Function Of Eukaryotic Translation Elongation Factor 1A(eEF1A) During Infection By Wheat Soil-borne Viruses

Plant viruses have limited genome size,can encode a few proteins,thus they must recruit host factors for survival and replication in infected cells.e EF1A is crucial for the elongation of protein translation in eukaryotes.e EF1A is highly conserved in various species and has been shown to regulate the replication of plant RNA viruses.The genomes of CWMV and WYMV consists of two positive-strand RNAs which are critical for viral replication and translation.Some progress has been made in the molecular biology of CWMV and WYMV,but few host factors were identified to participate in CWMV,WYMV or other furovirus infection in plants.In order to explore the molecular mechanism of the host factors in furovirus infection,here,we investigate the role of host factor e EF1A in CWMV and WYMV infection.Through a series of evidences,we prove that e EF1A is an important host factor for CWMV and WYMV infection.The main results in this study are listed as follows:1.CWMV and WYMV infection induce expression of e EF1A in T.aestivum（Ta）and N.benthamiana（Nb）plants.A BLAST analysis reveal that the amino acid sequences of e EF1As from many species share approximately 98%sequence identity,indicating that e EF1As are highly conserved in various species.CWMV infected N.benthamiana plants were sampled and analysed through q RT-PCR、Western blot and Northern blot assays during 7-28 dpi.The accumulation of CWMV m RNA、protein and genomic RNA and the expression of e EF1A increased with the prolongation of CWMV infection time.2.CWMV/WYMV was inoculated in e EF1A-silenced N.benthamiana by TRV-induced gene silencing（VIGS）.QRT-PCR、Western blot and Northern blot were used to detect the multiplication of CWMV in Nbe EF1A-silenced plants,the accumulation of CWMV m RNA、protein and genomic RNA,replication and translation efficiency of CWMV were significantly reduced.The results of transient over-expression of Nbe EF1A in N.benthamiana was on the contrary.The accumulation of WYMV protein was significantly reduced in Nbe EF1A-silenced plants,whereas transient over-expression of Nbe EF1A enhances WYMV protein accumulation in N.benthamiana.Based on these findings,we conclude that e EF1A is required for CWMV and WYMV infection in plants.3.We first verifiy that there is no protein-protein interaction between e EF1A and CWMV replication-associated proteins in vivo and in vitro.The binding of Nbe EF1A to the CWMV genomic RNA is analyzed by EMSA,and the results showed that Nbe EF1A cannot bind to CWMV（-）3’-UTR and（+）/（-）5’-UTR of RNA1 and RNA2、CWMV^301–781 of RNA1 and（+/–）CWMV^681–981 of RNA2 were randomly selected,only the（+）3’-UTR of CWMV RNA1 and RNA2 specifically binds to Nbe EF1A.4.MST analyses show that the TLS in the CWMV RNA1/RNA2 3’-UTR is essential for the binding between Nbe EF1A and CWMV genomic RNAs.In order to investigate the role of TLS during CWMV infection,TLS deletion mutants were constructed:CWMVΔR1,CWMVΔR2,respectively.Comparing with wild-type（WT）CWMV and CK plants,the accumulation of CWMV m RNA、protein and genomic RNA,replication and translation efficiency of CWMV in CWMVΔR1 and CWMVΔR2-infected plants were significantly reduced,and the symptoms were more similar to those of CK plants.Taken together,our analyses demonstrated that the conserved TLS in the 3’-UTR is a key domain for Nbe EF1A binding CWMV genomic RNAs,which is crucial for CWMV infection.5.RNA secondary structure prediction of the conserved 3’terminal region of the CWMV3’-UTR revealed a t RNA-like structure（TLS）consisting of six SLs.To elucidate the key domains of e EF1A binding to CWMV 3’-UTR,we prepared corresponding deletion mutant transcripts for these six SLs:ΔSL-1,ΔSL-2,ΔSL-3,ΔSL-4,ΔSL-5 andΔSL-6.The binding affinity of e EF1A with CWMV RNA2 3’-UTR was analyzed by EMSA and MST,respectively,the results showed that SL-6 was the key to binding e EF1A to CWMV RNA2 3’-UTR.Furthermore,the complementary mutant 3’-UTR^{Ins（auguuca）},deletion mutant m3’-UTR^Δ3561-3567 and point mutant m3’-UTR^ugaacau were constructed for SL-6,compare with WT CWMV,the mutant m3’-UTR^Δ3561-3567 and m3’-UTR^ugaacau was significantly decreased virus accumulation,replication and translational efficiency,while the complementary mutant approached WT CWMV.This finding suggests that the mutation in SL-6 of CWMV 3’-UTR interferes with its ability to bind to e EF1A and leads to a decrease in viral infectivity.