A Double Mutation in the Conserved Motifs of the Helper Component Protease of Papaya Leaf Distortion Mosaic Virus for the Generation of a Cross-Protective Attenuated Strain.

Potyviral helper component protease (HC-Pro), as a major determinant of symptom expression in susceptible plants, is a likely target candidate in the production of attenuated strains for cross-protection. In this study, single or double mutations of Lys (K) to Glu (E) in the Lys-Ile-Thr-Cys motif and Arg (R) to Ile (I) in the Phe-Arg-Asn-Lys motif of the HC-Pro from the severe papaya leaf distortion mosaic virus strain DF (PLDMV-DF) reduced symptom expression and virus accumulation in infected papaya (Carica papaya) plants. The papaya plants infected with the attenuated double mutant of PLDMV-EI presented as symptomless. PLDMV-EI provided effective protection against PLDMV-DF infection in three papaya cultivars and had no effect on plant growth and development. Our result showed that PLDMV-EI is a promising mild strain for the practical use of cross-protection in the field.

Profile of siRNAs derived from green fluorescent protein (GFP)-tagged Papaya leaf distortion mosaic virus in infected papaya plants.

We used green fluorescent protein (GFP)-tagged Papaya leaf distortion mosaic virus (PLDMV-GFP) to track PLDMV infection by fluorescence. The virus-derived small interfering RNAs (vsiRNAs) of PLDMV-GFP were characterized from papaya plants by next-generation sequencing. The foreign GFP gene inserted into the PLDMV genome was also processed as a viral gene into siRNAs by components involved in RNA silencing. The siRNAs derived from PLDMV-GFP accumulated preferentially as 21- and 22-nucleotide (nt) lengths, and most of the 5'-terminal ends were biased towards uridine (U) and adenosine (A). The single-nucleotide resolution map revealed that vsiRNAs were heterogeneously distributed throughout the PLDMV-GFP genome, and vsiRNAs derived from the sense strand were more abundant than those from the antisense strand. The hotspots were mainly distributed in the P1 and GFP coding region of the antisense strand. In addition, 979 papaya genes targeted by the most abundant 1000 PLDMV-GFP vsiRNAs were predicted and annotated using GO and KEGG classification. Results suggest that vsiRNAs play key roles in PLDMV-papaya interactions. These data on the characterization of PLDMV-GFP vsiRNAs will help to provide insight into the function of vsiRNAs and their host target regulation patterns.

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.

Gene expression profiling of papaya (Carica papaya L.) immune response induced by CTS-N after inoculating PLDMV.

Plant immune regulation is a defensive strategy of plants for protection against pathogen invasion, and Chitosan-N (CTS-N) can induce plant autoimmunity regulation mechanisms. CTS-N was found to induce an immunomodulatory response in papaya against Papaya leaf-distortion mosaic virus (PLDMV). To date, the gene expression profile of CTS-N-induced papaya immunomodulatory response has not been reported. Here, the transcriptional map of papaya leaf genes were subjected to three treatments, viz., non-viral inoculation without CTS-N treatment (CK), virus inoculation without CTS-N treatment (CG), and virus inoculation of 1 g/L treatment (B). These were studied by pot culture experiment. Comparison of the B group with the CG group revealed 732 upregulated and 510 downregulated genes. Comparison of the CG group with the CK group revealed 909 upregulated and 1024 downregulated genes. To determine gene function, gene ontology (GO) analysis was performed, where 480 biological process genes, 256 molecular function genes, and 343 cell composition genes were differentially expressed. Kyoto Encyclopedia of Genes and Genomes (KEGG) results revealed that the top three pathways were phenylpropane biosynthesis, starch and sucrose metabolism, and plant hormone signal transduction. Real-time Quantitative PCR (qPCR) results were consistent with the transcriptome results, with a correlation coefficient of 0.87. The results of the transcriptional group showed that genes associated with plant resistance were induced by CTS-N-treatment in papaya. The chitinase gene was related to the plant disease process. Related genes in plant hormone signal transduction pathways are associated with plant resistance, and six differentially expressed genes were correlated with enhanced immune resistance in papaya.

Generation of stable infectious clones of plant viruses by using Rhizobium radiobacter for both cloning and inoculation.

A novel Rhizobium radiobacter (synonym Agrobacterium tumefaciens)-mediated approach was developed to generate stable infectious clones of plant viruses. This method uses R. radiobacter for both cloning and inoculation of infectious clones, bypassing the requirement of cloning in E. coli to avoid the instability. Only three steps are included in this method: (i) construct viral genome-encoding plasmids in vitro by one-step Gibson assembly; (ii) transform the assembled DNA products into R. radiobacter; (iii) inoculate plants with the R. radiobacter clones containing the viral genome. Stable infectious clones were obtained from two potyviruses papaya ringspot virus (PRSV) and papaya leaf distortion mosaic virus (PLDMV) using this method, whereas attempts utilizing "classical" E. coli cloning system failed repeatedly. This method is simple and efficient, and is promising for a wide application in generation of infectious clones of plant virus, especially for those which are instable in E. coli.

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.

Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.

Detection of Papaya leaf distortion mosaic virus by reverse-transcription loop-mediated isothermal amplification.

Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32×10(-6) µg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV.