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.

In plants, eukaryotic translation elongation factor 1B (eEF1B) is composed of three subunits, eEF1Bα, eEF1Bβ and eEF1B γ. Two subunits are nucleotide exchange subunits (eEF1Bα and eEF1Bβ) and one is a structural protein (eEF1Bγ). In the previous study, eEF1B was identified as a common host factor for several RNA viruses. To test which subunit of eEF1B is essential for Potato virus X (PVX) replication, the virus-induced gene silencing (VIGS) for eEF1Bα, β or γ was performed in Nicotiana benthamiana and green fluorescent protein (GFP)-tagged PVX was inoculated. PVX-GFP accumulation was decreased when eEF1Bβ or γ subunit was silenced, whereas eEF1Bα had no effect on PVX-GFP accumulation in inoculated leaves. Targeting induced local lesions in genome (TILLING) was performed using a Capsicum annuum EMS population to test whether mutations in eEF1Bβ subunit affect virus infection in pepper. We obtained 81 eEF1Bβ mutant lines consisted of 16,759 individuals. These mutant lines are being tested to validate the function of eEF1B β in PVX replication.