Identification, Generation Of Infectious Full-length CDNA Clones Of Papaya Leaf Distortion Mosaic Virus From Hainan (PLDMV-DF)and Its Use For GFP Expression

Papaya (Carica papaya L.) is one of the tropical fruits with economic importance in Hainan of China. Papaya leaf distortion mosaic virus (PLDMV), recently occurred in a papaya plantations from PRSV-resistant transgenic papaya in Dongfang, Hainnan besides Papaya ringspot virus (PRSV) and Papaya mosaic virus (PaMV). These three viral disease produce similar symptoms in papaya, such as mosaic, yellow-green leaf discoloration and distortion on leaves, water-soaking streaks on petioles, and ring-spots on fruits, making it difficult to distinguish among PLDMV, PRSV, and PapMV without further testing in the field. This study developed a sensitive and rapid method to detection of these three viruses by RT-LAMP (reverse transcription loop mediated isothermal amplification) and multiplex RT-PCR. Presently, the pathological and molecular characters associated with PLDMV are still unclear because of rare studies on it, and construction of full-length infectious cDNA clones is critical for the functional analysis of RNA viruses. Therefore, this thesis was determined the full-length genomic sequence of PLDMV Dongfang isolate (PLDMV-DF), and was successfully constructed full-length cDNA infectious of PLDMV-DF and viral expression vector for GFP. It will play an important role in further study the viral determinants involved in virus replication, local and systemic movement and symptom development, and interactions between viral and host factors during virus infection in plants.The main results were as follows:(1) PLDMV-DF identification and host range studiesThis is the first reported PLDMV-DF isolates in Hainan, China, and identified its host range, PLDMV-DF isolate can infect both transgenic papaya infection non-transgenic papaya, but can not infect Cucurbita pepo and Nicotiana benthamiana.(2) The full length genomic sequence of PLDMV-DF isolate was determinedThe full length genomic sequence of PLDMV-DF isolate was determined (total10153nucleotieds (nt), not including the poly(A) tail) and published in GenBank (Accession number:JX974555), flanked with5’-and3’-untranslated regions (UTR) of134and209-nt. It is containing a long open reading frame (ORF) encoding3269amino acids and a small ORF (pipo, Pretty Interesting Potyviridae ORF) encoding75amino acids. Based on the results of alignment, the full-length nucleotide sequences and amino acid squences similarity were94.3-94.9%and95.9-96.3%, respectively, betaween DF and J56P, KS, CZ isolate. To further understand the relationships between PLDMV-DF and PLDMV isolates from Japan and Taiwan, phylogenetic trees were constructed using a multiple alignment of the complete nucleotide sequences of PLDMV isolates and the amino acid sequences of their polyproteins. Notably, the PLDMV-DF isolate shares the most recent common ancestor of the Japan-J56p isolate rather than the Taiwan isolates, though the geographic distance between hainan and Taiwan is shorter than that between hainan and Japan.(3) Development and validation of a RT-LAMP and multiplex RT-PCR for detection of three papaya virusesIn this study, a specific, sensitive, and rapid RT-LAMP assay was developed for detection of PLDMV, PRSV and PaMV, and its potential application for the diagnosis of PLDMV in the field was evaluated. Meanwhile, a multiplex RT-PCR assay was established to detect simultaneously these three viruses. The multiplex RT-PCR was applied successfully for detection of three viruses from341field samples collected from18counties of Hainan Island, China. Rates of single infections were186/341(54.5%),93/341(27.3%), and3/341(0.9%), for PRSV, PLDMV, and PapMV, respectively;59/341(17.3%) of the samples were co-infected with PRSV and PLDMV, which is the first time being reported in Hainan Island.(4) Construction of a full-length cDNA infectious clone of PLDMV-DF transcribed in vitro through in-fusion methodIn order to stabilize full length clone of PLDMV-DF in E.coli cells, we introducing two introns in the P3and/or Cl-encoding regions to obtain two infectious clones of pT7-FL-In2(intron2is inserted into the P3(3709/3710nt) region) and pT7-FL-In2/IV2(intron2and intron IV2are inserted into the P3(3709/3710nt) and CI (5028/5029nt), respectively). In three separate experiments, the capped in vitro transcript derived from pT7-FL-In2and pT7-FL-In2/FV2was shown to be infectious in the systemic host papaya by mechanical inoculation, while higer infection efficiency (73.7%-75.0%) was obtained than uncapped transcript (58.8%-61.1%).(5) Development of an effective and fast Agrobacterium-mediated inoculation system for the cloned cDNA constructs of PLDMV-DF by yeast homologous recombination systemIn this study, we improved the yeast homologous recombination system, and successfully constructed two types of PLDMV-DF infectious clone p35S-FL and p35S-FL-In2(intron2is inserted into the P3(3709/3710nt)) by agroinoculation, and obtained four agrobacterium clones of p35S-FL39-12, p35S-FL39-34, p35S-FL-In22-1, and p35S-FL-In22-3. These clones agro-inoculated with silencing suppressor vector of pBI121-HC-Pro has more infection efficiency (85.7%-91.4%) than agroinoculated independently (64.7%-69.4%), and symptoms development in advanced2-3days. (6) Development of GFP-tagged cDNA clones of PLDMV-DF and stable systemic expression of GFP in papaya through a modified yeast homologous recombination systemsUtilizing the optimized yeast recombinanation method, we have successfully constructed the viral expression vector of PLDMV-DF (p35S-FL-P1/GFP-8, p35S-FL-P1/GFP-9, p35S-FL-P1/GFP-In2-5, p35S-FL-GFP/CP-23-1, p35S-FL-GFP/CP-38-1and p35S-FL-GFP/CP-38-2), by insertion of the marker gfp gene between the coding regions for P1and HC-Pro and between NIb and CP. The clone of p35S-FL-P1/GFP-9, p35S-FL-GFP/CP-38-1and p35S-FL-GFP/CP-38-2, were highly infectious efficiency of73.5-93.9%, and strong GFP expression singal were examined under a confocal fluorescence microscopy and under a hand-held UV light, and the result of western blot is positive. However, the clone of p35S-FL-P1/GFP-9could be used to an attenuated strains for cross-protection against severe PLDMV infection, because of a single-site mutations in the P1coding region. p35S-FL-GFP/CP-23-1mutations affect GFP expression, but does not affect its infectivity (66.7%), result to symptom development delayed30-40days, and gfp gene could be lost from insertion site at90days after inoculation.(7) Determination of the silencing efficiency of three ihpRNAs in papaya protoplasts that infected with GFP-tagged cDNA clones of PLDMV-DFWe used four constucts for silencing of GFP-tagged cDNA clones of PLDMV: namely, pRNAi-CP879, pRNAi-CP400, pRNAi-CP326, and pRNAi-CPi4o, which target the CP region. Four versions of ihpRNA expression vectors has a marked drop in GFP expression compared to the control. The silencing efficiency of the pRNAi-CP879, pRNAi-CP400, and pRNAi-CP326constructs was similar to but higher than that for pRNAi-CP140with a shorter fragment of CP gene. The above results indicated the length of the target fragment might affect the silencing efficiency.