Efficient Protein Expression and Virus-Induced Gene Silencing in Plants Using a Crinivirus-Derived Vector

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.

Two Crinivirus-Conserved Small Proteins, P5 and P9, Are Indispensable for Efficient Lettuce infectious yellows virus Infectivity in Plants

“…The GFP ORF was then ligated into their N- or C-termini as an in-frame coding sequence. Constructs including the knockout mutants (P5X, P9X) and the deletion mutants (P5Δ, P9Δ) were generated from the LIYV RNA2 infectious clone [ 27 ], the P5X-GFP and P9X-GFP constructs were modified from the GFP-tagged LIYV vector [ 28 ], and the P5 truncation mutants were derived from pEAQHT-P5GFP (P5 O Δ:GFP, P5 TMS Δ:GFP, P5 IN Δ:GFP) or from the LIYV RNA2 infectious clone (P5 O Δ, P5 TMS Δ, P5 IN Δ). The mutagenesis was introduced into the existing constructs through In-Fusion Cloning using primers to incorporate the mutations outside the unwanted region and then re-circularize into desired clones.…”

“…Four- to six-leaf stage Hc-Pro transgenic N. benthamiana plants and Agrobacterium tumefaciens strain GV3101 were used for transient expression analysis and virus inoculation as described before [ 27 , 28 ]. The subcellular localization of the GFP fusion proteins within agroinfiltrated N. benthamiana epidermal cells was examined using a Leica TCS SP2 inverted confocal microscope (Leica Microsystems, Wetzlar, Germany) under a 63× water immersion objective, with excitation/emission filter wavelengths: 488/497 to 520 nm for GFP and 561/585 to 615 nm for RFP.…”

“…To test the effects of the LIYV mutants on lettuce plants ( Lactuca sativa L.), virions were purified from agroinfected N. benthamiana plants, acquired by whiteflies through membrane-feeding, and transmitted to two- to three-leaf stage young lettuce plants as previously described [ 28 , 30 ].…”

“…RT-PCR and RT-qPCR were performed as described before [ 28 ]. The P5 and P9 mutations were checked by RT-PCR using primer sets LIYV-P5_F (5′-ATAGCAGTAGCTGTCGCAAACC-3′)/LIYV-P5_R (5′-ACTTTTTATTGATAAAATACTTATTACAAATT-3′) and LIYV-P9_F (5′-CATCACGGAAGATAGGAGTGAG-3′)/LIYV-P9_R (5′-TGTCAGCTTCATGATCCCTG-3′) which amplify sequences flanking the P5 and P9 genes, respectively.…”

“…The P5 and P9 mutations were checked by RT-PCR using primer sets LIYV-P5_F (5′-ATAGCAGTAGCTGTCGCAAACC-3′)/LIYV-P5_R (5′-ACTTTTTATTGATAAAATACTTATTACAAATT-3′) and LIYV-P9_F (5′-CATCACGGAAGATAGGAGTGAG-3′)/LIYV-P9_R (5′-TGTCAGCTTCATGATCCCTG-3′) which amplify sequences flanking the P5 and P9 genes, respectively. The relative LIYV RNA accumulation level in N. benthamiana plants was determined by RT-qPCR as mentioned by Qiao, et al [ 28 ], while for tests with lettuce plants, the lettuce TIP41-like protein (TIP41) transcripts quantified with TIP41_F: 5′-GAGAGATTTGCTGGAGGGAAACTA-3′, TIP41_R: 5′-CCTTTGACTGATGATGTTTGGA-3′ and TIP41_Probe: 5′-TCCGCATTCATGGCTGGGAGAT-3′ were used as an internal control. To quantify the transcript levels of the stress-related N. benthamiana genes, qPCR was performed employing a SYBR Green supermix with gene specific primers, and the amplified 18S rRNA was used as an internal control.…”