Candidate effectors contribute to race differentiation and virulence of the lentil anthracnose pathogen Colletotrichum lentis.

BACKGROUND

The hemibiotroph Colletotrichum lentis, causative agent of anthracnose on Lens culinaris (lentil) was recently described as a new species. During its interaction with the host plant, C. lentis likely secretes numerous effector proteins, including toxins to alter the plant's innate immunity, thereby gaining access to the host tissues for nutrition and reproduction.

RESULTS

In silico analysis of 2000 ESTs generated from C. lentis-infected lentil leaf tissues identified 15 candidate effectors. In planta infection stage-specific gene expression waves among candidate effectors were revealed for the appressorial penetration phase, biotrophic phase and necrotrophic phase. No sign of positive selection pressure [ω (dN/dS) < 1] in effectors was detected at the intraspecific level. A single nucleotide polymorphism in the ORF of candidate effector ClCE6, used to develop a KASPar marker, differentiated perfectly between pathogenic race 0 and race 1 isolates when tested on 52 isolates arbitrarily selected from a large culture collection representing the western Canadian population of C. lentis. Furthermore, an EST encoding argininosuccinate lyase (Arg) was identified as a bacterial gene. A toxin protein ClToxB was further characterized as a potential host-specific toxin through heterologous in planta expression. The knock-down of ClToxB transcripts by RNAi resulted in reduced virulence, suggesting that ClToxB is a virulence factor. In silico analysis of the ClToxB sequence and comparative genomics revealed that ToxB is unlikely a foreign gene in the C. lentis genome. Incongruency between established species relationships and that established based on gene sequence data confirmed ToxB arose through evolution from a common ancestor, whereas the bacterial gene Arg identified in C. lentis was horizontally transferred from bacteria.

CONCLUSIONS

EST mining and expression profiling revealed a set of in planta expressed candidate effectors. We developed a KASPar assay using effector polymorphism to differentiate C. lentis races. Comparative genomics revealed a foreign gene encoding a potential virulence factor Arg, which was horizontally transferred from bacteria into the genus Colletotrichum. ClToxB is further characterized as a host-specific toxin that is likely to contribute to quantitative differences in virulence between the races 0 and 1.