Differentiation of pathogenic races of the lentil anthracnose pathogen C.lentis using effectors
The genus Colletotrichum has been under revision for the last decade. It contains species of high economic importance as pathogens of major crops, but also includes species that are model organisms to study host-pathogen interactions. The hemibiotroph Colletotrichum lentis, causative agent of anthracnose on Lens culinaris (lentil) was recently described as a new species. In the host cell, C. lentis initially generates thick, biotrophic primary hyphae restricted to one epidermal cell before switching to the necrotrophic phase by developing thin filamentous secondary hyphae that invade other host cells.
During its interaction with the host plant, C. lentis likely secretes numerous effector proteins including toxins to manipulate the plant’s innate immunity thereby gaining access to the host tissues for nutrition. In silico analysis of expressed sequence tags generated from C. lentis-infected lentil leaflet tissues identified 15 candidate effectors. No sign of positive selection pressure was observed at the intraspecific level, suggesting that C. lentis effectors are under stabilizing selection. In planta infection stage-specific gene expression waves among candidate effectors were revealed for the appressorial penetration phase, biotrophic phase and necrotrophic phase through RT-qPCR. Homologs of 10 out of 15 C. lentis candidate effectors were identified in several other species of Colletotrichum with more than 65% peptide sequence identities across species. Similarly, a toxin protein ClToxB encoded by a single copy gene with four characteristic cysteine residues and further characterized as a potential a host-specific toxin through heterologous in planta expression, had homologs of extensive similarity in five species including three Colletotrichum spp., suggesting that effectors of different, but related species are not as unique as previously thought. In silico analysis of ClToxB sequence and comparative genomics revealed that ToxB is unlikely an alien gene in the C. lentis genome. Congruency between established species relationships and that established based on ToxB sequence data confirmed it arose through evolution from a common ancestor, whereas the bacterial gene Arg identified in ESTs of C. lentis and encoding argininosuccinate lyase was horizontally transferred from bacteria as previously shown for other Colletotrichum species. The knock-down of ClToxB transcripts by RNAi resulted in reduced virulence, suggesting that ClToxB is a virulence factor.
Two pathogenic races (virulent race 0 and less virulent race 1) were described in the Canadian population of C. lentis. A silent single nucleotide polymorphisms in the open reading frame of candidate effector ClCE6 was used to develop Kompetitive Allele Specific PCR marker, which differentiated perfectly between race 0 isolates and race 1 isolates, suggesting that it may be co-segregating with the virulence governing locus/loci, hence can be used to determine the race identity of C. lentis isolates. Race indexing of C. lentis isolates is important not only for monitoring the population dynamics of the pathogen, but also for screening germplasm under field conditions.
Candidate effectors contribute to race differentiation and virulence of the lentil anthracnose pathogen Colletotrichum lentis.
Bhadauria V, MacLachlan R, Pozniak C, Banniza S
BMC Genomics. 2015 Aug 22