Rapid identification of the Leptosphaeria maculans avirulence gene AvrLm2 using an intraspecific comparative genomics approach

Abstract

Five avirulence genes from Leptosphaeria maculans, the causal agent of blackleg of canola (Brassica napus), have been identified previously through map-based cloning. In this study, a comparative genomic approach was used to clone the previously mapped AvrLm2. Given the lack of a presence-absence gene polymorphism coincident with the AvrLm2 phenotype, 36 L. maculans isolates were resequenced and analysed for single-nucleotide polymorphisms (SNPs) in predicted small secreted protein-encoding genes present within the map interval. Three SNPs coincident with the AvrLm2 phenotype were identified within LmCys1, previously identified as a putative effector-coding gene. Complementation of a virulent isolate with LmCys1, as the candidate AvrLm2 allele, restored the avirulent phenotype on Rlm2-containing B. napus lines. AvrLm2 encodes a small cysteine-rich protein with low similarity to other proteins in the public databases. Unlike other avirulence genes, AvrLm2 resides in a small GC island within an AT-rich isochore of the genome, and was never found to be deleted completely in virulent isolates.

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 AvrLm2 gene was also found on SC_6 in a cluster with AvrLm1 and AvrLm6. It is a 699 bp gene that encodes a 232 aa protein (Ghanbarnia et al, 2014), located on a GC island in the middle of an AT isochore. Similar to other known avirulence genes, this gene too encodes a small secreted protein and does not match other proteins when searched in databases.…”

“…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…”

“…Fudal et al (2009) and Van de Wouw et al (2010) also found evidence of RIP mutations at the AvrLm6 locus. AvrLm2 was diversified by the effect of three SNPs in its coding sequence (Ghanbarnia et al, 2014). In the case of AvrLm4-7, Rlm4 virulence is conferred via a single amino acid substitution in AvrLm4, whereas Rlm7 virulence is conferred through RIP mutations and locus deletion of Avr7 (Daverdin et al, 2012;Parlange et al, 2009;Van de Wouw and Howlett, 2012).…”

“…In such a scenario, the pathogen needs to overcome resistance and does so because of its high evolutionary potential. The detectable avirulence gene is quickly rendered unrecognisable through methods such as deletion, RIP mutation, point mutation or by conditionally losing the chromosome that contains the avirulence gene (Gout et al, 2006b;Fudal et al, 2007;Fudal et al, 2009;Balesdent et al, 2013;Ghanbarnia et al, 2014;Van de Wouw et al, 2014). This led us to hypothesise that the gene loss tool could highlight candidate effectors that are being lost in the sample set as compared to the reference.…”