SummaryLeptosphaeria maculans , a Dothideomycete causing stem canker on oilseed rape ( Brassica napus ), develops gene-for-gene interactions with its host plants. To date, nine resistance genes ( Rlm1-9 ) have been identified in Brassica spp. The corresponding nine avirulence genes ( AvrLm1-9 ) in L. maculans have been mapped at four independent loci, thereby revealing two clusters of three and four linked avirulence genes. Here, we report the completion of map-based cloning of AvrLm1 . AvrLm1 was genetically delineated within a 7.3 centimorgan interval corresponding to a 439 kb BAC contig. AvrLm1 is a single copy gene isolated within a 269 kb non-coding, heterochromatin-like region. The region comprised a number of degenerated, nested copies of four long-terminal repeat (LTR) retrotransposons, including Pholy and three novel Gypsy-like retrotransposons. AvrLm1 restored the avirulent phenotype on Rlm1 cultivars following functional complementation of virulent isolates. AvrLm1 homologues were not detected in other Leptosphaeria species or in known fungal genomes including the closely related species Stagonospora nodorum . The predicted AvrLm1 protein is composed of 205 amino acids, of which only one is a cysteine residue. It contains a peptide signal suggesting extracellular localization. Unlike most other fungal avirulence genes, AvrLm1 is constitutively expressed, with a probable increased level of expression upon plant infection, suggesting the absence of tight regulation of AvrLm1 expression.

SummaryLeptosphaeria maculans is the ascomycete responsible for one of the most damaging diseases of oilseed rape (Brassica napus), stem canker of crucifers. Both avirulence (AvrLm) genes in the fungus and resistance (Rlm) genes in the plant are genetically clustered. Using a map-based cloning strategy, we delineated a 238 kb region containing the AvrLm7 locus. Structural features of the region were reminiscent of those previously found on another chromosome for genomic regions encompassing AvrLm1 and AvrLm6, i.e. GC-equilibrated, gene-rich isochores alternating with AT-rich, recombinationdeficient, gene-poor isochores. These latter corresponded to mosaics of degenerated and truncated transposable elements. AvrLm7 is the only gene located within a 60 kb AT-rich isochore. It induced resistance responses in plants harbouring either Rlm7 or Rlm4, and was thus renamed AvrLm4-7. It encodes a 143-amino-acid cysteine-rich protein, predicted to be secreted, and strongly induced during early stages of plant infection. Sequencing and restriction analyses of AvrLm4-AvrLm7 or avrLm4-AvrLm7 alleles in L. maculans field isolates, and targeted point mutagenesis strongly suggested that one single base mutation, leading to the change of a glycine to an arginine residue, was responsible for the loss of AvrLm4 specificity whereas AvrLm7 recognition was unaltered.

Map-based cloning of avirulence genes of the AvrLml-2-6 cluster was recently undertaken in Leptosphaeria maculans and led to the identification of AvrLm1. The ensuing chromosome walk toward AvrLm6 resulted in the delineation of a 562-kb bacterial artificial chromosome (BAC) clone contig in an avirulent isolate. Following sequencing of the contig and sequence comparison with a virulent isolate, four AvrLm6 candidate genes were identified. Complementation of the virulent isolate with the four candidates was performed and one gene was found to fully restore the avirulent phenotype on Rlm6 oilseed rape genotypes. AvrLm6 was found to be located in the same genome context as AvrLml, because it is a solo gene surrounded by 85 and 48 kb of degenerated repeats on its 5' and 3' sides, respectively. AvrLm6 is an orphan gene encoding a small, potentially secreted, cysteine-rich protein. Comparison of AvrLm1 and AvrLm6 expressions by quantitative reverse-transcription polymerase chain reaction revealed that both genes are highly overexpressed during primary leaf infection. Using RNA interference, decreasing expression of AvrLm6 was shown to result in virulence toward Rlm6 genotypes whenever the expression was reduced by more than 60% compared with the wild-type isolate.

SummaryExtending the durability of plant resistance genes towards fungal pathogens is a major challenge. We identified and investigated the relationship between two avirulence genes of Leptosphaeria maculans, AvrLm3 and AvrLm4-7. When an isolate possesses both genes, the Rlm3-mediated resistance of oilseed rape (Brassica napus) is not expressed due to the presence of AvrLm4-7 but virulent isolates toward Rlm7 recover the AvrLm3 phenotype.Combining genetic and genomic approaches (genetic mapping, RNA-seq, BAC (bacterial artificial chromosome) clone sequencing and de novo assembly) we cloned AvrLm3, a telomeric avirulence gene of L. maculans. AvrLm3 is located in a gap of the L. maculans reference genome assembly, is surrounded by repeated elements, encodes for a small secreted cysteinerich protein and is highly expressed at early infection stages.Complementation and silencing assays validated the masking effect of AvrLm4-7 on AvrLm3 recognition by Rlm3 and we showed that the presence of AvrLm4-7 does not impede AvrLm3 expression in planta. Y2H assays suggest the absence of physical interaction between the two avirulence proteins.This unusual interaction is the basis for field experiments aiming to evaluate strategies that increase Rlm7 durability.

Summary Interactions between Leptosphaeria maculans, causal agent of stem canker of oilseed rape, and its Brassica hosts are models of choice to explore the multiplicity of ‘gene‐for‐gene’ complementarities and how they diversified to increased complexity in the course of plant–pathogen co‐evolution. Here, we support this postulate by investigating the AvrLm10 avirulence that induces a resistance response when recognized by the Brassica nigra resistance gene Rlm10. Using genome‐assisted map‐based cloning, we identified and cloned two AvrLm10 candidates as two genes in opposite transcriptional orientation located in a subtelomeric repeat‐rich region of the genome. The AvrLm10 genes encode small secreted proteins and show expression profiles in planta similar to those of all L. maculans avirulence genes identified so far. Complementation and silencing assays indicated that both genes are necessary to trigger Rlm10 resistance. Three assays for protein–protein interactions showed that the two AvrLm10 proteins interact physically in vitro and in planta. Some avirulence genes are recognized by two distinct resistance genes and some avirulence genes hide the recognition specificities of another. Our L. maculans model illustrates an additional case where two genes located in opposite transcriptional orientation are necessary to induce resistance. Interestingly, orthologues exist for both L. maculans genes in other phytopathogenic species, with a similar genome organization, which may point to an important conserved effector function linked to heterodimerization of the two proteins.