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

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.     

Position of the reduced mycorrhizal colonisation (<Emphasis Type="Italic">Rmc</Emphasis>) locus on the tomato genome map

Our research aims to investigate the molecular communication between land plants and arbuscular mycorrhizal (AM) fungi in the establishment of symbiosis. We have identified a mutation in the facultative AM host tomato, which we named rmc. Plants that are homozygous for rmc no longer host most AM fungi. The mutation also affects the interaction of tomato with root knot nematode and Fusarium wilt. However, the function/s encoded by the intact Rmc locus is/are unknown. To clone and sequence the gene or genes that comprise the Rmc locus, we have initiated a positional cloning project. In this paper, we report the construction of mapping populations and use of molecular markers from the published genome map to identify the location of Rmc on tomato chromosome 8. Nucleotide binding site-leucine rich repeat resistance genes, reported to reside in the same region of that chromosome, provided insufficient differences to develop cleaved amplified polymorphic sequence markers. Therefore, we were unable to map these sequences in relation to rmc. Our results potentiate future work to identify the Rmc function and to determine the genetic basis for the multiple plant-microbe interaction functions that the rmc mutation has defined.     

Genetic mapping of the <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> avirulence gene corresponding to the <Emphasis Type="Italic">LepR1</Emphasis> resistance gene of <Emphasis Type="Italic">Brassica napus</Emphasis>

AvrLepR1 of the fungal pathogen Leptosphaeria maculans is the avirulence gene that corresponds to Brassica LepR1, a plant gene controlling dominant, race-specific resistance to this pathogen. An in vitro cross between the virulent L. maculans isolate, 87-41, and the avirulent isolate, 99-56, was performed in order to map the AvrLepR1 gene. The disease reactions of the 94 of the resulting F₁ progenies were tested on the canola line ddm-12-6s-1, which carries LepR1. There were 44 avirulent progenies and 50 virulent progenies suggesting a 1:1 segregation ratio and that the avirulence of 99-56 on ddm-12-6s-1 is controlled by a single gene. Tetrad analysis also indicated a 1:1 segregation ratio. The AvrLepR1 gene was positioned on a genetic map of L. maculans relative to 259 sequence-related amplified polymorphism (SRAP) markers, two cloned avirulence genes (AvrLm1 and AvrLm4-7) and the mating type locus (MAT1). The genetic map consisted of 36 linkage groups, ranging in size from 13.1 to 163.7 cM, and spanned a total of 2,076.4 cM. The AvrLepR1 locus was mapped to linkage group 4, in the 13.1 cM interval flanked by the SRAP markers SBG49-110 and FT161-223. The AvrLm4-7 locus was also positioned on linkage group 4, close to but distinct from the AvrLepR1 locus, in the 5.4 cM interval flanked by FT161-223 and P1314-300. This work will make possible the further characterization and map-based cloning of AvrLepR1. A combination of genetic mapping and pathogenicity tests demonstrated that AvrLepR1 is different from each of the L. maculans avirulence genes that have been characterized previously.     

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

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.     

The Brassica napus receptor‐like protein RLM2 is encoded by a second allele of the LepR3/Rlm2 blackleg resistance locus

Leucine‐rich repeat receptor‐like proteins (LRR‐RLPs) are highly adaptable parts of the signalling apparatus for extracellular detection of plant pathogens. Resistance to blackleg disease of Brassica spp. caused by Leptosphaeria maculans is largely governed by host race‐specific R‐genes, including the LRR‐RLP gene LepR3. The blackleg resistance gene Rlm2 was previously mapped to the same genetic interval as LepR3. In this study, the LepR3 locus of the Rlm2 Brassica napus line ‘Glacier DH24287’ was cloned, and B. napus transformants were analysed for recovery of the Rlm2 phenotype. Multiple B. napus, B. rapa and B. juncea lines were assessed for sequence variation at the locus. Rlm2 was found to be an allelic variant of the LepR3 LRR‐RLP locus, conveying race‐specific resistance to L. maculans isolates harbouring AvrLm2. Several defence‐related LRR‐RLPs have previously been shown to associate with the RLK SOBIR1 to facilitate defence signalling. Bimolecular fluorescence complementation (BiFC) and co‐immunoprecipitation of RLM2‐SOBIR1 studies revealed that RLM2 interacts with SOBIR1 of Arabidopsis thaliana when co‐expressed in Nicotiana benthamiana. The interaction of RLM2 with AtSOBIR1 is suggestive of a conserved defence signalling pathway between B. napus and its close relative A. thaliana.     

Molecular approaches to understanding mycorrhizal symbioses

Molecular analyses of plant–microbe interactions have become common place in the last two decades. Although there are philosophical considerations about the application of a reductionist approach to some areas of research, the collaborative interface (e.g. molecular ecology) can provide specialised insight to the generalist, whilst adding broader relevance to the research of the specialist. However, the expense of this discipline has tended to restrict research to work on model host–microbe interactions. Molecular techniques were embraced early on by a few pioneers from the field of mycorrhizal research. Despite some high profile research, the number of molecular mycorrhizal publications has only recently begun to escalate. However the extent of literature now has exceeded the capacity for a comprehensive short review. In this paper we will briefly discuss the use of model species for molecular research and explore the range of questions that are being addressed using molecular techniques, whilst minimising use of specific jargon, to maximise the usefulness of this review to a non specialist audience. Our primary focus is on arbuscular mycorrhizal symbiosis, to complement the papers by Tagu et al., Podila et al. and Chalot et al. (all this volume), who have addressed aspects of research on ectomycorrhizal symbioses. Here we include specific citations from research groups around the world, along with reference to more detailed reviews, to provide a taste of the current excitement in this fundamental and rapidly evolving research field.