Optimising Agrobacterium-mediated transformation of Corynespora cassiicola to deliver DsRed

Abstract

Corynespora cassiicola is a cosmopolitan fungus with a broad host range. One of the hosts often severely impacted is the rubber tree, Hevea brasiliensis on which it causes devastating leaf defoliation. To date, control of the disease is mainly focused on the application of fungicides, but this only provides short-term solution. Functional genomics through transformation allows evaluation of the disease effectors which may eventually lead to more precise control measures to curb the disease. A transformation system for C. cassiicola mediated by Agrobacterium tumefaciens was successfully established with resistance to hygromycin and DsRed as markers. The protocol was optimised with regard to Agrobacterium strains, C. cassiicola isolates, and other parameters. Combination of isolate CLN23 and A. tumefaciens strain LBA4404 carrying pCAMDsRed gave the highest number of transformants. The optimum fungal age, temperature for incubation and co-cultivation period were 7 days old, 28 °C and 4 days, respectively. However, these parameters are highly subjected to interaction between the variables. The transformants carried the DsRed reporter gene, and demonstrated readily apparent red fluorescence, which allowed disease development in planta to be observed. This robust transformation system should pave the way for future genetic manipulations of C. cassiicola.