Helicase domain encoded by Cucumber mosaic virus RNA1 determines systemic infection of Cmr1 in pepper.
The Cmr1 gene in peppers confers resistance to Cucumber mosaic virus isolate-P0 (CMV-P0). Cmr1 restricts the systemic spread of CMV strain-Fny (CMV-Fny), whereas this gene cannot block the spread of CMV isolate-P1 (CMV-P1) to the upper leaves, resulting in systemic infection. To identify the virulence determinant of CMV-P1, six reassortant viruses and six chimeric viruses derived from CMV-Fny and CMV-P1 cDNA clones were used. Our results demonstrate that the C-terminus of the helicase domain encoded by CMV-P1 RNA1 determines susceptibility to systemic infection, and that the helicase domain contains six different amino acid substitutions between CMV-Fny and CMV-P1(.) To identify the key amino acids of the helicase domain determining systemic infection with CMV-P1, we then constructed amino acid substitution mutants. Of the mutants tested, amino acid residues at positions 865, 896, 957, and 980 in the 1a protein sequence of CMV-P1 affected the systemic infection. Virus localization studies with GFP-tagged CMV clones and in situ localization of virus RNA revealed that these four amino acid residues together form the movement determinant for CMV-P1 movement from the epidermal cell layer to mesophyll cell layers. Quantitative real-time PCR revealed that CMV-P1 and a chimeric virus with four amino acid residues of CMV-P1 accumulated more genomic RNA in inoculated leaves than did CMV-Fny, indicating that those four amino acids are also involved in virus replication. These results demonstrate that the C-terminal region of the helicase domain is responsible for systemic infection by controlling virus replication and cell-to-cell movement. Whereas four amino acids are responsible for acquiring virulence in CMV-Fny, six amino acid (positions at 865, 896, 901, 957, 980 and 993) substitutions in CMV-P1 were required for complete loss of virulence in 'Bukang'.
Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Korea.
Won-Hee
WH
Kang
Jang-Kyun
JK
Seo
Bong Nam
BN
Chung
Kook-Hyung
KH
Kim
Byoung-Cheorl
BC
Kang
D060054:Agrobacterium / Q000821:virology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002212:Capsicum / Q000821:virology; D017799:Cucumovirus / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D060467:Disease Resistance / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D009154:Mutation; D010935:Plant Diseases / Q000821:virology; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry*; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D014777:Virus Diseases / Q000821:virology
D000596:Amino Acids; D018076:DNA, Complementary; D012367:RNA, Viral; D049452:Green Fluorescent Proteins; D020365:RNA Helicases