Abstract
Citrus tristeza virus (CTV), the largest non-segmented plant RNA virus, has several peculiar features, among which is the production of a 5′-terminal long non-coding RNA (lncRNA) termed low-molecular-weight tristeza 1 (LMT1). In this study, we found that p33, a unique viral protein that performs multiple functions in the virus infection cycle, specifically binds LMT1, both in vivo and in vitro. These results were obtained through the expression of p33 under the context of the wild type virus infection or along with a mutant CTV variant that does not produce LMT1 as well as via ectopic co-expression of p33 with LMT1 in Nicotiana benthamiana leaves followed by RNA immunoprecipitation and rapid amplification of cDNA ends assays. Further experiments in which a recombinant p33 protein and an in vitro transcribed full-length LMT1 RNA or its truncated fragments were subjected to an electrophoretic mobility shift assay demonstrated that p33 binds to at least two distinct regions within LMT1. To the best of our knowledge, this is the first report of a plant virus protein binding to a lncRNA produced by the same virus. The biological significance of the interaction between these two viral factors is discussed.
Highlights
Citrus tristeza virus (CTV), a member of the family Closteroviridae, is the largest nonsegmented plant RNA virus that is responsible for two of the most destructive viral diseases of citrus: quick decline and stem pitting [1,2,3,4,5,6,7,8]
The p33 protein tagged with the green fluorescent protein (GFP) was co-expressed with CTV in N. benthamiana plants through infiltration with an A. tumefaciens culture transformed with a binary vector carrying a fusion of the p33 open reading frames (ORFs) with that of GFP positioned under the Cauliflower mosaic virus 35S promoter along with a culture transformed with a vector carrying a cDNA
reverse transcription-PCR (RT-PCR) using post-RIP GFP-p33-bound RNA obtained via co-immunoprecipitation of extracts from GFP:p33- and CTV-red fluorescent protein (RFP)-bearing samples resulted in the amplification of only the fragment corresponding to the 50 -proximal region of the CTV genome (Figure 1B; “Elute”)
Summary
Citrus tristeza virus (CTV), a member of the family Closteroviridae, is the largest nonsegmented plant RNA virus that is responsible for two of the most destructive viral diseases of citrus: quick decline and stem pitting [1,2,3,4,5,6,7,8]. Apart from the genomic RNA (gRNA) and its negative-sense complimentary copy, CTV produces more than 30 sub-genomic RNAs (sgRNAs) and a number of different defective RNAs upon replication in the infected cells [9,10,11,12]. Among those are the two positive-sense 50 -coterminal non-coding sgRNAs termed low-molecular-weight tristeza 1. LMT1 (~750 nts) is generated through replication by initiation at the 30 end of the negative strand copy of gRNA and termination at the respective controller element [11,15]. We showed that LMT1 plays a role in counter-acting the host defense responses and is required for citrus infection [16]
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