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

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.

Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

A set of host proteins interacting with papaya ringspot virus NIa-Pro protein identified in a yeast two-hybrid system.

UNLABELLED: The protein-protein interactions between viral and host proteins play an essential role in plant virus infection and host defense. The potyviral nuclear inclusion protein a protease (NIa-Pro) is involved in various steps of viral infection. In this study, the host proteins interacting with papaya ringspot virus (PRSV) NIa-Pro were screened in a Carica papaya L. plant cDNA library using a Sos recruitment two-hybrid system (SRS). We confirmed that the full-length EIF3G, FBPA1, FK506BP, GTPBP, MSRB1, and MTL from papaya can interact specifically with PRSV NIa-Pro in yeast, respectively. These proteins fufill important functions in plant protein translation, biotic and abiotic stress, energy metabolism and signal transduction. In this paper, we discuss possible functions of interactions between these host proteins and NIa-Pro in PRSV infection and their role in host defense. KEYWORDS: Sos recruitment two-hybrid system; papaya ringspot virus; NIa-Pro; protein-protein interaction.