Heterochromatin-Like Regions as Ecological Niches for Avirulence Genes in the Leptosphaeria maculans Genome: Map-Based Cloning of AvrLm6

Abstract

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.

Population diversity and genomics of the fungal pathogen of canola Leptosphaeria maculans

“…The products of these genes are known as effector proteins, and these carry out functions such as plant defence suppression and host cellwall modifications upon infection (Rouxel et al, 2011). Of the eleven L. maculans Avr genes identified, six (AvrLm1, AvrLm6, AvrLm4-7, AvrLm11, AvrLm2 and AvrLm3) (Fudal et al, 2007;Gout et al, 2006b;Parlange et al, 2009;Balesdent et al, 2013;Ghanbarnia et al, 2014;Plissonneau et al, 2015), have been characterised (Table 1.1). A seventh AvrLm gene, AvrLmJ1, virulent on Brassica juncea cultivars, has also been characterised .…”

“…The AvrLm1 protein itself contains a single cysteine residue and is composed of 205 amino acids. The region comprising AvrLm6 is 133 kb long, non-coding and contains long-terminal repeat retrotransposons (Fudal et al, 2007). Both genes generate small protein products with a single N-terminal signal peptide.…”

“…On the other hand, effectors damaging to pathogen fitness can just as easily be lost. Four of the known effector genes, AvrLm1, AvrLm6, AvrLm4-7 and AvrLm11 are hosted in AT-rich isochores of the blackleg genome (Gout et al, 2006b;Fudal et al, 2007;Parlange et al, 2009;Balesdent et al, 2013;Grandaubert et al, 2014b).…”

“…Previous studies have established that L. maculans possesses a high evolutionary potential and rapidly evolves to overcome host resistance (Chapter 3; Barrins et al, 2004;Rouxel et al, 2011;Travadon et al, 2011;Dilmaghani et al, 2012;Zander et al, 2013;Grandaubert et al, 2014b;Patel et al, 2015). Gene loss via deletion is an effective method of adaptation employed by several fungal pathogens like Magnaporthe oryzae (Couch et al, 2005), Blumeria graminis (Wicker et al, 2013), including L. maculans (Gout et al, 2006b;Fudal et al, 2007). We hypothesised that one of the major causes for gene loss in this pathogen would be deletion in known effector genes and other pathogenicity related or unknown effectors, to overcome host recognition.…”

“…Initially, map-based cloning was used to identify AvrLm1 and AvrLm6 (Gout et al, 2006b;Fudal et al, 2007), which are clustered on SC_6, along with AvrLm2 (Ghanbarnia et al, 2014), in a region affected by RIP mutations. In isolates virulent on cultivars containing Rlm1 and Rlm6, the loci containing AvrLm1…”