Effect Of Ubiquitin Conjugating Enzyme (E2) On Accumulation Of Rice Stripe Virus (RSV) In Laodelphax Striatellus And Its Mechanism

As an important pest in rice production in Asia,Laodelphax siriatellus(small brown planthopper,SBPH)can transmit Rice stripe virus and cause great field loss.RSV invasion is accompanied by the interaction of several components with the virus in SBPH.These components may resist or promote virus infection,replication and transmission.In previous studies,33 differential proteins were identified in the salivary glands of infected and non-infected SBPH including ubiquitin-related proteins,such as ubiquitin-60S ribosomal protein L40 precursor.Ubiquitin(Ub)is activated by ubiquitin activating enzyme(E1)and then connected to ubiquitin conjugating enzyme(E2)by sulfur ester bond.Subsequently,Ub is labeled to the target protein under the catalysis of ubiquitin ligase enzyme(E3),and the labeled target protein is finally recognized and degraded by 26s proteasome.This ubiquitination process regulates the degradation of over 80%of proteins in eukaryotic cells,which is involved in almost all life activities,and also plays an important role in host-virus relationships.This paper mainly studied the effect of E2,the ubiquitin conjugating enzyme,on the accumulation of RSV in SBPH and explored its mechanism1.Gene cloning and expression pattern of E2 in SBPHAccording to the transcriptome sequences,four different E2 sequences in SBPH were obtained by RACE-PCR,with lengths of 1089bp,1209bp,854bp,1303bp respectively.The protein structure of the four sequences matched four different E2 subtype models of E2 A,E2 E,E2 G2,E2 H,and contained ubiquitin binding domain UBC with length of 154,188,144,184 amino acids,respectively.After the establishment of a phylogenetic tree,it was found that the four subtypes of E2 were highly differentiated,which may have different functions.Then,the expression pattern of the four subtypes were detected by real-time PCR.Four subtypes of E2 were expressed in different ages and tissues of SBPH and may be generally involved in various life activities.The expression levels of E2 A.E2 E and E2 H were higher in the midgut,while the expression of E2 G2 was higher in the ovaries,and the expression peaks of all four appeared 3 days after emergence.This suggests that E2 may play different roles in the corresponding tissues and ages.2.Inhibition of RSV by E2 in SBPH and its mechanismReal-time PCR was used to detect the gene expression of four subtypes of infected and non-infected SBPH,and it was found that the four subtypes of E2 in the infected SBPH were highly expressed,suggesting that the four subtypes played a certain role in the process of RSV infection of SBPH.Further,RNAi was used to silence four subtypes,and the results showed that only silencing E2 E inreased RSV load in SBPH.Then,real-time PCR,immunoblot analysis and confocal immunofluorescence were used for the further study on E2 E.We found that the inhibition of E2 E could significantly up-regulate the gene and protein expression of RSV Cp,and increased Cp content was also detected in the salivary glands,ovaries and midgut,which proved that E2 E of SBPH could inhibit the replication and accumulation of RSV.Since E2 E did not interact with Cp,Sp,NS2,NS3,NSVC4 and RdRp encoded by RSV,the yeast two-hybrid and GST pull-down were used to verify that E2 E could interact with a lab-cloned E3 ubiquitin ligase,which could interact with RSV.Our results suggest that E2 E may inhibit the accumulation of RSV by activating the antiviral related reaction of SBPH,or by degrading the virus under the catalysis of E3,rather than directly affecting the accumulation of RSV through interaction.More experiments are needed to verify this hypothesis.