Infectious cDNA clones of the crinivirus Tomato chlorosis virus are competent for systemic plant infection and whitefly-transmission

Abstract

Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) causes important emergent diseases in tomato and other solanaceous crops. ToCV is not transmitted mechanically and is naturally transmitted by whiteflies. The ToCV genome consists of two molecules of linear, positive-sense RNA encapsidated into long flexuous virions. We present the construction of full-length cDNA clones of the ToCV genome (RNA1 and RNA2) fused to the SP6 RNA polymerase promoter and under the control of the CaMV 35S promoter. RNA1 replicated in the absence of RNA2 in Nicotiana benthamiana and tomato protoplasts after inoculation with cDNA-derived in vitro transcripts. Agroinfiltration of RNA1 and RNA2 under the 35S promoter resulted in systemic infection in N. benthamiana plants. In addition, tomato plants were infected by grafting with agroinfected N. benthamiana scions, showing the typical ToCV symptoms. The viral progeny generated in tomato was transmissible by the whitefly Bemisia tabaci.

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

“…Furthermore, Northern blot analyses were conducted using DIG-labelled specific RNA probes for RNA1 (566 nt within the P22 gene) and RNA2 (698 bp within the CP gene), which were synthesized using DIG RNA labelling mix (Roche) with specific primers P26/P27 and P28/P29 (Table S1). Consistent with LIYV (Klaassen et al, 1996;Wang et al, 2009), LCV (Mongkolsiriwattana et al, 2011) and ToCV (Orílio et al, 2014), CCYV RNA1 showed strong replication at 1-day post-infection (dpi) (Fig. 2c).…”

“…Unlike previously reported crinivirus infectious clones of LIYV, LCV and ToCV, which are fully active in the host plants after grafting or virion transmission by whiteflies from agroinfected N. benthamiana plants (Wang et al, 2009;Chen et al, 2012;Orílio et al, 2014), our CCYV infectious clone successfully established direct infection in the host plant C. sativus, as well as N. benthamiana , indicating a potentially wide range of host applications. To our knowledge, our infectious clone of CCYV represents the first report of successful infection of a crinivirus in the natural host plant and in N. benthamiana.…”

P22 of tomato chlorosis virus, an RNA silencing suppressor, is naturally expressed in the infected plant

“…The p22 protein encoded in the RNA1, as well as the major (CP) and minor (CPm) coat proteins encoded in the RNA2 of ToCV are implicated as RNA silencing suppressors (RSS) (14). Until now, the CP is the only viral protein being detected in ToCV-infected tomato plants (15). In vitro assays demonstrated that the p22 protein is bound preferentially to the long dsRNAs, thus protecting these dsRNAs from dicer cleavage (16).…”

“…The full-length cDNAs of ToCV RNA1 and 2 were cloned under a double 35S promoter in the binary vector pCass-RZ (18) to produce the two constructs pCaToCR1 and pCa-ToCR2 for ToCV RNA1 and RNA2, respectively. Biological activity of the constructs pCa-ToCR1 and pCa-ToCR2 was demonstrated through agroinfection of N. benthamiana plants according to reported protocols (15).…”

Two proteins of Cucurbit chlorotic yellows virus, P59 and P9, are self-interacting

“…Data on the function of crinivirus P59 and P9 are limited due to the lack of an efficient reverse genetics system. To date, several full-length infectious clones of criniviruses have been constructed and will promote the study of crinivirus genotypes as well as the interactions of P59 and P9 in planta [14][15][16].…”