Development of a GFP expression vector for Cucurbit chlorotic yellows virus

BACKGROUND: Cucurbit chlorotic yellows virus (CCYV), a bipartite crinivirus, causes chlorotic leaf spots and yellowing symptoms on cucurbit leaves. We previously developed an infectious clone of CCYV. Limited work has been conducted on the construction of a crinivirus green fluorescence protein (GFP) expression vector to date. FINDING: We constructed a CCYV GFP expression vector using the “add a gene” strategy based on CCYV RNA2 cDNA constrcut. Three resultant clones, pCCYVGFP(SGC), pCCYVGFP(CGC), and pCCYVGFP(CGS,) were constructed with different promoters used to initiate GFP and CP expression. At 25 dpi GFP fluorescence was detectable not only in leaf veins but also in the surrounding cells. pCCYVGFP(CGC)-infected cucumber leaves exhibited cell spread at 25 dpi, whereas pCCYVGFP(SGC) and pCCYVGFP(CGS) were mainly found in single cells. Further observation of pCCYVGFP(CGC) GFP expression at 30 dpi, 40 dpi, and 50 dpi showed phloem-limited localization in the systemic leaves. CONCLUSIONS: We developed of a CCYV GFP expression vector that will be useful for further study of CCYV movement in cucurbits.