have led to spermatization of C. ribicola receptive hyphae by C. comandraepycniospores, resulting in the formation of hybrid aecia. An important question is whether these hybrids have a different host range that could potentially extend its geographic range in areas where the telial host, Ribes spp. L., is not abundant. The hybrid rust Melampsora ×columbiana Newcombe was shown to exhibit virulence against certain hybrid poplar clones that had previously been reported as resistant against both parental rusts (M. medusae Thuem. and M. occidentalisJacks) and abundant pathogenic variation has been observed. Furthermore, the ability to colonize unexpected hosts could provide fitness advantages over parental species, as was observed inPhytophthora spp. pathogenic on alder. Host range and virulence assays should be conducted to assess the potential impact of this hybrid.
Three members of the Puccinia genus, P. triticina (Pt), P. striiformis f.sp. tritici (Pst), and P. graminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; by comparison repeats occupy 31.5% for Pst and 36.5% for Pgt. We find all three genomes are highly heterozygous, with Pst (5.97 SNPs/kb) nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt. Of 1,358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate enzyme degradation. Two allelic homeodomain, HD1 and HD2, pairs and three pheromone receptor (STE3) mating-type genes were identified in each dikaryotic Puccinia species. The HD proteins were active in a heterologous Ustilago maydis mating assay and host induced gene silencing of the HD and STE3 alleles reduced wheat host infection.
White pine blister rust is caused by the fungal pathogen Cronartium ribicola J.C. Fisch (Basidiomycota, Pucciniales). This invasive alien pathogen was introduced into North America at the beginning of the 20th century on pine seedlings imported from Europe and has caused serious economic and ecological impacts. In this study, we applied a population and landscape genetics approach to understand the patterns of introduction and colonization as well as population structure and migration of C. ribicola. We characterized 1,292 samples of C. ribicola from 66 geographic locations in North America using single nucleotide polymorphisms (SNPs) and evaluated the effect of landscape features, host distribution, and colonization history on the structure of these pathogen populations. We identified eastern and western genetic populations in North America that are strongly differentiated. Genetic diversity is two to five times higher in eastern populations than in western ones, which can be explained by the repeated accidental introductions of the pathogen into northeastern North America compared with a single documented introduction into western North America. These distinct genetic populations are maintained by a barrier to gene flow that corresponds to a region where host connectivity is interrupted. Furthermore, additional cryptic spatial differentiation was identified in western populations. This differentiation corresponds to landscape features, such as mountain ranges, and also to host connectivity. We also detected genetic differentiation between the pathogen populations in natural stands and plantations, an indication that anthropogenic movement of this pathogen still takes place. These results highlight the importance of monitoring this invasive alien tree pathogen to prevent admixture of eastern and western populations where different pathogen races occur.
Several obligate biotrophic phytopathogens, namely oomycetes and fungi, invade and feed on living plant cells through specialized structures known as haustoria. Deploying an arsenal of secreted proteins called effectors, these pathogens balance their parasitic propagation by subverting plant immunity without sacrificing host cells. Such secreted proteins, which are thought to be delivered by haustoria, conceivably reprogram host cells and instigate structural modifications, in addition to the modulation of various cellular processes. As effectors represent tools to assist disease resistance breeding, this short review provides a bird’s eye view on the relationship between the virulence function of effectors and their subcellular localization in host cells.
Many organisms such as bacteria, fungi, oomycetes, nematodes and insects grow, feed and/or reproduce in close association with plant hosts. To establish such intimate interactions, symbionts (either mutualistic or parasitic) secrete effectors into host tissues, which are molecules that modulate plant cell structures and processes (Win et al., 2012a). This last decade, advances in genomics have revealed that symbionts possess dozens to hundreds of effectors. Currently, the field is moving rapidly from effector identification towards effector characterization, which provides a better understanding of how these effectors promote the establishment of a successful relationship with host plants. The 30th New Phytologist Symposium clearly illustrated this theme, as an international panel of c. 150 scientists was brought together to discuss current efforts to decipher effector functions within a wide range of biological systems. The remote location of the meeting in the Sierra Nevada mountains of California, USA, promoted lively discussions between participants during and after the sessions, but also via social networks (the whole conference was covered by a twitter feed, #30NPS tag, available on http://storify.com/KamounLab/30th-new-phytologist-symposium-immunomodulation-by).
The Symposium consisted of five sessions: pathogenomics, effector secretion and trafficking, induction and suppression of host immunity, structural biology of effectors and their targets, and emerging systems. In this meeting report, we focus on three main aspects or ‘take home messages’ discussed during the conference: (1) the identification of effector partners in plants; (2) recent advances facilitated by live-cell imaging and structural biology; and (3) emerging systems in the field.
Rust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology.
To support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs.
The current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence.
Wide variation and overlap in morphological characters have led to confusion in species identification within the fungal rust genus Melampsora. The Melampsora species with uredinial–telial stages on white poplar and aspens are especially prone to misidentification. This group includes the Melampsora populnea species complex and the highly destructive pine twisting rust, Melampsora pinitorqua, which alternates between hosts in Populus section Populus and Pinus. Our objective was to compare morphologically based identification to genetic material extracted from Melampsora species pathogenic to aspen and white poplar. We compared morphometric traits and DNA barcodes obtained from internal transcribed spacer (ITS), large ribosomal RNA subunit (28S), and mitochondrial cytochrome oxidase 1 (CO1) sequences to delimit within this taxonomically difficult group. Eight different Melampsora species were initially defined based on host specificity and morphometric data. DNA barcodes were then overlaid on these initial species definitions. The DNA barcodes, specifically those defined on ITS and 28S sequences, provided a highly accurate means of identifying and resolving Melampsora taxa. We highlighted species misidentification in specimens from Canadian herbaria related to either Melampsora medusae f. sp. tremuloidae or Melampsora aecidioides. Finally, we evidenced that the north-American species found on Populus alba, M. aecidioides is closely related but distinct from the four species of the M. populnea complex (Melampsora larici-tremulae,Melampsora magnusiana, Melampsora pinitorqua, and Melampsora rostrupii) found in Eurasia.
We report the discovery, characterization and validation of 118 single nucleotide polymor- phisms (SNPs) for poplar leaf rust Melampsora medusae f. sp. deltoidae identified using a gene-targeted approach in an expressed sequence tag (EST) library. We developed a genotyping assay using the iPLEXTM primer extension method for two multiplex assays of 28 and 22 SNPs.
The goal of this research was to develop coding genetic markers for the white pine blister rust (WPBR) causal agent, Cronartium ribicola , without bias for populations from eastern or western North America. Eight markers were used to genotype 80 individuals, using the SSCP (Single-Strand Conformation Polymorphism) technique, allowing to study genetic variability within these populations.
This study confirmed the presence of a barrier to gene flow between populations located east and west of the Prairies, populations from the Rockies having grouped with western populations. The genetic differentiation between eastern and western populations is highly significant and nucleotide diversity was three times higher in eastern populations.
Furthermore, an hybrid between C. ribicola and Cronartium comandrae , causal agent of comandra blister rust, is reported for the first time.
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