In the present study, we developed an efficient and reproducible Agrobacterium-facilitated transformation procedure for chilli using cotyledonary and hypocotyl explants. Our study demonstrated that the explants derived from hypocotyls of young seedlings of chilli and cotyledonary explants obtained from tomato seedlings are highly amenable for genetic transformation in chilli / tomato through Agrobacterium-based approach. Subsequently, we made use of the same protocols to develop CgCOM1i transgenic plants of chilli and tomato.
Anthracnose disease is caused by the ascomycetes fungal species Colletotrichum, which is responsible for heavy yield losses in chilli and tomato worldwide. Chemical fungicides are traditionally used to control anthracnose disease, but with limited success. In the present study we assessed the potential of Host-Induced Gene Silencing (HIGS) approach to target the Colletotrichum gloeosporioides COM1 (CgCOM1) gene involved in fungal conidial and appresorium development, to restrict the fungal infection in chilli and tomato. To aid this study, stable transgenic lines of chilli and tomato were developed using the RNAi construct of the fungal pathogen gene CgCOM1 employing Agrobacterium-mediated transformation. Transgenic plants were characterized by molecular and gene expression analyses. Generation of specific CgCOM1 siRNA in the transgenic chilli and tomato RNAi lines was confirmed by stem-loop RT-PCR. Fungal challenge assays on leaves and fruits showed that the CgCOM1i transgenic lines attained a high degree of resistance against infection by C. gleoesporioides, as evidenced by the inhibition of progression anthracnose disease up to 90% as compared to vector control and wild type plants. RT-qPCR analysis revealed barely any CgCOM1 transcripts in the C. gloeosporioides infected tissues, indicating near complete silencing of CgCOM1 gene expression in the pathogen. Microscopic studies on Cg-challenged leaves of chilli-CgCOM1i lines revealed highly suppressed germ tube growth and appressoria formation in C. gloeosporioides, indicating incapacitation of infecting ability of the fungal pathogen.
Current study showed that HIGS is a powerful tool to control anthracnose disease caused by C. gloeosporioides in chilli and tomato. This technique could also be useful in developing resistant crops against various other fungal diseases.
Key Words of the Research: Anthracnose, Colletotrichum gloeosporioides, CgCOM1, Host Induced Gene Silencing, RNAi