Stéphane Hacquard

Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses

Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy). We report here genome and transcriptome analyses of Colletotrichum higginsianum infecting Arabidopsis thaliana and Colletotrichum graminicola infecting maize. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded in C. higginsianum. Genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy. Our findings show that preinvasion perception of plant-derived signals substantially reprograms fungal gene expression and indicate previously unknown functions for particular fungal cell types.

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.

Sequential Delivery of Host-Induced Virulence Effectors by Appressoria and Intracellular Hyphae of the Phytopathogen Colletotrichum higginsianum

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death.

Microbiota and Host Nutrition across Plant and Animal Kingdoms

Plants and animals each have evolved specialized organs dedicated to nutrient acquisition, and these harbor specific bacterial communities that extend the hosts metabolic repertoire. Similar forces driving microbial community establishment in the gut and plant roots include diet/soil-type, host genotype, and immune system as well as microbe-microbe interactions. Here we show that there is no overlap of abundant bacterial taxa between the microbiotas of the mammalian gut and plant roots, whereas taxa overlap does exist between fish gut and plant root communities. A comparison of root and gut microbiota composition in multiple host species belonging to the same evolutionary lineage reveals host phylogenetic signals in both eukaryotic kingdoms. The reasons underlying striking differences in microbiota composition in independently evolved, yet functionally related, organs in plants and animals remain unclear but might include differences in start inoculum and niche-specific factors such as oxygen levels, temperature, pH, and organic carbon availability.

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.

Root Endophyte Colletotrichum tofieldiae Confers Plant Fitness Benefits that Are Phosphate Status Dependent.

Summary A staggering diversity of endophytic fungi associate with healthy plants in nature, but it is usually unclear whether these represent stochastic encounters or provide host fitness benefits. Although most characterized species of the fungal genus Colletotrichum are destructive pathogens, we show here that C. tofieldiae (Ct) is an endemic endophyte in natural Arabidopsis thaliana populations in central Spain. Colonization by Ct initiates in roots but can also spread systemically into shoots. Ct transfers the macronutrient phosphorus to shoots, promotes plant growth, and increases fertility only under phosphorus-deficient conditions, a nutrient status that might have facilitated the transition from pathogenic to beneficial lifestyles. The host’s phosphate starvation response (PSR) system controls Ct root colonization and is needed for plant growth promotion (PGP). PGP also requires PEN2-dependent indole glucosinolate metabolism, a component of innate immune responses, indicating a functional link between innate immunity and the PSR system during beneficial interactions with Ct.

Mosaic genome structure of the barley powdery mildew pathogen and conservation of transcriptional programs in divergent hosts

Significance Powdery mildew fungi are widespread plant pathogens with an obligate biotrophic lifestyle causing devastating damage to many crops. Blumeria graminis f. sp. hordei (Bgh) infects only barley and is engaged in an evolutionary arms race with the host immune system. Genome sequencing of Bgh isolates revealed an isolate-specific mosaic of monomorphic and polymorphic DNA blocks, suggesting a mechanism that provides a large standing genetic variation in virulence polymorphisms. Detailed Bgh transcriptome profiles during early pathogenesis on barley and immunocompromised Arabidopsis revealed a conserved Bgh transcriptional program despite ∼200 million years of reproductive isolation of these hosts. Barley powdery mildew, Blumeria graminis f. sp. hordei (Bgh), is an obligate biotrophic ascomycete fungal pathogen that can grow and reproduce only on living cells of wild or domesticated barley (Hordeum sp.). Domestication and deployment of resistant barley cultivars by humans selected for amplification of Bgh isolates with different virulence combinations. We sequenced the genomes of two European Bgh isolates, A6 and K1, for comparative analysis with the reference genome of isolate DH14. This revealed a mosaic genome structure consisting of large isolate-specific DNA blocks with either high or low SNP densities. Some of the highly polymorphic blocks likely accumulated SNPs for over 10,000 years, well before the domestication of barley. These isolate-specific blocks of alternating monomorphic and polymorphic regions imply an exceptionally large standing genetic variation in the Bgh population and might be generated and maintained by rare outbreeding and frequent clonal reproduction. RNA-sequencing experiments with isolates A6 and K1 during four early stages of compatible and incompatible interactions on leaves of partially immunocompromised Arabidopsis mutants revealed a conserved Bgh transcriptional program during pathogenesis compared with the natural host barley despite ∼200 million years of reproductive isolation of these hosts. Transcripts encoding candidate-secreted effector proteins are massively induced in successive waves. A specific decrease in candidate-secreted effector protein transcript abundance in the incompatible interaction follows extensive transcriptional reprogramming of the host transcriptome and coincides with the onset of localized host cell death, suggesting a host-inducible defense mechanism that targets fungal effector secretion or production.

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.

Survival trade-offs in plant roots during colonization by closely related beneficial and pathogenic fungi

The sessile nature of plants forced them to evolve mechanisms to prioritize their responses to simultaneous stresses, including colonization by microbes or nutrient starvation. Here, we compare the genomes of a beneficial root endophyte, Colletotrichum tofieldiae and its pathogenic relative C. incanum, and examine the transcriptomes of both fungi and their plant host Arabidopsis during phosphate starvation. Although the two species diverged only 8.8 million years ago and have similar gene arsenals, we identify genomic signatures indicative of an evolutionary transition from pathogenic to beneficial lifestyles, including a narrowed repertoire of secreted effector proteins, expanded families of chitin-binding and secondary metabolism-related proteins, and limited activation of pathogenicity-related genes in planta. We show that beneficial responses are prioritized in C. tofieldiae-colonized roots under phosphate-deficient conditions, whereas defense responses are activated under phosphate-sufficient conditions. These immune responses are retained in phosphate-starved roots colonized by pathogenic C. incanum, illustrating the ability of plants to maximize survival in response to conflicting stresses.