Two agroinfection-compatible fluorescent protein-tagged infectious cDNA clones of papaya leaf distortion mosaic virus facilitate the tracking of virus infection

Abstract

Papaya leaf distortion mosaic virus (PLDMV, the genus Potyvirus) is an emerging threat to papaya production. Here, agroinfection-compatible fluorescent protein-tagged PLDMV infectious cDNA clones driven by the Cauliflower mosaic virus 35S promoter were successfully constructed using one-step Gibson assembly. The clones were directly transformed into Agrobacterium tumefaciens to prevent potential problems such as plasmid instability during propagation in Escherichia coli. Ninety-five percent of papaya seedlings infected with PLDMV-GFP or PLDMV-mCherry developed systemic symptoms typical of those caused by wild-type PLDMV. Green and mCherry red fluorescence was observed in leaves, stems, and roots of infected papaya plants. The fluorescent protein-tagged agroinfectious PLDMV cDNA clones were stable in papaya for more than 90 days and during six serial passages at 30-day intervals. The availability of these infectious clones will contribute to research on PLDMV-host interactions and can be applied in the papaya breeding program for PLDMV resistance.

An efficient papaya leaf distortion mosaic potyvirus vector for virus-induced gene silencing in papaya

“…Potyvirus-based vectors usually employ fusion strategies. Specifically, target fragments may be fused into the ORF of a large polyprotein at the N-terminus of P1 , between the P1 and HC-Pro cistrons or between the NIb and CP cistrons 27 , 37 , 38 . In this study, we inserted the NC frame between the NIb and CP cistrons of PLDMV-E to facilitate rapid cloning of the desired gene fragments for silencing.…”

“…In this study, we inserted the NC frame between the NIb and CP cistrons of PLDMV-E to facilitate rapid cloning of the desired gene fragments for silencing. To enable infection by PLDMV-NC after a target fragment has been cloned into the NC frame, the inserted fragment should be translated as part of the potyviral polyprotein during infection, and the resultant expressed protein insertion should be released from the viral polyprotein precursor by original and additional duplication of NIa-Pro proteolytic sites 25 , 38 . Therefore, the base pairs of the target sequence cloned into an NC frame should be multiples of three and cannot contain any stop codons.…”

“…NC was applied to the pPLDMV-NC constructs to obtain the inserts of interest. The GFP fragment (nts 1428–1775 of GFP ; GenBank accession: MK896905) was amplified from the pPLDMV-GFP plasmid 38 and cloned into pPLDMV-NC to generate pPLDMV-ΔGFP as a nontarget control for VIGS.…”

Development of cassava common mosaic virus-based vector for protein expression and gene editing in cassava

“…The SGP4 sequence in pCsCMV2-NC included 53 nts downstream of the CP start codon. For the VOX plasmids, the GFP coding sequence (GenBank accession: MK896905) was PCR-amplified from the pPLDMV-GFP plasmid [ 63 ] and inserted into the pCsCMV-NC, pCsCMV1-NC, and pCsCMV2-NC vectors through NC to generate pCsCMV-GFP, pCsCMV1-GFP, and pCsCMV2-GFP, respectively. In addition, the P. ananatis carotenoid biosynthesis gene crtB (GenBank accession: D90087) was synthesized (Sangon Biotech) and subsequently inserted into pCsCMV2-NC to construct pCsCMV2-crtB.…”