The Role Of D SatelliteRNA Derived Small RNA In Host Gene Silencing And Disease Development

Cucumber mosaic virus(CMV)associated with D satellite RNA(D-sat RNA)causes systemic necrosis in tomato.Previous reports have shown that three nucleotides near its 3’end determine its necrogenecity in tomato.They are the nucleotides at positions 285,290 and 292.When they are simultaneously mutated,the mutant sat RNA,Dm-sat RNA does not cause the plant death.The unpublished data from our laboratory has shown that RDR6 and SGS3 are involved in the biogeneration of D-sat RNA-Srna,and its accumulation is closely correlated with the lethal infection of D-sat RNA in tomato.Other studies have found a s RNA derived from Y-sat RNA can cause the tobacco yellow leaf by interfering the expression of magnesium protoporphyrin chelatase subunit I(CHl I).Therefore,this thesis explores the possible role of D-sat RNA-s RNA in silencing host gene expression and causing the catastrophic lethal disease.At first we constructed s RNA and m RNA degradation libraries respectively using HD-adapters for high throughput sequencing,and the apical young stem from tomato plants infected with CMV,CMV/D-sat RNA,CMV/Dm-sat RNA was harvested as well as the un-infected Mock sample for RNA exraction.Sequence analysis of these libraries resulted all the reads of D-sat RNA-Srna,including the s RNA containing the above three necrogenic nucleotides(D-lethal-vs RNA).Combining all D-lethal-vs RNA with the corresponding m RNA degradation libraries,we used Paresnip2 software to predict the possible target m RNAs.The analysis resulted in five tomato m RNAs including SCC1P2,ERF4,CSF,EIN2 and SCMF that might be cleaved through D-lethal-vs RNA targeting.To verify it,we fused the targeting sequence of these putative targets with e GFP respectively,which was transiently expressed together with corresponding D-lethal-vs RNA in Nicotiana benthamiana leaves.In the meantime,the recombinant e GFP constructs were expressed in N.benthamiana leaves infected with CMV/D-sat RNA or CMV/Dm-sat RNA.The results indicate that SCC1P2 can be silenced by D-sat RNA-s RNA and its targeting sequences were tomato specific.To explore the role of D-sat RNA-induced host gene silencing in its pathogenecity,we used a VIGS vector,Tobacco rattle virus(TRV)vector and an overexpression vector Potato Virus X(PVX)to express plus and minus strand of D-sat RNA in tomato,but the infected plants did not develop lethal necrosis.In conclusion,D-lethal-vs RNA could mediate the cleavage of SCC1P2 m RNA,but if these molecular events are associated with the occurrence of tomato death remains to be further clarified.