Infectious clones of the crinivirus cucurbit chlorotic yellows virus are competent for plant systemic infection and vector transmission

Abstract

Cucurbit chlorotic yellows virus (CCYV), a recently identified bipartite crinivirus, causes economic losses in cucurbit plants. CCYV is naturally transmitted only by whitefly Bemisia tabaci. Here we constructed full-length cDNA clones of CCYV (RNA1 and RNA2) fused to the T7 RNA polymerase promoter and the cauliflower mosaic virus 35S promoter. CCYV replicated and accumulated efficiently in Cucumis sativus protoplasts transfected with in vitro transcripts. Without RNA2, RNA1 replicated efficiently in C. sativus protoplasts. Agroinoculation with the infectious cDNA clones of CCYV resulted in systemic infection in the host plants of C. sativus and Nicotiana benthamiana. Virus derived from the infectious clones could be transmitted between cucumber plants by vector whiteflies. This system will greatly enhance the reverse genetic studies of CCYV gene functions.

Generation of stable infectious clones of plant viruses by using Rhizobium radiobacter for both cloning and inoculation

“…Infectious RNA can be produced via in vitro or in vivo transcription. Performing in vitro transcription is inconvenient and expensive, so more and more infectious clones of plant viruses have been generated using R. radiobacter -mediated transfection (Ambros et al, 2011;Park et al, 2017;Shi et al, 2016;Wang et al, 2015;Wieczorek et al, 2015;Zheng et al, 2015). We developed the method for generation of infectious clones of plant viruses by using R. radiobacter for cloning and inoculation, which is convenient and not expensive.…”

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

“…Use of binary vector backbones with reduced copy number origins lessens foreign DNA loads and potential bacterial toxicity of the viral sequences. Binary vectors with single-, low-or medium-copy origins are available (Hamilton et al, 1996;Pasin et al, 2017;Xiang et al, 1999) and have been used to generate infectious clones of members of families with large genomes, such as Potyviridae, Rhabdoviridae and Closteroviridae (Ambr os et al, Lellis et al, 2002;Pasin et al, 2017Pasin et al, , 2018Prokhnevsky et al, 2002;Shi et al, 2016;Wang et al, 2015). Choice of binary vector origin was also shown to affect Agrobacterium transformation efficiency in both stable transformation and transient expression assays (Pasin et al, 2017;Zhi et al, 2015).…”

A Semipersistent Plant Virus Differentially Manipulates Feeding Behaviors of Different Sexes and Biotypes of Its Whitefly Vector

“…To obtain CCYV-infected plant cultures, cucumber plants at 1–2 true-leaf stage were inoculated with Agrobacterium tumefaciens -mediated CCYV clones [ 41 ]. Plants were then left to grow for 30 days, and infection status was determined by symptom of chlorotic leaf spots and yellowing, and subsequently confirmed by real-time RT-PCR.…”