Sandrine Gélisse’s research while affiliated with University of Paris-Saclay and other places

Background Septoria tritici blotch (STB) is one of the most damaging wheat diseases worldwide, and the development of resistant cultivars is of paramount importance for sustainable crop management. However, the genetic basis of the resistance present in elite wheat cultivars remains largely unknown, which limits the implementation of this strategy. A collection of 285 wheat cultivars originating mostly from France was challenged with ten Zymoseptoria tritici isolates at the seedling stage. The collection was further evaluated in seven field trials across France using artificial inoculation. Results Genome-wide association study resulted in the detection of 57 wheat QTL, among which 40 were detected at the seedling stage. Three quarters of these QTL were in genomic regions previously reported for to confer resistance to Z. tritici, but 10 QTL are novel and may be of special interest as new sources of resistance. Some QTL colocalise with major Stb resistance genes, suggesting their presence in the French elite winter wheat germplasm. Among them, the three QTL with the strongest effect colocalize with Stb6, Stb9 and Stb18. There was minimal overlap between the QTL detected at the seedling and adult plant stages, with only 1 out of 20 seedling QTL also being detected in field trials inoculated with the same isolate. This suggests that different resistance genes are involved at the seedling and adult plant stages. Conclusion This work reveals the highly complex genetic architecture of French wheat resistance to STB and provides relatively small QTL intervals, which will be valuable for identifying the underlying causative genes and for marker-assisted selection.

Monitoring virulent strains within pathogen populations is crucial to improve host resistance deployment strategies. Such monitoring increasingly involves field pathogenomics studies of molecular polymorphisms in pathogen genomes based on high‐throughput screening technologies. However, it is not always straightforward to predict virulence phenotypes from these polymorphisms, and in planta phenotyping remains necessary. We developed a method for ‘bulk phenotyping on checkerboard microcanopies of wheat near‐isogenic lines’ (BPC) for estimating the frequency of virulence against a resistance gene in mixed populations of the fungal pathogen Zymoseptoria tritici , the causal agent of Septoria tritici blotch (STB) in wheat, without the need for strain‐by‐strain pathogen phenotyping. Our method involves the uniform inoculation of a microcanopy of two wheat lines—one with the target resistance gene and the other without it—with a multistrain mixture of isolates representative of the population to be characterized, followed by the differential quantification of infection points (lesions). Using Stb16q , a wheat resistance gene that has recently broken down in Europe, we found a robust correlation between the ratio of the mean number of lesions on each wheat line and the frequency of virulent strains in the inoculum. Using pairs of virulent and avirulent strains, as well as synthetic populations consisting of 10 virulent strains and 10 avirulent strains mixed in different proportions, we validated the principle of the method and established standard curves at virulence frequencies close to those observed in natural conditions. We discuss the potential of this method for virulence monitoring in combination with molecular methods.

Monitoring virulent strains within fungal pathogen populations is crucial to improve host resistance deployment strategies. Such monitoring increasingly involves field pathogenomics studies of molecular polymorphisms in genomes based on high-throughput screening technologies. However, it is not always straightforward to predict virulence phenotypes from these polymorphisms and in planta phenotyping remains necessary. We developed a method for "bulk phenotyping on checkerboard microcanopies of wheat near-isogenic lines" (BPC) for estimating the frequency of virulence against an Stb gene in populations of Zymoseptoria tritici, the causal agent of Septoria tritici blotch in wheat, without the need for strain-by-strain phenotyping. Our method involves the uniform inoculation of a microcanopy of two wheat lines - one with the resistance gene and the other without it - with a multi-strain cocktail representative of the population to be characterized, followed by the differential quantification of infection points (lesions). Using Stb16q, a resistance gene that has recently broken down in Europe, we found a robust correlation between the ratio of the mean number of lesions on each wheat line and the frequency of virulent strains in the inoculum. Using pairs of virulent and avirulent strains, and synthetic populations consisting of 10 virulent strains and 10 avirulent strains mixed in different proportions, we validated the principle of the method and established standard curves at virulence frequencies close to those observed in natural conditions. We discuss the potential of this method for virulence monitoring in combination with recently developed molecular methods.

Zymoseptoria tritici is the fungal pathogen responsible for Septoria tritici blotch on wheat. Disease outcome in this pathosystem is partly determined by isolate-specific resistance, where wheat resistance genes recognize specific fungal factors triggering an immune response. Despite the large number of known wheat resistance genes, fungal molecular determinants involved in such cultivar-specific resistance remain largely unknown. We identified the avirulence factor AvrStb9 using association mapping and functional validation approaches. Pathotyping AvrStb9 transgenic strains on Stb9 cultivars, near isogenic lines and wheat mapping populations, showed that AvrStb9 interacts with Stb9 resistance gene, triggering an immune response. AvrStb9 encodes an unusually large avirulence gene with a predicted secretion signal and a protease domain. It belongs to a S41 protease family conserved across different filamentous fungi in the Ascomycota class and may constitute a core effector. AvrStb9 is also conserved among a global Z. tritici population and carries multiple amino acid substitutions caused by strong positive diversifying selection. These results demonstrate the contribution of an ‘atypical’ conserved effector protein to fungal avirulence and the role of sequence diversification in the escape of host recognition, adding to our understanding of host-pathogen interactions and the evolutionary processes underlying pathogen adaptation.

Septoria leaf blotch is a foliar wheat disease controlled by a combination of plant genetic resistances and fungicides use. R-gene-based qualitative resistance durability is limited due to gene-for-gene interactions with fungal avirulence (Avr) genes. Quantitative resistance is considered more durable but the mechanisms involved are not well documented. We hypothesize that genes involved in quantitative and qualitative plant-pathogen interactions are similar. A bi-parental population of Zymoseptoria tritici was inoculated on wheat cultivar ‘Renan’ and a linkage analysis performed to map QTL. Three pathogenicity QTL, Qzt-I05-1, Qzt-I05-6 and Qzt-I07-13, were mapped on chromosomes 1, 6 and 13 in Z. tritici, and a candidate pathogenicity gene on chromosome 6 was selected based on its effector-like characteristics. The candidate gene was cloned by Agrobacterium tumefaciens-mediated transformation, and a pathology test assessed the effect of the mutant strains on ‘Renan’. This gene was demonstrated to be involved in quantitative pathogenicity. By cloning a newly annotated quantitative-effect gene in Z. tritici that is effector-like, we demonstrated that genes underlying pathogenicity QTL can be similar to Avr genes. This opens up the previously probed possibility that ‘gene-for-gene’ underlies not only qualitative but also quantitative plant-pathogen interactions in this pathosystem.

Crop pathogens pose severe risks to global food production due to the rapid rise of resistance to pesticides and host resistance breakdowns. Predicting future risks requires monitoring tools to identify changes in the genetic composition of pathogen populations. Here we report the design of a microfluidics-based amplicon sequencing assay to multiplex 798 loci targeting virulence and fungicide resistance genes, and randomly selected genome-wide markers for the fungal pathogen Zymoseptoria tritici. The fungus causes one of the most devastating diseases on wheat showing rapid adaptation to fungicides and host resistance. We optimized the primer design by integrating polymorphism data from 632 genomes of the same species. To test the performance of the assay, we genotyped 192 samples in two replicates. Analysis of the short-read sequence data generated by the assay showed a fairly stable success rate across samples to amplify a large number of loci. The performance was consistent between samples originating from pure genomic DNA as well as material extracted directly from infected wheat leaves. In samples with mixed genotypes, we found that the assay recovers variations in allele frequencies. We also explored the potential of the amplicon assay to recover transposable element insertion polymorphism relevant for fungicide resistance. As a proof-of-concept, we show that the assay recovers the pathogen population structure across French wheat fields. Genomic monitoring of crop pathogens contributes to more sustainable crop protection and yields.

Zymoseptoria tritici is the fungal pathogen responsible for Septoria tritici blotch on wheat. Disease outcome in this pathosystem is partly determined by isolate-specific resistance, where wheat resistance genes recognize specific fungal factors triggering an immune response. Despite the large number of known wheat resistance genes, fungal molecular determinants involved in such cultivar-specific resistance remain largely unknown. We identified the avirulence factor AvrStb9 using association mapping and functional validation approaches. Pathotyping AvrStb9 transgenic strains on Stb9 cultivars, near isogenic lines and wheat mapping populations, showed that AvrStb9 interacts with Stb9 resistance gene, triggering a strong immune response. AvrStb9 encodes an unusually large avirulence gene with a predicted secretion signal and a protease domain. It belongs to a S41 protease family conserved across different filamentous fungi in the Ascomycota class and may constitute a core effector. AvrStb9 is also conserved among a global Z. tritici population and carries multiple amino acid substitutions caused by strong positive diversifying selection. These results demonstrate the contribution of an ‘atypical’ conserved effector protein to fungal avirulence and the role of sequence diversification in the escape of host recognition, adding to our understanding of host-pathogen interactions and the evolutionary processes underlying pathogen adaptation.

Little is known about the impact of host immunity on sexual reproduction in fungal pathogens. In particular, it is unclear whether crossing requires both sexual partners to infect living plant tissues. We addressed this issue in a three-year experiment investigating different scenarios of Zymoseptoria tritici crosses according to the virulence (‘vir’) or avirulence (‘avr’) of the parents against a qualitative resistance gene. Co-inoculations (‘vir × vir’, ‘avr × vir’, ‘avr × avr’) and single inoculations were performed on a wheat cultivar carrying the Stb16q resistance gene (Cellule) and a susceptible cultivar (Apache), in the greenhouse. We assessed the intensity of asexual reproduction by scoring disease severity, and the intensity of sexual reproduction by counting the ascospores discharged from wheat residues. As expected, disease severity was more intense on Cellule for ‘vir × vir’ co-inoculations than for ‘avr × vir’ co-inoculations, with no disease for ‘avr × avr’. However, all types of co-inoculation yielded sexual offspring, whether or not the parental strains caused plant symptoms. Parenthood was confirmed by genotyping (SSR markers), and the occurrence of crosses between (co-)inoculated and exogenous strains (other strains from the experiment, or from far away) was determined. We showed that symptomatic asexual infection was not required for a strain to participate in sexual reproduction, and deduced from this result that avirulent strains could be maintained asymptomatically “on” or “in” leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually. In two of the three years, the intensity of sexual reproduction did not differ between the three types of co-inoculation in Cellule, suggesting that crosses involving avirulent strains are not anecdotal. We discuss the possible mechanisms explaining the maintenance of avirulence in Z. tritici populations and the potential impact of particular resistance deployments such as cultivar mixtures for limiting resistance breakdown.

Crop pathogens pose severe risks to global food production due to the rapid rise of resistance to pesticides and host resistance breakdowns. Predicting future risks requires monitoring tools to identify changes in the genetic composition of pathogen populations. Here we report the design of a microfluidics-based amplicon sequencing assay to multiplex 798 loci targeting virulence and fungicide resistance genes, and randomly selected genome-wide markers for the fungal pathogen Zymoseptoria tritici . We optimized the primer design by integrating polymorphism data from 632 genomes of the same species. To test the performance of the assay, we genotyped 192 samples, including replicates, of Z. tritici. The fungus causes one of the most devastating diseases on wheat showing rapid adaptation to fungicides and host resistance. Analysis of the short-read sequence data generated by the assay showed a fairly stable success rate across samples to amplify a large number of loci. The performance was consistent between samples originating from pure genomic DNA as well as material extracted directly from infected wheat leaves. In samples with mixed genotypes, we found that the assay recovers variations in allele frequencies. We explored also the potential of the amplicon assay to recover transposable element insertion polymorphism relevant for fungicide resistance. As a proof-of-concept, we show that the assay recovers the pathogen population structure across French wheat fields. Genomic monitoring of crop pathogens contributes to more sustainable crop protection and yields.

Little is known about the impact of host immunity on sexual reproduction in fungal pathogens. In particular, it is unclear whether crossing requires both sexual partners to infect living plant tissues. We addressed this issue in a three-year experiment investigating different scenarios of Zymoseptoria tritici crosses on wheat according to the virulence (‘vir’) or avirulence (‘avr’) of the parents against a qualitative resistance gene. Co-inoculations (‘vir × vir’, ‘avr × vir’, ‘avr × avr’) and single inoculations were performed on a cultivar carrying the resistance gene (Cellule) and a susceptible cultivar (Apache), in the greenhouse. We assessed the intensity of asexual multiplication by scoring disease severity, and the intensity of sexual reproduction by counting the ascospores discharged from wheat residues. As expected, disease severity was more intense on Cellule for ‘vir × vir’ co-inoculations than for ‘avr × vir’ co-inoculations, with no disease for ‘avr × avr’. However, all types of co-inoculation yielded sexual offspring, whether or not the parental strains caused plant symptoms. Parenthood was confirmed by genotyping (SSR markers), and the occurrence of crosses between (co-)inoculated and exogenous strains (other strains from the experiment, or from far away) was determined. We found that symptomatic asexual infection was not required for a strain to participate in sexual reproduction, and that avirulent strains could be maintained asymptomatically “on” or “in” leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually. In two of the three years, the intensity of sexual reproduction did not differ significantly between the three types of co-inoculation in Cellule, suggesting that crosses involving avirulent strains are not anecdotal. We discuss the possible mechanisms explaining the maintenance of avirulence in Z. tritici populations and supporting the potential efficacy of cultivar mixtures for limiting resistance gene breakdown. Highlights Avirulent Zymoseptoria tritici strains can reproduce sexually in wheat plants carrying the corresponding resistant gene. Symptomatic infection of plant tissues is not essential for a strain to reproduce sexually. Avirulent strains can be maintained asymptomatically “on” or “in” leaf tissues of plants carrying the corresponding resistant gene for long enough to reproduce sexually. Crosses of virulent strains with virulent and avirulent strains in a plant host carrying the corresponding resistance gene can produce offspring with similar population sizes. Several possible scenarios for sexual crosses can explain the maintenance of avirulence in Zymoseptoria tritici populations evolving in a wheat canopy, particular in cultivar mixtures.