Molecular bases of the R-Avr interaction in the model forest pathosystem Populus-Melampsora through an integrative biology approach
PI: Sébastien Duplessis – UMR: UMR 1136 Interactions Arbres/Micro-organismes (EcoGenomic Team)
Co-applicants: Pascal Frey, Fabien Halkett, Stéphane De Mita (UMR 1136 IAM, Ecology Team), Arnaud Hecker, Nicolas Rouhier (UMR 1136 IAM, Protein Engineering Team)
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Context — The strong impact of rust diseases on the productivity of many agrosystems is urging us to understand the mechanisms of interaction between rust fungi and their host plants in order to design durable resistance strategies. Our knowledge of rust fungus biology is still limited, mostly because of the obligate biotrophic status of these pathogens, which impedes the development of functional approaches. Thanks to recent advances made in genomics, it is now possible to dissect complex genetic traits such as disease resistance in non-model species. The availability of the genome sequence of both the poplar tree Populus trichocarpa and the poplar leaf rust fungus Melampsora larici-populina, is a great opportunity to decipher the genetic bases of tree-biotrophic fungus interactions.
Objectives —The present project aims to study the mechanisms of interaction between molecular determinants responsible for resistance (R genes) in poplar and virulence (effectors, Avr genes) in the rust fungus M. larici-populina.
Approach — We propose to develop an integrative biology approach bridging population genomics, functional genomics and protein biochemistry to identify the key determinants of fungal virulence and to validate the interaction between R and AVR proteins.
Key results —
- 88 M. larici-populina isolates from a large collection sampled over 20 years in France around a major event of rust resistance breakdown were selected and multiplicated on poplar leaves. After DNA isolation, genomes were obtained through Illumina sequencing. Single Nucleotide Polymorphisms (SNPs) were identified in these genomes by comparison to the M. larici-populina 98AG31 isolate reference genome available at the Joint Genome Institute.
- Structure of targeted populations and demographic events related to M. larici-populina virulence 7 emergence were determined by population genetics.
- Based on the population structure knowledge, a Genome Scan approach was applied to identify genomic regions under selection possibly after the virulence 7 emergence event.
Main conclusions / key discussion points —
- Populations structure analysis indicates that virulence 7 isolates appeared in 1994 from populations adapted to other poplar resistances before Resistance 7 deployment. This genetic group was subjected to a bottleneck followed by a demographic expansion allowing diffusion to the whole french territory.
Future perspectives — Candidate genes are now scrutinized to determine the gene(s) more likely responsible for virulence 7 (e.g. expression during interaction with the host plant). Recombinant protein(s) corresponding to the most promising genes will be produced in Escherichia coli and will be infiltrated in poplar cultivars carrying Resistance 7 to validate the R-Avr interaction (hypersensitive response). If successful, the fungal protein function will be studied in greater detail (localization and protein target(s) in host cells) and the corresponding poplar Resistance protein will be searched for by co-immunoprecipitation and pull-down.
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Photo caption
Credits : Pascal Frey, Benjamin Petre & Sébastien Duplessis (INRA)
The poplar leaf rust disease caused by Melampsora larici-populina is marked by the formation of orange pustules on the lower side of infected leaves that are sporogenous structures producing thousands of spores spreading the disease. Pictures here illustrate the disease symptoms at the leaf scale, in close-up showing spores agregates on the leaf surface and fungal infection structures stained by toluidine blue within the leaf parenchyma.
