Effects Of Citrus Triszeta Virus On Storage Performance Of Citrus Fruit And Its Silencing Suppression Mechanism

Citrus is the most important fresh fruit in southern China.Quality maintenance and safety control in postharvest stage are indispensable part in the citrus production.In the past half century,the use of chemical sterilization to treat fruits during production has achieved obvious antiseptic preservation effect,but resulted in drug resistance among pathogens and posed the potential threat human health and the ecological environment due to pesticide residues on fruit,which have received a lot of attention recently.Thus,the emerging green preservation technologies,aimed for reducing the amount of chemicals,have been paid much attention and are replacing the traditional post-harvest preservation methods in industry.The fruits are independent living individuals after harvest when material and signal exchange are cut off from the tree.In view of this,the internal immune system resists infection of exogenous pathogen during postharvest storage and transportation.Citrus tristeza virus(CTV)is virus species widely identified in citrus plants,and pathogenicity of CTV in different strains showed significant differences.While a severe CTV strain can cause rapid senescence or death of tree,the mid strain does not generally produce evident symptoms on the host plants,which indicates a moderate effect from CTV mild strains in the immunity response.In theory,enhancing the intrinsic immunity of fruits can benefit postharvest green production,but little is known about the intrinsic immune regulation of citrus fruits in postharvest stage.In this study,the effects of CTV on the postharvest storage performance of citrus fruits were evaluated by using two materials,one with while the other without CTV,but both with the similar genetic background and grown in similar field environment.Further,the molecular regulation mechanism of VSR,the key inhibitors of host immunity from CTV mild strain N4,was analyzed.The results are as follows:1.Presence of CTV in citrus fruit induced immunity in fruits but affected their quality and postharvest storage performance.The infection by CTV enhanced the expression of resistance related genes like RDR1,Pectatelyase5,Gluanase,PR-3 and NBS-LRR in citrus postharvest fruits,increase their immune resistance to penicillium digitatum,and thus reduced the rot rate.However,the CTV infection led to a lighter pigmentation of the fruit peel and a significantly increased weight loss rate.Moreover,the infection of CTV lead to decreased total soluble solids(TSS),but a significantly higher content of titratable acid(TA)in citrus fruits;More importantly,the contents ofγ-Aminobutyric acid(GABA),asparagine and glutamic acid were totally higher in citrus fruits peel with CTV during the whole stage.The above results indicated that CTV infection in citrus fruits might improve the immunity of citrus fruits by enhancing the expression of the resistance-related genes,but resulted in metabolic changes of sugars,acids,and amino acids in the fruit,as well as affected the postharvest storage performance of citrus fruits.2.Silencing suppressor(VSR)p20,p23 encoded by CTV mild strain N4 can inhibit the production of s RNA in RNAi pathway,and CTV-N4-CP can inhibit systemic silencing in graft scion induced by silencing signal.CTV-N4 p20 and p23 can inhibit the gene silencing of Green fluorescent protein(GFP)during transient expression(S-PTGS),as well as GFP silencing induced by double-stranded RNA(ds RNA)and micro RNA(mi RNA).Furthermore,p20 and p23 can inhibit the accumulation of GFP s RNA during transient GFP expression and secondary si RNA when GFP silencing was induced by ds RNA,but they were not be able to inhibit the accumulation of mi RNA and primary si RNA during mi RNA and ds RNA induced GFP silencing.In addition,CP of CTV-N4 can inhibit the systemic silencing in scion induced by silencing signal,but it cannot inhibit signal transportation.The above results indicate that three VSRs of CTV-N4 can inhibit RNA silencing with different mechanisms,which provided new insights on how CTV manipulate citrus immune system.3.The silencing inhibition function and protein stability of p20 are affected by putative host factors and infection of pathogens,while that of p23 are affected by different conserved amino acids of its own protein sequence.When p20 was cloned into an overexpression vector producing N-terminal tagged protein,it could not be detected and had no silencing inhibitory function;but when cloned into overexpressing vector producing C-terminal tagged protein,it could be detected and its silencing inhibition function was strong.However,when the N-terminal tagged p20 were expressed from the Tobacoo Rattle Virus(TRV)vector,the p20 protein could be detected,the size of which is larger than that of the C-terminal tagged p20 protein.Our data indicated that the N-terminal of p20 is cleaved by an unkown enzyme in hosts during transient expression,and this enzyme may be inhibited by virus infection.By mutating the conserved amino acids in p23,we found that protein stability and VSR function of p23 protein were greatly affected;and by co-expressing with heterologous VSR p19,we found that 2 conserved amino acids only affect its VSR activity but not protein stability.The above studies provided bases for further investigation of interaction between CTV mild strain and Citrus,as well as potential application of CTV mild strain in citrus fruit postharvest processing.