Cloning the Cercospora zeina Ecp2 effector gene for Agrobacterium-mediated transient transformation

Abstract

Maize (Zea mays) is a staple crop in Africa that is under severe threat to disease by pathogenic organisms. Grey leaf spot (GLS) caused by Cercospora zeina drastically limits the yield and quality of maize produced. Not enough is understood about how C. zeina causes GLS, but it is known that it is a maize-specific hemibiotrophic fungus. Proteins called effectors are essential for the virulence of pathogens such as Cladosporium fulvum. Extracellular protein 2 (Ecp2), an effector identified in some Dothideomycete fungi, has an unknown function but has been shown to play a role in the virulence of the fungi. The aim of this study was to clone C. zeina Ecp2 (CzEcp2) into a binary vector for agroinfiltration of Nicotiana spp. The C. zeina genome and RNA sequence data (in planta and in vitro) were searched for a candidate Ecp2 gene. The complete CzEcp2 sequence (with the fungal signal peptide) and the mature sequence (lacking the fungal signal peptide) were cloned into the pTRAkc-ERH binary vector. pTRAkc-ERHCzEcp2 EB (fungal signal peptide) and pTRAkc-ERHCzEcp2 NB (LPH signal peptide) were respectively transformed into Agrobacterium tumefaciens GV3101 (pSOUP+pMP90). Phytophthora infestans INF1 was used as the positive control for HR expression. Untransformed Agrobacterium and the pTRAkc-ERH empty vector were used as negative controls. Nicotiana benthamiana, Nicotiana tabacum cv. Petit Havana and Nicotiana tabacum cv. LA Burley were then transiently agroinfiltrated. The plants were monitored for a hypersensitive response (HR) for 10 days. CzEcp2 expression did not result in HR for the three Nicotiana spp., but chlorosis was observed. INF1 caused a HR in all three Nicotiana spp. and the negative controls did not cause any changes. The lack of HR where CzEcp2 was expressed, may be due to lack of CzECP2 transport and recognition, a delayed HR or that the T-DNA was not adequately transferred into the host cells.