Functional characterisation of the Cercospora zeina crp1 gene in pathogenesis

Abstract

Blue light and the perception thereof plays a fundamental role in the survival of all organisms

in changing environments. Cercospora zeina, is the causal agent of grey leaf spot (GLS) disease of maize plants in Southern Africa. The disease is of great economic importance as annual yield losses of up to 65% are experienced by South African farmers. The White Collar-1 protein (WC-1) has been characterised in Neurospora crassa and found to entrain the fungal circadian clock and regulate growth and development of the pathogen. In this study we set out to identify the orthologue of the WC-1 protein in C. zeina, and determine to what level blue light plays a role in the development of GLS in maize. The infection strategy followed by C. zeina is understudied at a molecular level, and therefore in order to effectively control the disease in the field, we need to understand the mechanisms used by the pathogen to infect and colonise

the host.

The orthologue of the WC-1 protein was identified in the draft genome sequence of C. zeina and compared to WC-1 from other fungi. Blue light photoreceptors homologous to the WC-1 protein all contain a modified PAS domain, light-oxygen-voltage (LOV) domain to which the FAD chromophore binds to activate the protein. Phylogenetic relationships of WC-1 homologues from three fungal phyla (Ascomycota, Basidiomycota and Zygomycota) were inferred using a maximum likelihood approach. The results indicated that the Basidiomycota WC-1 homologues underwent independent evolution compared to the other two phyla. The orthologues of the other four circadian clock proteins, WC-2, FRQ, FWD-1 and FRH were identified in the genome sequences of C. zeina and C. zeae-maydis. Furthermore, a previously unidentified putative blue light photoreceptor of the cryptochome family, CRY-DASH, was identified in C. zeina and C. zeae-maydis that might also regulate blue light responses in the

pathogens.

We functionally characterised the CRP1 protein in C. zeina using a split marker approach, which replaces the gene if interest with a hygromycin resistance gene (hygR). Putative Δcrp1 knock-outs were generated and cultured on V8® media supplemented with hygromycin. Putative knock-outs were screened for the presence of the complete hygromycin resistance gene followed by verification that the hygR gene inserted into the correct genomic location.

Southern hybridisation using an internal hygR fragment revealed only a single copy of the inserted gene with no ectopic insertions. Although the genetic screens clearly showed that the endogenous crp1 gene was replaced by the hygR gene, expression of the gene was still detected in Δcrp1 knock-outs, although at reduced levels compared with the wild type C. zeina possibly due to the formation of a heterokaryon. The reduced expression of the crp1 gene in the knock-out lines still showed clear phenotypic differences compared to the wild type fungus. Phenotypic characterisation of the putative Δcrp1 knock-outs indicated that the crp1 gene plays a role in light dependent repression of conidia formation. The crp1 gene is also believed to play a role in the way melanin is deposited (i.e. to which cell layer the melanin granules are transported) and in the production of melanin in constant light. Furthermore, the potential level of pathogenesis of the Δcrp1 knock-outs was decreased based on the significant reduction in the formation of a hyphal network over the stomata used to infiltrate the host plant. In conclusion, all the components of the fungal circadian clock are conserved within C. zeina. We successfully disrupted the C. zeina crp1 the homologue of WC-1, the limiting component of the circadian clock in fungi using a split marker approach. The crp1 gene was found, even at reduced expression levels, to play a role in light dependent spore production, secondary metabolism of melanin and the pathogenesis through reduced stomatal tropism.