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.

Carotenoids are vital pigments responsible for yellow, orange and red color in plants. In Capsicum, capsanthin-capsorubin synthase (CCS), phytoene synthase (PSY), β-Carotene hydroxylase (CRTZ-2) and lycopene β-cyclase (LCYB) were identified to be involved in the carotenoids synthesis pathway. Previously molecular markers based on the CCS and PSY genes have been developed to distinguish fruit colors in pepper. However these markers can distinguish fruit colors of limited pepper genotypes. Therefore, there is need of developing additional markers for accurate prediction of fruit colors using molecular markers. In this study carotenoids contents of 16 pepper accessions were analyzed and the CCS, PSY, CRTZ-2, LCYB genes were sequenced to identify the genes affecting the fruit color. Among all the analyzed carotenoids, capsanthin was accumulated in much higher amount in red and orange fruits (1100-2500 mAU·min and 30-500 mAU·min respectively) while violaxanthin (20-1200 mAU·min) was accumulated more in yellow fruits. Sequence analysis revealed that deletions and two frame shift mutations in CCS gene for yellow accessions. Frame shift mutations of the PSY gene were detected in two orange accessions. These results show that mutations in CCS and PSY genes affect the fruit colors of pepper, and markers can be developed using mutations of these genes.