Isolation and Characterization of Pepper Genes Involved in CMV-P1 Infection

Abstract

Capsicum annuum Bukang is a resistant variety to Cucumber mosaic virus isolate-P0 (CMV-P0), CMV-P1 can overcome the CMV resistance of Bukang due to mutations in Helicase (Hel) domain of CMV RNA1. To identify host factors involved in CMV-P1 infection, a yeast two-hybrid system derived from C. annuum Bukang cDNA library was used. A total of 156 potential clones interacting with the CMV-P1 RNA helicase domain were isolated. These clones were confirmed by β-galactosidase filter lift assay, PCR screening and sequence analysis. Then, we narrowed the ten candidate host genes which are related to virus infection, replication or virus movement. To elucidate functions of these candidate genes, each gene was silenced by virus induced gene silencing in Nicotiana benthamiana. The silenced plants were then inoculated with green fluorescent protein (GFP) tagged CMV-P1. Virus accumulations in silenced plants were assessed by monitoring GFP fluorescence and enzyme-linked immunosorbent assay (ELISA). Among ten genes, silencing of formate dehydrogenase (FDH) or calreticulin-3 (CRT3) resulted in weak GFP signals of CMV-P1 in the inoculated or upper leaves. These results suggested that FDH and CRT3 are essential for CMV infection in plants. The importance of FDH and CRT3 in CMV-P1 accumulation was also validated by the accumulation level of CMV coat protein confirmed by ELISA. Altogether, these results demonstrate that FDH and CRT3 are required for CMV-P1 infection in plants.