Periodic Reporting for period 1 - EvolMAX (Molecular determinants of host adaptation in fungal-plant pathogens: origins and evolution of virulence effectors by genomic, phylogenetic and association mapping studies)
Reporting period: 2020-01-01 to 2021-12-31
Emerging fungal pathogens are an increasing threat to ecosystems, global health, and food security. Increasing global trade, expansion of monocultures and climate change facilitate more pathogen encounters and opportunities for infecting new potential hosts. Host shifts and plant resistance breakdowns largely contribute to the emergence of new plant pathogens, resulting in major diseases outbreaks and yield losses. Despite the growing number of studies on invasive fungi, our understanding of how these fungal pathogens evolve, switch hosts and become virulent is far from complete. Host shifts are thought to be mediated by molecular changes at small proteins secreted by pathogens. These proteins, known as effectors, manipulate key plant defence mechanisms, promote infection and represent key elements of fungal virulence. Knowledge of the mechanisms underlying rapid evolution of pathogens and their change in virulence during host shifts is crucial to design efficient and sustainable disease management strategies. The proposed research focus on a major model for the study of plant-pathogen interactions, the ascomycete fungus Pyricularia oryzae (syn. Magnaporthe oryzae). This destructive fungal pathogen, causing up to 30% of rice production losses globally, infects diverse cereals and grasses. The specific objectives of EvolMAX were to:
• Identify virulence genes of P. oryzae involved in adaptation to new rice varieties.
• Investigate effector repertoire diversity and evolution in P. oryzae lineages associated with various cereal hosts to better understand host shifts.
The proposed research thus focused on processes related to host adaptation within lineages, and at wider scales between lineages and species. At the rice variety level, we identified two major candidate genes involved in virulence for three rice varieties using genome-wide association mapping. At wider scale, we compiled the most unique and robust dataset of all P. oryzae available genomes infecting all host species to understand the molecular basis of host adaptation.
• Identify virulence genes of P. oryzae involved in adaptation to new rice varieties.
• Investigate effector repertoire diversity and evolution in P. oryzae lineages associated with various cereal hosts to better understand host shifts.
The proposed research thus focused on processes related to host adaptation within lineages, and at wider scales between lineages and species. At the rice variety level, we identified two major candidate genes involved in virulence for three rice varieties using genome-wide association mapping. At wider scale, we compiled the most unique and robust dataset of all P. oryzae available genomes infecting all host species to understand the molecular basis of host adaptation.
Tasks carried out during the course of EvolMAX and the main results are as follows:
To identify effector genes, we have sequenced 78 P. oryzae isolates from one recombining population of the rice-infecting lineage using short-reads. Using full-genome data, we confirmed previous lines of evidence that the studied population is mainly sexual whereas the rice-infecting lineage is mostly clonal throughout its distribution range. Moderate to high levels of recombination found for this population enabled the identification of loci involved in virulence using a genome-wide association mapping approach (GWAS). In addition, we performed gene annotation, and identified secreted proteins.
We performed pathogenicity tests to assess isolate virulence profiles. We used 64 isolates of the studied recombining population and inoculated their spores onto 16 rice varieties and isogenic lines which differed from each other by different major resistance genes. To identify loci involved in virulence, we tested for associations between genotypes and virulence profiles of the 64 P. oryzae isolates for each rice variety or line tested. Out of 16 rice varieties or isogenic lines, six had balanced scores. For the rice variety Toride1, the pathogen’s gene that triggers a response that prevents infection was already known (AVR-Pizt) and therefore, the analysis for this variety represented a control GWAS. The target gene was correctly found within the genomic region associated with virulence, validating the power of this approach. For three other varieties and isogenic lines (LTH, Maratelli, IRBL19-A), a shared genomic region was associated with virulence. In the genomic region of interest, homologous relationships for core genes were identified. To escape recognition, adapted pathogens generally loose or modify their effectors that match to the plant resistant genes present in the host population. Consistent with this expectation, we compared patterns of absence/presence but also haplotypes of these orthologous genes to virulence profiles to pinpoint precisely the genes involved. This approach allowed us to identify two candidate genes that were also predicted as effectors. By using a P. oryzae population with substantial recombination, we were able to conduct in a timely manner what is, to our knowledge, the first proper genome-wide study of virulence phenotypes in P. oryzae. Functional validation and further analysis of the candidate genes will be the next step in collaboration with S. Cesari and T. Kroj at the Plant Health Institute of Montpellier.
For comparative genomics of effector repertoires among lineages associated with different cereal hosts, we compiled whole-genome raw data for 642 P. oryzae isolates from 28 plant genus. Half (321) of the 642 isolates were sequenced in our lab at the Plant Health Institute of Montpellier and the remainder is from public databases. Raw genomic data included short-read and long-read sequencing data for 595 and 47 unique isolates, respectively. Genome assemblies of short-read sequencing data were performed for the 595 isolates so far.
Regarding the dissemination of the action, three abstracts at conferences and a seminar at the Plant Health Institute of Montpellier have been presented. Two high impact peer-reviewed articles are expected. Photographs of our experiments were shared with National Research Institute for Agriculture, Food and Environment (INRAE) to illustrate research conducted in the Institute. A photograph showing the rice sprouts used in our pathogenicity tests was awarded a prize. Also, we made a video to show to the general public our daily scientific work and how we tackle major agricultural and environmental challenges. Additional outreach activities included exhibitions at the French Science Festival, discussions about the project with high-school students at two events and with the lay public at the European Researcher’s nights in 2020 and 2021.
To identify effector genes, we have sequenced 78 P. oryzae isolates from one recombining population of the rice-infecting lineage using short-reads. Using full-genome data, we confirmed previous lines of evidence that the studied population is mainly sexual whereas the rice-infecting lineage is mostly clonal throughout its distribution range. Moderate to high levels of recombination found for this population enabled the identification of loci involved in virulence using a genome-wide association mapping approach (GWAS). In addition, we performed gene annotation, and identified secreted proteins.
We performed pathogenicity tests to assess isolate virulence profiles. We used 64 isolates of the studied recombining population and inoculated their spores onto 16 rice varieties and isogenic lines which differed from each other by different major resistance genes. To identify loci involved in virulence, we tested for associations between genotypes and virulence profiles of the 64 P. oryzae isolates for each rice variety or line tested. Out of 16 rice varieties or isogenic lines, six had balanced scores. For the rice variety Toride1, the pathogen’s gene that triggers a response that prevents infection was already known (AVR-Pizt) and therefore, the analysis for this variety represented a control GWAS. The target gene was correctly found within the genomic region associated with virulence, validating the power of this approach. For three other varieties and isogenic lines (LTH, Maratelli, IRBL19-A), a shared genomic region was associated with virulence. In the genomic region of interest, homologous relationships for core genes were identified. To escape recognition, adapted pathogens generally loose or modify their effectors that match to the plant resistant genes present in the host population. Consistent with this expectation, we compared patterns of absence/presence but also haplotypes of these orthologous genes to virulence profiles to pinpoint precisely the genes involved. This approach allowed us to identify two candidate genes that were also predicted as effectors. By using a P. oryzae population with substantial recombination, we were able to conduct in a timely manner what is, to our knowledge, the first proper genome-wide study of virulence phenotypes in P. oryzae. Functional validation and further analysis of the candidate genes will be the next step in collaboration with S. Cesari and T. Kroj at the Plant Health Institute of Montpellier.
For comparative genomics of effector repertoires among lineages associated with different cereal hosts, we compiled whole-genome raw data for 642 P. oryzae isolates from 28 plant genus. Half (321) of the 642 isolates were sequenced in our lab at the Plant Health Institute of Montpellier and the remainder is from public databases. Raw genomic data included short-read and long-read sequencing data for 595 and 47 unique isolates, respectively. Genome assemblies of short-read sequencing data were performed for the 595 isolates so far.
Regarding the dissemination of the action, three abstracts at conferences and a seminar at the Plant Health Institute of Montpellier have been presented. Two high impact peer-reviewed articles are expected. Photographs of our experiments were shared with National Research Institute for Agriculture, Food and Environment (INRAE) to illustrate research conducted in the Institute. A photograph showing the rice sprouts used in our pathogenicity tests was awarded a prize. Also, we made a video to show to the general public our daily scientific work and how we tackle major agricultural and environmental challenges. Additional outreach activities included exhibitions at the French Science Festival, discussions about the project with high-school students at two events and with the lay public at the European Researcher’s nights in 2020 and 2021.
Comparative genomic analyses of the largest and highest-quality set of P. oryzae genomes, representing a comprehensive set of the pathogen’s hosts, will provide a greater understanding of the ecoevolutionary factors underlying the diversification of P. oryzae and its effectors, a key knowledge underlying fungal adaptation to new host plants. Altogether, the identification of new candidate genes involved in the adaptation of P. oryzae to major resistance traits in rice varieties will be directly used in epidemiological surveillance and plant breeding. As the identification of effectors is necessary for the identification of plant immune receptors, susceptibility genes removal from rice or other cereal plants may lead to reduced susceptibility to blast disease.