Comparing a pathogenicity effector gene family from two cercospora species by agroinfiltration into tobacco

Abstract

Cercospora zeina is the causal agent of grey leaf spot (GLS) of maize, which causes devastating crop yield losses. The fungus secretes molecules termed effectors to overcome host defences, obtain nutrients from the host and negatively affect the host. In this study, two effectors in C. zeina and their orthologs from Cercospora zeae-maydis were identified as good candidates to study based on in silico analyses. This included analyses by EffectorP 3.0, Localizer 1.0, ApoplastP 1.0 SignalP 6.0. The two candidate effectors in C. zeina were named CzCSEP4 and CzCSEP20, and in C. zeae-maydis termed CzmCSEP4 (89% identity to CzCSEP4, on the protein level) and CzmCSEP20 (94% identity to CzCSEP20, on the protein level). These genes were expected to cause a hypersensitive response in Nicotiana benthamiana in the form of cell death. The genes were amplified from C. zeina or from C. zeae-maydis cDNA and their native signal peptides were replaced with an apoplastic-specific signal peptide. These effectors were then cloned into a binary vector system, transformed into Agrobacteria, and infiltrated into N. benthamiana. In N. benthamiana the positive control of INF1 (Phytophthora infestans) showed necrosis, whereas the two C. zeina candidate effectors did not (p>0.05, t-test). The effectors were demonstrated to have structural similarities to the Fulvia fulva Ecp2 effector’s Hce2 (Homologs of C. fulvum Ecp2) domain. Effectors containing this domain were able to elicit a hypersensitive response in several tobacco species, including N. tabacum, and could not elicit a response in N. benthamiana. These genes were then tested in N. tabacum and showed a hypersensitive response in the infiltrated areas that was significantly different to the Agrobacterium only control (p<0.05, t-test). In conclusion, these genes can be termed secreted effector proteins, thereby proving that these genes are likely to form part of an effector gene family and may play a role in promoting pathogenicity of the fungi in the host.