Leptosphaeria maculans AvrLm9: a new player in the game of hide and seek with AvrLm4‐7

Abstract

Blackleg disease of Brassica napus caused by Leptosphaeria maculans (Lm) is largely controlled by the deployment of race-specific resistance (R) genes. However, selection pressure exerted by R genes causes Lm to adapt and give rise to new virulent strains through mutation and deletion of effector genes. Therefore, a knowledge of effector gene function is necessary for the effective management of the disease. Here, we report the cloning of Lm effector AvrLm9 which is recognized by the resistance gene Rlm9 in B. napus cultivar Goéland. AvrLm9 was mapped to scaffold 7 of the Lm genome, co-segregating with the previously reported AvrLm5 (previously known as AvrLmJ1). Comparison of AvrLm5 alleles amongst the 37 re-sequenced Lm isolates and transgenic complementation identified a single point mutation correlating with the AvrLm9 phenotype. Therefore, we renamed this gene as AvrLm5-9 to reflect the dual specificity of this locus. Avrlm5-9 transgenic isolates were avirulent when inoculated on the B. napus cultivar Goéland. The expression of AvrLm5-9 during infection was monitored by RNA sequencing. The recognition of AvrLm5-9 by Rlm9 is masked in the presence of AvrLm4-7, another Lm effector. AvrLm5-9 and AvrLm4-7 do not interact, and AvrLm5-9 is expressed in the presence of AvrLm4-7. AvrLm5-9 is the second Lm effector for which host recognition is masked by AvrLm4-7. An understanding of this complex interaction will provide new opportunities for the engineering of broad-spectrum recognition.

The Brassica napus Wall-Associated Kinase-Like (WAKL) gene Rlm9 provides race-specific blackleg resistance

“…Further ddPCR analysis of the T 1 plants derived from transgenic line NLA68 (one heterozygous transgene insertion at T 0 ) allowed for selection of a T 1 plant carrying a single homozygous insertion which was self-fertilized to produce homozygous T 2 seed (hereafter referred to as Westar:Rlm9). Further testing of Westar:Rlm9 with additional transgenic isolates carrying virulence genes matching other A07 blackleg R genes (2367:AvrLm1, 2367:AvrLm3, 2367:AvrLm4-7, 2367:AvrLm7) produced only susceptible interactions (data not shown) as previously demonstrated (Ghanbarnia et al, 2018). This reconfirmed both the identity of BnaA07g20220D as Rlm9 and the specificity of the Rlm9 -AvrLm5-9 interaction.…”

“…Stb6 confers resistance to races of the apoplastic fungal pathogen Zymoseptoria tritici which produce the matching effector AvrStb6, though resistance is semi-dominant and conferred without a typical hypersensitive response (HR) (Zhong et al, 2017;Saintenac et al, 2018). In contrast Rlm9 induces a clear, dominant HR at the site of infection, responding to the presence of the L. maculans avirulence protein AvrLm5-9 (Ghanbarnia et al, 2018). The emergence of WAKL proteins as new players in race-specific resistance brings with it many fundamental questions.…”

“…Recently, we reported the cloning of L. maculans effector AvrLm5-9 (Ghanbarnia et al, 2018). As previously reported recognition of AvrLm5-9 and AvrLm3 by their cognate R proteins, Rlm9 and Rlm3 is masked in the presence of AvrLm4-7 and this masking effect is neither due to direct interaction between these effector proteins nor due to the suppression of their transcription (Plissonneau et al, 2016;Ghanbarnia et al, 2018). To examine whether AvrLm5-9 directly interacts with Rlm9, we cloned the extracellular region of Rlm9 in the prey vector pGADT7 and AvrLm5-9 lacking the signal peptide sequences in the bait vector pGBKT7 for yeast two-hybrid assay.…”

“…They form a tight genetic cluster on chromosome A07 and may possibly be allelic variants of the same R locus (Larkan et al, 2016). The corresponding avirulence (Avr) genes AvrLm3, AvrLm4-7 and AvrLm5-9 have been cloned from Lm and all encode small cysteine-rich secreted proteins (Parlange et al, 2009;Plissonneau et al, 2016;Ghanbarnia et al, 2018). Recognition of both AvrLm3 and AvrLm5-9 by Rlm3 and Rlm9, respectively, is masked in the presence of AvrLm4-7.…”

“…Recognition of both AvrLm3 and AvrLm5-9 by Rlm3 and Rlm9, respectively, is masked in the presence of AvrLm4-7. However AvrLm4-7 neither interferes with the expression of, nor directly interacts with, AvrLm3 or AvrLm5-9, nor do AvrLm3 and AvrLm5-9 interact (Ghanbarnia et al, 2018). To investigate this complex system of pathogen recognition we have pursued the cloning of Brassica R genes from the Rlm3/4/7/9 gene cluster.…”