A two genes - for - one gene interaction between Leptosphaeria maculans and Brassica napus.

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.

Quantitative resistance increases the durability of qualitative resistance to Leptosphaeria maculans in Brassica napus.

It has frequently been hypothesized that quantitative resistance increases the durability of qualitative (R-gene mediated) resistance but supporting experimental evidence is rare. To test this hypothesis, near-isogenic lines with/without the R-gene Rlm6 introduced into two Brassica napus cultivars differing in quantitative resistance to Leptosphaeria maculans were used in a 5-yr field experiment. Recurrent selection of natural fungal populations was done annually on each of the four plant genotypes, using crop residues from each genotype to inoculate separately the four series of field trials for five consecutive cropping seasons. Severity of phoma stem canker was measured on each genotype and frequencies of avirulence alleles in L. maculans populations were estimated. Recurrent selection of virulent isolates by Rlm6 in a susceptible background rendered the resistance ineffective by the third cropping season. By contrast, the resistance was still effective after 5 yr of selection by the genotype combining this gene with quantitative resistance. No significant variation in the performance of quantitative resistance alone was noted over the course of the experiment. We conclude that quantitative resistance can increase the durability of Rlm6. We recommend combining quantitative resistance with R-gene mediated resistance to enhance disease control and crop production.

Repeat-induced point mutation (RIP) as an alternative mechanism of evolution toward virulence in Leptosphaeria maculans.

Three avirulence genes, AvrLm1, AvrLm6, and AvrLm4-7, were recently identified in Leptosphaeria maculans and found to be localized as solo genes within large noncoding, heterochromatin-like regions mainly composed of retrotransposons, truncated and degenerated by repeat-induced point mutation (RIP). The Rlm6 resistance gene has been overcome within 3 years in outdoor experiments in France and, here, we investigate the molecular basis of evolution toward virulence at the AvrLm6 locus. A region of 235 kb was sequenced in a virulent isolate and showed the deletion of AvrLm6 and three divergent mosaics of retrotransposons. AvrLm6 was found to be absent from 66% of 70 virulent isolates, with multiple events of deletion. The sequencing of virulent alleles in 24 isolates revealed a few cases of point mutations that had created stop codons in the sequence. The most frequent mutation events, however, were RIP, leading to the modification of 4 to 9% of the bases compared with the avirulent allele and generating 2 to 4 stop codons. Thus, RIP is described for the first time as an efficient mechanism leading to virulence and the multiple patterns of mutation observed suggest that multiple RIP events could occur independently in a single field population during 1 year.

Molecular phylogeny of the Leptosphaeria maculans-L. biglobosa species complex.

Leptosphaeria maculans (anamorph Phoma lingam), the ascomycete causing stem canker of crucifers, is a species complex that can be separated into at least seven distinct subgroups using a combination of biochemical and molecular criteria. In the present study sequences of the entire ITS region, including the 5.8S rDNA, of 38 isolates representing the seven subgroups, along with specimens from culture collections, were analysed, compared to those of closely related Leptosphaperia species, and the phylogeny inferred using parsimony and distance analyses. A well-supported clade encompassed all isolates of the seven subgroups along with L. conferta, a known saprobe of dried crucifer stems. The L. maculans isolates were further separated into two well-supported clades corresponding to L. maculans s. str. and the recently named L. biglobosa. Parsimony and distance analyses further separated groups within both species, usually corresponding to specific host plants or geographic origin, e.g. L. maculans 'brassicae' from cultivated Brassica, L. maculans 'lepidii'. from Lepidium sp., L. biglobosa 'brassicae', from various Brassica species, L. biglobosa 'thlaspii' from Thlaspi arvense, L. biglobosa 'erysimii' from Erysimum sp., and L. biglobosa 'canadensis' mostly found in central Canada. The oldest L. maculans specimens maintained in international collections clustered with either L. maculans 'brassicae', L. biglobosa 'brassicae', or a still different group closely related to L. biglobosa 'thlaspii'. The evolutionary relationships between the seven infraspecific groups are discussed in terms of phytopathological relevance and species isolation linked with specific life cycle, geographic isolation or host specificity.