Cross Protection Against Papaya Ringspot Virus(PRSV) By Attenuated PRSV

Cross protection of attenuated mutants is an important means of controlling plant virus diseases. A nonpathogenic mild strain is a major limiting factor for controlling plant viruses by cross protection. Alignment of the full or part amino acid sequences of mild and severe strains of potyviruses, the sequences analyses of the gene P1and HC-Pro revealed that eight point mutations of parental isolate PRSV-LM at I309, K481,K598, H728,F753, L-754, N787 and D944 were potentially associated with the attenuated symptom. Four full-length cDNA infectious clone mutants of PRSV-LM were successfully constructed by site-directed mutagenesis and Gibson Assembly cloning method which separately contained two mutations (at L754 and N787), four mutations ( at I309, K481,F753 and D944),six mutations (at I309, K481,F753, L754, N787 and D944),and eight mutations (at I309, K481,K598,R728,F753,L754,N787,and D944),respectively named as pGprsvm2,pGprsvm4,pGprsvm6 and pGprsvm8. Meanwhile, another servere strain named as pPRSV-LM-GFP/CP is consructed with the gfp gene inserted to genome of pPRSV-LM betwween nib and cp gene.System infectivity developed on non-transgenic papaya after inoculation with that five clones were monitored by reverse transcription-polymerase chain reaction(RT-PCR)and visual observation. Symptom severity on papaya of the pGprsvm2 was similar to the parental isolate PRSV-LM and pPRSV-LM-GFP/CP,which had bright fluorescent green in fluorescence microscopy. With the exception of pGprsvm2, the symptoms induced by three other mutants (pGprsvm4, pGprsvm6 and pGprsvm8) compared with the parental PRSV-LM showed milder symptom severity. By contrast, pGprsvm8 induced mildest symptom. On the other way, the anlysis of the association between the symptoms developed on the non-transgenic papaya agroinoculated with four kinds of attenuated mutants and the eight point mutations located at the gene P1and HC-Pro, revealed that two amino acids change (at K598→E and R728→I) were critical for the attenuated symptom.Cross protection effectiveness were evaluated by reinoculated with severe strains pPRSV-LM as challenge virus at different time intervals of 0 day, 20 days, 30 days and 40 days after protective inoculation with attenuated mutants of pGprsvm4, pGprsvm6 and pGprsvm8. The time of challenge inoculation affected cross protection effectiveness.If the challenge inoculation was made at 0 day after the initial inoculation, no cross protection were observed.Cross protection was more effective against severe strains pPRSV-LM when the time interval increase to 20 days or more, which compared with the parental PRSV-LM showed more milder symptom severity. pGprsvm6 and pGprsvm8 as the protectant virus compared with attenuated mutants pGprsvm4 shew more effectiveness of cross protection,developed milder symptom severity than that of pGprsvm4 as protectant virus and challenge inoculation with pPRSV-LM .What’s more,pGprsvm6 and pGprsvm8 as protectant virus and reinoculated with severe strains pPRSV-LM-GFP/CP at different time intervals of 10 day, 20 days, 30 days and 40 days. In the experimental group which mild strain pGprsvm6 as the protectant virus and reinoculated with severe strains pPRSV-LM-GFP/CP at different time intervals,incomplete cross protection were observed as the symptoms of mosaic and chlorosis developed, which means mild strain pGprsvm6 protecting papaya from being infected with challenge virus pPRSV-LM-GFP/CP after initial inoculation.Complete or high degree of cross protection was observed when the attenuated mutants pGprsvm8 as the protectant virus and reinoculated with challenge virus pPRSV-LM-GFP/CP at different time intervals without conspicuous symptoms develpoed. Fluorescent green in fluorescence microscopy and under UV light were not observed yet. On the other hand, RT-PCR anlysis of papaya chanllenged with pPRSV-LM-GFP/CP after the initial inoculation with attenuated mutants and sequencing anlysis revealed the existence of protectant virus, but not challenge virus, which mean challenge virus pPRSV-LM-GFP/CP could not infect papaya after initial inoculation.