Papaya Leaf Distortion Mosaic Virus Mild Mutants And Evaluation Of Their Effects On Cross Protection

Papaya is one of the most important tropical economic fruit trees in Hainan,and Papaya ringspot virus?PRSV?has been the main limiting factor affecting the development of papaya industry.Since our research team newly discovered Papaya leaf distortion mosaic virus?PLDMV?on transgenic PRSV papaya plants in 2012,the PLDMV hazard has been increasing year by year,gradually increasing to threaten the planting of papaya in Hainan.Destructive diseases have become a problem that needs to be solved urgently in the production of papaya in China.At present,PLDMV is still difficult to prevent and control by means of chemical control in cultivation techniques.Although Taiwan Zhongxing University has developed anti-PLDMV and PRSV anti-transgenic papayas,there are issues such as ecological risks and food safety of transgenic plants,which limits the popularization and application of transgenic papayas.To explore a green prevention and control PLDMV method that is environmentally friendly,efficient and convenient,and has broad application prospects.This study adopted a cross-protection strategy to conduct research on the prevention and control of PLDMV.Cross-protection as an effective prevention and control method for plant virus diseases has been successfully applied to the prevention and treatment of citrus decay,zucchini mosaic disease,and papaya ring spot disease.At present,there is no report on cross protection of PLDMV.Using cross protection to prevent and control PLDMV is the key factor to obtain attenuated mutants with cross protection effect.In this study,site-directed mutation technique and Gibson splicing were used.Method was used to screen the attenuated virus mutants with potential application and to verify its cross protection.The main results as follows:1.Through analysis of the full-length and partial amino acid sequences of 69 potato virus Y viruses?strong and weak strains?,the pathogenicity-related amino acid mutation sites were screened from P1 and HC-Pro genes of the wild-type virulent strain p35S-PLDMV-GFP,which are:Q₂₂₆?S,A₄₁₃?Q,K₅₃₃?E,N₆₄₉?A,R₆₆₃?I,D₆₇₉?K,F₆₈₈?L,I₇₃₁G₇₃₂?DE,T₇₉₂?A and E₈₇₈?N.2.Using the site-directed mutagenesis technique and the method established in this study group to quickly construct agrovirus-infectious plant virus-infective clones,artificial point mutations rapidly constructed five species containing one mutation site(R₆₆₃),two mutation sites(R₆₆₃ and T)?792?),and four mutation sites(N₆₄₉,D₆₇₉,I₇₃₁,and G₇₃₂),Seven mutation sites(Q₂₃₂,A₄₁₃,K₅₃₃,R₆₆₃,F₆₈₈,T₇₉₂,and E?878?)and 11 mutation sites(Q₂₃₂,A₄₁₃,K₅₃₃,N₆₄₉,R₆₆₃,D₆₇₉,F₆₈₈,T₇₉₂,I₇₃₁,G₇₃₂,and E₈₇₈),in which Five p3 5 S-PLDMV-GFP full-length cDNA clone attenuated mutants were p35S-PLDMV-GFP-A,p35S-PLDMV-GFP-B,p35S-PLDMV-GFP-C,p35S-PLDMV-GFP-D and p35S-PLDMV-GFP-E.3.Five attenuated mutants were inoculated with Papaya plants by Agrobacterium inoculation method.The observation of the disease,RT-PCR,ELISA and fluorescence detection results showed that:After 60 days of inoculation,five attenuated mutants were able to infect Papaya systemically,and the infection efficiency was over 80%;There were no obvious symptoms on the papayas infected by the five attenuated mutants,but slight water-stained spots were observed on the stems,and green fluorescence with different intensities was observed on the leaves and water-stained sections;The virus accumulation of five attenuated mutants in papaya was significantly lower than that of wild-type virulent strains,but there was no significant difference in virus accumulation between the five attenuated mutant clones.It shows that five attenuated mutants can be used as attenuated strains for subsequent cross protection experiments.4.Five attenuated mutants were used to inoculate the papaya plants 30 days later and were challenged with the PLMDV virulent strain p35S-PLDMV-mCherry carrying the mCherry reporter gene.Preliminary cross-protection studies showed that:After 60 days of challenge,all five attenuated mutants showed cross-protection,while the attenuated mutants p35S-PLDMV-GFP-D and p35S-PLDMV-GFP-E were more attenuated than the other three attenuated mutants?p35S-PLDMV-GFP-A,p35S-PLDMV-GFP-B and p35S-PLDMV-GFP-C?showed better cross-protection effect.The attenuated mutants p35S-PLDMV-GFP-D and p35S-PLDMV-GFP-E may have complete cross-protection and can be used for field cross protection and control of PLDMV.