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BIOGER

BIOlogie et GEstion des Risques en agriculture - Champignons Pathogènes des Plantes

Main Results

Main results

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Population studies
  • Design of molecular tools to differentiate different entities previously collectively named L. maculans. Exploitation of world-wide collections maintained at INRA (IBCN, IMASCORE) to demonstrate the existence of a species complex, the  L. maculans-L. biglobosa species complex, encompassing at least seven genetically distinct entities. Use  of genomics and comparative genomics to show that each of these actually is a distinct species.
  • Setting up of networks, protocols and tools to characterize L. maculans race structure in various parts of the world (Europe, Canada, Australia, USA & South America).
  • Sampling and characterization of L. maculans isolates adapted to cabbage : these have uncommon life traits such as importance of asexual multiplication in their life cycle (Coll. O. Moreno-Rico, UNI-Aguascalientes).
  • Phylogeographic studies aiming at retracing the origin and spread of L. maculans between continents (coll. T. Giraud & P. Gladieux, ESE Orsay). 
  • Setting up of systematic and dynamic survey of races when novel resistance sources are used in the agronomical practice (Rlm1, Rlm7) (collaboration CETIOM).
  • Developmement of molecular markers used for race identification in the field and dynamic survey of emergence and dissemination of new races (coll. CETIOM).
Genetics of the interactions
  • Formal genetic approches used to identify 11 avirulence (AvrLm) genes in L. maculans.
  • Resistance of oilseed rape at the leaf stage depends on major genes (Rlm genes). The resistance genes prevent the fungus from entering and subsequent colonisation of the plant tissues. They are very efficient to control the disease when the corresponding avirulent race of the fungus is prevalent in the field population.
  • Quantitative genetic approaches were used to identify Quantitative Trait Loci (QTLs) involved in fungal aggressiveness (quantitative aspects of fungal pathogenicity).
  • Development of improved differential sets on both the fungus and plant sides (strains of L. maculans and Brassica sp. genotypes) to analyse fungal populations and determine resistance genes occurring in plant genotypes.
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Genomics and comparative genomics
  • The L. maculans chromosomes are structured into isochores (alternating large genomic region with homogeneous GC content and abrupt transition between each compartment), a chromosome structure that was never observed in fungi before.
  • Avirulence genes as well as ca. one hundred of genes encoding putative effectors (Small Secreted Proteins) are located within AT-rich isochores, enriched in truncated and degenerated TEs and poor in coding sequences.
  • This genome location has consequences in terms of gene innovation and evolution under resistance gene selection. It favours non-conventional evolutionary mechanisms such as RIP (Repeat-Induced Point) mutations, large-size deletions etc...
  • We resequenced three isolates of L. maculans with contrasted geographic origin (AIP Bioressources; coll. M. Links & H. Bohran, AAF Saskatoon, Canada). The isolates show a very low amount of SNPs and mainly differ in their gene content, of which the most impacted are genes encoding putative effectors. Depending on the isolates, 12 to 40% of putative effector genes are isolate-specific. 
  • Comparative and evolutionary genomic approaches within the species complex (ANR FungIsochores, Coll. BJ Howlett & R Lowe, Uni-Melbourne, Australia) indicate that TEs are involved in chromosome rearangments, in gene diversification, and in adaptation to host plant. L. maculans like other filamentous phytopathogens has a two-speed genome of which the plastic part contributes to pathogenicity and adaptation.
Genomics, T-DNA insertion library and pathogenicity
  •  A L. maculans T-DNA library comprising of 5000 transformants has been obtained and a systematic analysis of insertion patterns in the genome was performed. More than 300 T-DNA tags were located in the genome : T-DNA integration is correlated with gene density and favored T-DNA targets are TATA-box within promoters and gene introns. Gene Ontology analysis indicates the T-DNA has a favored target towards genes expressed in the culture conditions corresponding to those of agrotransformation, i.e. germinating conidia.
  • Reproducible pathogenicity defects at the cotyledon stage have been observed in ca. 3% of the transformants. Only half of the altered phenotypes could be attributed to the T-DNA insertion.
  • The exploitation of the mutant library allowed us to identify three new genes involved in pathogenicity : (i) Lmgpi15, involved in biosynthesis of GPI (Glycosylphosphatidyl-inositol) anchors, (ii) Lmpma1, a plasma membrane H+-ATPase involved in regulation of intracellular pH (iii) and Lmepi, an UDP-glucose-epimerase involved in galactose metabolism. These data point to the importance of primary metabolism in the infection and tissue colonisation strategies of the fungus.
  • Using genome data, we identified and annotated 355 genes encoding putative transcription factors (TFs) of which 129 were up-regulated during the first stages of plant infection. Eight of these are specific to L. maculans. We initiated a functional analysis of TFs in L. maculans and found that RNAi silencing of an AT-hook TF was responsible for pathogenicity defects and reduced expression of two effector genes.
  • Using genome data, we have built a pipeline to identify simple sequence repeats such as minisatellites (MS) ou microsatellites in order to generate genetic markers to saturate the genetic map. The enriched genetic map has been used to map QTLs following a cross between two isolates with different levels of aggressiveness.
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Functional analysis of Avr genes
  • Three avirulence genes, AvrLm1, AvrLm6 and AvrLm4-7 have been cloned using positional cloning strategies at a time when genome data were not available. The availability of genome data and high density genetic map favoured the cloning of a series of other avirulence genes such as AvrLm11, the cognate avirulence of a B. rapa resistance. This gene is located on a Conditionnally Dispensable Chromosome of L. maculans.
  • Until now, all avirulence genes of L. maculans are located within large AT isochores and are up-regulated upon the first stages of plant infection. They encode Small-Secreted Proteins (SSPs) enriched in cystein residues. Their function is difficult to predict since they lack known domains and usually have no paralogs or recognisable orthologs in other fungi.
  • A repertoire of SSP-encoding genes has been established and a bioinformatic analysis suggested the encoded proteins may contain degenerated RxLR-like motifs. Such is the case for two avirulence proteins of L. maculans, AvrLm6 and AvrLm4-7. Like effector proteins of other filamentous phytopathogens, AvrLm6 and AvrLm4-7 can be translocated within plant or animal cells in the absence of the fungus. One possible mechanism for translocation involving binding to extracellular Pi3P (phosphatidylinositol-3-phosphate) has been proposed (coll. B. Tyler & S. Kale, Oregon State University) and is widely debated in the research community.
  • The 3-D crystal structure of AvrLm4-7 has been determined (coll. H. van Tilbeurgh, IBBMC, Orsay). However, it was insufficient to determine intrinsic function of the protein. Linking the 3-D structure and mutations occuring in field isolates when submitted to the Rlm7 selection, allowed us to identify two regions of the protein involved in interaction with Rlm4 or Rlm7, and one region of the protein involved in translocation to plant host cells. 
Genome structure and its incidence on effector gene expression and adaptation to host
  • Genes encoding avirulence effectors show a specific expression pattern, with repression of expression in vitro and a peak of over-expression in the first stages of tissue colonisation (7 dpi). The TE-rich genome environment contributes to create an heterochromatin landscape, including histone methylation in promoters of genes (coll. M. Freitag, Oregon State University). The shift towards pathogenicity depends on chromatin decondensation allowing whose determinism is unknown.
  • We set up a 5-year experiment to evaluate evolution of L. maculans field populations submitted to the novel Rlm7 selection. In addition we also compared cropping practices between two locations : Grignon (minimum tillage) and Versailles ("cautious" cropping practices) (coll. INRA Agronomie, Grignon). Analysis of the molecular events responsible for loss of the avirulent phenotype surprisingly allowed us to identify all recorded molecular events responsible for the loss of avirulence function identified in the literature for fungal phytopathogens, plus a couple of new events, while this was investigated in a very small, 0.25 ha field. Sequences of events showed RIP mutations were prevalent at the origin of the experiment, whilst partial or complete deletions of the gene predominate at the end of the experiment. This suggests that an immediate response to the selection corresponds to RIP inactivation, due to the TE-rich genome environment promoting gene duplication and/or RIP “leakage”, and the reproduction regime of the fungus. Mutation events and resistance breakdown were greatly favoured by minimum tillage cropping practices.
  • A marked fitness deficit is linked to the loss of AvrLm4-7, while a much lesser impact is associated with the loss of AvrLm1 (coll. B. Fitt, Rothamsted Research). This is consistent with the rapid (less than 4 years) "breakdown" of resistance gene Rlm1 in the field in France and Australia. In contrast, the use of Rlm7 for more than 10 years still has not led to such a "breakdown" in France.
  • Involvement of other avirulence genes in fungal fitness produce contrasted results with some genes having no measurable effect on fungal fitness, others favouring the development of leaf lesions and others reducing size of leaf lesions (ANR Génoplante AvirLep ; coord. M.H. Balesdent). These data indicate a gene-dependent fitness cost for avirulence effectors of L. maculans. Such data can be exploited to evaluate a priori the dispensability of an avirulence gene and thus deduce the corresponding durability of the cognate resistance gene. They also can be integrated in modelling approaches by providing a "fitness cost" to be integrated in the model.
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