Pascal Frey

Obligate biotrophy features unraveled by the genomic analysis of rust fungi

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.

Obligate Biotrophy Features Unraveled by the Genomic Analysis of the Rust Fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici

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.

High‐throughput microsatellite isolation through 454 GS‐FLX Titanium pyrosequencing of enriched DNA libraries

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high‐throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next‐generation sequencing on 454 GS‐FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11 497 to 34 483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high‐quality genetic markers for nonmodel organisms.

Poplar Peroxiredoxin Q. A Thioredoxin-Linked Chloroplast Antioxidant Functional in Pathogen Defense

Peroxiredoxins are ubiquitous thioredoxin- or glutaredoxin-dependent peroxidases, the function of which is to destroy peroxides. Peroxiredoxin Q, one of the four plant subtypes, is a homolog of the bacterial bacterioferritin comigratory proteins. We show here that the poplar (Populus tremula x Populus tremuloides) protein acts as a monomer with an intramolecular disulfide bridge between two conserved cysteines. A wide range of electron donors and substrates was tested. Unlike type II peroxiredoxin, peroxiredoxin Q cannot use the glutaredoxin or cyclophilin isoforms tested, but various cytosolic, chloroplastic, and mitochondrial thioredoxins are efficient electron donors with no marked specificities. The redox midpoint potential of the peroxiredoxin Q catalytic disulfide is -325 mV at pH 7.0, explaining why the wild-type protein is reduced by thioredoxin but not by glutaredoxin. Additional evidence that thioredoxin serves as a donor comes from the formation of heterodimers between peroxiredoxin Q and monocysteinic mutants of spinach (Spinacia oleracea) thioredoxin m. Peroxiredoxin Q can reduce various alkyl hydroperoxides, but with a better efficiency for cumene hydroperoxide than hydrogen peroxide and tertiary butyl hydroperoxide. The use of immunolocalization and of a green fluorescence protein fusion construct indicates that the transit sequence efficiently targets peroxiredoxin Q to the chloroplasts and especially to those of the guard cells. The expression of this protein and of type II peroxiredoxin is modified in response to an infection by two races of Melampsora larici-populina, the causative agent of the poplar rust. In the case of an hypersensitive response, the peroxiredoxin expression increased, whereas it decreased during a compatible interaction.

Transcript Profiling of Poplar Leaves upon Infection with Compatible and Incompatible Strains of the Foliar Rust Melampsora larici-populina

To understand key processes governing defense mechanisms in poplar (Populus spp.) upon infection with the rust fungus Melampsora larici-populina, we used combined histological and molecular techniques to describe the infection of Populus trichocarpa × Populus deltoides ‘Beaupré’ leaves by compatible and incompatible fungal strains. Striking differences in host-tissue infection were observed after 48-h postinoculation (hpi) between compatible and incompatible interactions. No reactive oxygen species production could be detected at infection sites, while a strong accumulation of monolignols occurred in the incompatible interaction after 48 hpi, indicating a late plant response once the fungus already penetrated host cells to form haustorial infection structures. P. trichocarpa whole-genome expression oligoarrays and sequencing of cDNAs were used to determine changes in gene expression in both interactions at 48 hpi. Temporal expression profiling of infection-regulated transcripts was further compared by cDNA arrays and reverse transcription-quantitative polymerase chain reaction. Among 1,730 significantly differentially expressed transcripts in the incompatible interaction, 150 showed an increase in concentration ≥3-fold, whereas 62 were decreased by ≥3-fold. Regulated transcripts corresponded to known genes targeted by R genes in plant pathosystems, such as inositol-3-P synthase, glutathione S-transferases, and pathogenesis-related proteins. However, the transcript showing the highest rust-induced up-regulation encodes a putative secreted protein with no known function. In contrast, only a few transcripts showed an altered expression in the compatible interaction, suggesting a delay in defense response between incompatible and compatible interactions in poplar. This comprehensive analysis of early molecular responses of poplar to M. larici-populina infection identified key genes that likely contain the fungus proliferation in planta.

A comprehensive analysis of genes encoding small secreted proteins identifies candidate effectors in Melampsora larici-populina (poplar leaf rust).

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 the M. 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.

Chalara fraxinea is an invasive pathogen in France

Decline induced by Chalara fraxinea is an emerging disease that severely affects ash stands in Europe. The disease appears to have an invasive spread from East to West of Europe in the last decade. The teleomorphic stage, Hymenoscyphus pseudoalbidus, that occurs as apothecia on ash rachis in the litter was recently described. The origin of ash decline remains unclear as a cryptic species, H. albidus, a long-established fungus in Europe, could be present in the same niche, and as in Switzerland, H. pseudoalbidus was shown to have been present long before the recent epidemic outbreak. In France, the emerging disease is very recent and clearly restrained to Northeastern France. We thus collected isolates from infected hosts and from apothecia/ash rachis both inside and outside the infected area in France in order to compare them on the basis of pathogenicity towards ash seedlings and sequences of the ITS regions and of three single-copy genes. We showed that two population types exhibiting about 2% base pair polymorphism in the sequences analysed were present in Northern France. The first type, corresponding to H. pseudoalbidus, was present on rachis and infected hosts only in Northeastern France and showed strong pathogenicity towards ash seedlings in inoculation tests. By contrast, the second type, which corresponds to H. albidus, was present throughout Northern France and showed no pathogenicity towards ash seedlings. Our study confirms the results of Queloz et al. (2010) who presented molecular evidences for the existence of two cryptic species, H. albidus and H. pseudoalbidus. The results strongly suggest that Chalara fraxinea/H. pseudoalbidus is a recent invader in France.

The hrpB and hrpG Regulatory Genes of Ralstonia solanacearum Are Required for Different Stages of the Tomato Root Infection Process

hrp genes, encoding type III secretion machinery, have been shown to be key determinants for pathogenicity in the vascular phytopathogenic bacterium Ralstonia solanacearum GMI1000. Here, we show phenotypes of R. solanacearum mutant strains disrupted in the prhJ, hrpG, or hrpB regulatory genes with respect to root infection and vascular colonization in tomato plants. Tests of bacterial colonization and enumeration in tomato plants, together with microscopic observations of tomato root sections, revealed that these strains display different phenotypes in planta. The phenotype of a prhJ mutant resembles that of the wild-type strain. An hrpB mutant shows reduced infection, colonization, and multiplication ability in planta, and induces a defense reaction similar to a vascular hypersensitive response at one protoxylem pole of invaded plants. In contrast, the hrpG mutant exhibited a wild-type level of infection at secondary root axils, but the ability of the infecting bacteria to penetrate into the vascular cylinder was significantly impaired. This indicates that bacterial multiplication at root infection sites and transit through the endodermis constitute critical stages in the infection process, in which hrpB and hrpG genes are involved. Moreover, our results suggest that the hrpG gene might control, in addition to hrp genes, other functions required for vascular colonization.

Genomic Variation within Monilinia laxa, M. fructigena and M. fructicola, and Application to Species Identification by PCR

Brown rot and twig canker of fruit trees are caused by Monilinia laxa, M. fructigena and M. fructicola. The Internal Transcribed Spacer (ITS) between the 18S and the 28S rRNA genes of four M. laxa and four M. fructigena isolates collected in France was amplified by Polymerase Chain Reaction (PCR) using universal primers and sequenced. Multiple alignment of the ITS sequences and comparison with published sequences revealed very little intraspecific variation and a low interspecific polymorphism clustered in two regions. Species-specific PCR primers were designed to amplify a 356 bp fragment for each of the three species. The specificity of the three primer pairs was successfully tested with a collection of 17 M. laxa, 18 M. fructigena and 6 M. fructicola isolates collected from different hosts and different countries, unequivocally confirming the identification of each isolate based on morphological and cultural traits. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assays was also successfully confirmed with DNA extracted from different fungal species, either phylogenetically close to the genus Monilinia or commonly found on diseased fruits. Using this new reliable technique, doubtful isolates can be directly identified in a single PCR run. Moreover, detection and identification of the Monilinia species were successfully achieved directly on diseased fruits. This simple and rapid method can be particularly useful to detect M. fructicola which is a listed quarantine fungus in all European countries.

Melampsora larici-populina Transcript Profiling During Germination and Timecourse Infection of Poplar Leaves Reveals Dynamic Expression Patterns Associated with Virulence and Biotrophy

Melampsora larici-populina is responsible for poplar leaf rust disease and causes severe epidemics in poplar plantations in Europe. The poplar rust genome has been recently sequenced and, in order to find the genetic determinants associated with its biotrophic lifestyle, we generated a whole-genome custom oligoarray and analyzed transcript profiles of M. larici-populina during the infection timecourse in poplar leaves. Different stages were investigated during the asexual development of the rust fungus, including resting and germinating urediniospores and seven in planta stages in the telial host. In total, 76% of the transcripts were detected during leaf infection as well as in urediniospores, whereas 20% were only detected in planta, including several transporters and many small secreted proteins (SSP). We focused our analysis on gene categories known to be related to plant colonization and biotrophic growth in rust pathogens, such as SSP, carbohydrate active enzymes (CAZymes), transporters, lipases, and proteases. Distinct sets of SSP transcripts were expressed all along the infection process, suggesting highly dynamic expression of candidate rust effectors. In contrast, transcripts encoding transporters and proteases were mostly expressed after 48 h postinoculation, when numerous haustoria are already formed in the leaf mesophyll until uredinia formation, supporting their role in nutrient acquisition during biotrophic growth. Finally, CAZymes and lipase transcripts were predominantly expressed at late stages of infection, highlighting their importance during sporulation.