Abstract
Plant virus-based vectors are valuable tools for recombinant gene expression and functional genomics for both basic and applied research. In this study, Lettuce infectious yellows virus (LIYV) of the genus Crinivirus was engineered into a virus vector that is applicable for efficient protein expression and virus-induced gene silencing (VIGS) in plants. We examined gene replacement and “add a gene” strategies to develop LIYV-derived vectors for transient expression of the green fluorescent protein (GFP) reporter in Nicotiana benthamiana plants. The latter yielded higher GFP expression and was further examined by testing the effects of heterologous controller elements (CEs). A series of five vector constructs with progressively extended LIYV CP sgRNA CEs were tested, the longest CE gave the highest GFP expression but lower virus accumulation. The whitefly transmissibility of the optimized vector construct to other host plants, and the capability to accommodate and express a larger gene, a 1.8 kb β-glucuronidase (GUS) gene, were confirmed. Furthermore, the LIYV vector was also validated VIGS by silencing the endogenous gene, phytoene desaturase (PDS) in N. benthamiana plants, and the transgene GFP in N. benthamiana line 16c plants. Therefore, LIYV-derived vectors could provide a technical reference for developing vectors of other economically important criniviruses.
Highlights
Lettuce infectious yellows virus (LIYV) RNA1 is 8118 nt and encodes proteins associated with replication and alone is competent for replication: open reading frames (ORF) 1a and 1b code for the conserved domains of papain-like cysteine proteinase (PRO), methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp), P34 encoded by ORF2 is translated from its subgenomic RNA and is required for LIYV RNA2 replication [23,24]
The “add-a-gene” strategy, i.e., engineering of an autonomous expression cassette controlled by an additional subgenomic RNA (sgRNA) promoter or controller elements (CEs), is preferred in developing closterovirus-based vectors for stable and high-level expression [40]
Genes placed closer to the 30 terminus of the closterovirus genomic RNA tend to be expressed in greater amounts [36]
Summary
Plant virus-based vectors are important tools for gene expression and silencing in plants, and have been widely applied for both fundamental and applied research including tracking virus movement and distribution in plants [1,2,3], examining gene/protein functions by over-expression or silencing [4,5,6,7], triggering RNA interference (RNAi)-mediated plant protection against pathogens or insects [8,9,10,11,12], and producing commercial products such as enzymes, immunogens or antibodies [13,14,15,16,17]. The overall genomic organization is similar for all viruses in the genus, Lettuce infectious yellows virus (LIYV) is the type member [22]. RNA2 is 7193 nt and contains seven ORFs encoding proteins relevant to virion encapsidation, movement and vector
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