Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

Abstract

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

• Authors: Yoomi Choi, Min-Young Kang, Joung-Ho Lee, Won-Hee Kang, JeeNa Hwang, Jin-Kyung Kwon, Byoung-Cheorl Kang
• Journal: PloS one (PLoS One) Vol. 11 Issue 1 Pg. e0146320 ( 2016) ISSN: 1932-6203 [Electronic] United States
• PMID: 26751216 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Calreticulin
• DNA, Complementary
• Green Fluorescent Proteins
• Formate Dehydrogenases
• beta-Galactosidase
• RNA Helicases

Topics

• Agrobacterium (metabolism)
• Calreticulin (genetics)
• Capsicum (genetics)
• Cucumovirus (enzymology, genetics)
• DNA, Complementary (metabolism)
• Formate Dehydrogenases (genetics)
• Gene Expression Regulation, Plant
• Gene Library
• Gene Silencing
• Genes, Plant
• Green Fluorescent Proteins (metabolism)
• Plant Diseases (genetics)
• Plant Leaves (metabolism)
• Polymerase Chain Reaction
• Protein Binding
• Protein Structure, Tertiary
• RNA Helicases (metabolism)
• Sequence Analysis, DNA
• Two-Hybrid System Techniques
• beta-Galactosidase (metabolism)