Development of a GFP expression vector for Cucurbit chlorotic yellows virus
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Development of a GFP expression vector for Cucurbit chlorotic yellows virus Ying Wei1, Xiaoyu Han1, Zhenyue Wang1, Qinsheng Gu2, Honglian Li1, Linlin Chen1, Bingjian Sun1 and Yan Shi1* Abstract 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, pCCYVGFPSGC, pCCYVGFPCGC, and pCCYVGFPCGS, 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. pCCYVGFPCGC-infected cucumber leaves exhibited cell spread at 25 dpi, whereas pCCYVGFPSGC and pCCYVGFPCGS were mainly found in single cells. Further observation of pCCYVGFPCGC 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. Keywords: CCYV, Controller elements, GFP expression Cucurbit chlorotic yellows virus (CCYV), a recently discovered cucurbit-infecting crinivirus in the family Closteroviridae [1–7], is among the largest single-strand positive-sense RNA viruses [8]. The bipartite RNA genome comprises a 8607 nucleotide [nt] RNA1 and a 8041-nt RNA2. Recent studies on CCYV [9–12] have been hampered due to lack of reverse genetic tools. The construction of full-length infectious cDNA clones will facilitate the investigation of viral determinants of virus replication and movement, as well as the interactions between viral proteins and host factors. Our previous study developed two sets of full-length CCYV cDNA clones under the control of the T7 RNA polymerase promoter and 35S promoter [13]. Virus-based vectors are useful tools for the study of plant molecular biology. A number of plant virus vectors have been developed to express heterologous genes of interest in plants [14–18]. Only LIYV has been reported to construct a green fluorescence protein (GFP) vector for crinivirus using the ORF fusion strategy. Part of * Correspondence: [email protected] 1 College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, China Full list of author information is available at the end of the article
LIYV RNA1 encoded P34 protein was replaced by a GFP gene and further observation of Nicotiana benthamiana (Nb) leaves agro-infiltrated by the resultant clone displayed occasional single cells green fluorescence. Limited GFP expression has been observed in Nicotiana benthamiana plants, but in no other hosts. Cucumber is an economically important cultivated plant, and the available GFP expression vector of cucurbit viruses is limited [19]
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