The Mechanism Of Endogenous MiR159 In Response To Infection Of Tobacco Curly Shoot Virus And Its Betasatellite In Nicotiana Benthamiana

Tobacco curly shoot virus(TbCSV)is a monopartite virus belonging to the genus Begomovirus in the family Geminiviridae.TbCSV is an important pathogen causing leaf curl disease on tomato and tobacco plants.At present,some studies have been carried out on the detection and pathogencity of TbCSV and TbCSB.However,the effects of the virus on host endogenous genes,especially on endogenous miRNA which are helpful to find out the pathogenesis of the TbCSV and TbCSB,are still unclear.In this study,high-throughput sequencing and bioinformatics analysis were used to analyze the effects of infection of TbCSV/TbCSB on the expression of endogenous miRNA in Nicotiana benthamiana and the biological pathways regulated by these differentially expressed miRNAs.With the help of molecular biology technology,the effects of miRNA and its target genes on infection of TbCSV/TbCSB were analyzed.At the same time,the response of GA pathway to TbCSV/TbCSB infection was also analyzed.1.Effects of TbCSV/TbCSB infection on the endogenous miRNA expression of N.benthamianaSmall RNA libraries were constructed using total RNA from N.benthamiana leaves either infected with TbCSV/TbCSB or not.A total of 16,753,586 and 17,822,708 raw reads were generated via Illumina sequencing to represent these samples,of which only 5,936,641(20.38%)were in the infected library and 5,311,979(18.24%)in the non-infected library.When all the reads(18 to 30 nt)were analyzed,those with 24 nt were the most abundant and,6,119,284(44.04%)were found in the infected library and7,109,221(46.69%)were found in the non-infected library.After removing data of extremely low expression abundance,we obtained a total of 82 differentially expressed miRNAs,of which 40 were known miRNAs and 42 were predicted miRNAs.The GO functional annotation analysis of these differentially expressed miRNA target genes showed that these genes are mainly involved in plant metabolism,membrane formation,catalytic activity and substance binding.2.Effects of nbe-miR159 on TbCSV/TbCSB infection in N.benthamianaAnalysis of high-throughput sequencing results revealed that a large number of known miRNAs were differentially expressed.Among them,miR159 showed up-regulated expression in the treatment group(inoculated with TbCSV/TbCSB).In order to clarify the mechanism of the symptoms induced by the virus in N.benthamiana,we conducted research on some differentially expressed miRNAs.The expression levels of nbe-miR159 were detected after TbCSV/TbCSB infection.The results showed that the expression levels of nbe-miR159 were significantly less than those in the control group at 5 dpi,10 dpi,15 dpi,and 20 dpi.We found that the lowest expression level of nbe-miR159 in treated group is only about 20% of that in the control group at 10 dpi.At the same time,q PCR was used to detect the accumulation of TbCSV and TbCSB.The results showed that the accumulation of TbCSV and TbCSB increased from 5 dpi to 20 dpi.These results show that the accumulation of TbCSV and TbCSB in N.benthamiana were increased,and the expression level of nbe-miR159 was suppressed.When PVX and TRV vector were used to suppress the expression of nbe-miR159,N.benthamiana produced symptoms of leaf curling which were similar to the symptoms of TbCSV/TbCSB infection.And when N.benthamiana,in which the expression level of nbe-miR159 was suppressed by STTM method,was inoculated with TbCSV/TbCSB,we found not only the symptoms induced by the virus were aggravated,but also the accumulation of the TbCSV and TbCSB were increased.These results indicated that the expression level of nbe-miR159 was suppressed in the TbCSV/TbCSB infected N.benthamiana plants,and the accumulation of TbCSV and TbCSB were increased in the TbCSV/TbCSB infected N.benthamiana which the expression level of nbe-miR159 was suppressed by STTM method.3.Effects of nbe-miR159 target gene on TbCSV/TbCSB infection in N.benthamianaIn order to further demonstrate which downstream genes nbe-miR159 regulates to respond to virus infection.Firstly,the nbe-miR159 target genes were predicted with the help of bioinformatics prediction software ps RNATarget(http://plantgrn.noble.org/ps RNATarget).Two target genes(ID:Niben101Scf05078g08009,Niben8009;ID: Niben101Scf01383g07027,Niben7027)with the highest matching degree and score were selected for further research.That Niben8009 and Niben7027 are two target genes of nbe-miR159 which proved by co-injection experiment of fusing green florescent protein with overexpression of nbe-miR159.The expressions of Niben8009 and Niben7027 at 5 dpi,10 dpi,15 dpi,and20 dpi were detected respectively in TbCSV/TbCSB infected N.benthamiana.It was found that these two target genes had a negative correlation with the expression of nbe-miR159.Through PVX and TRV mediated systemic silencing of nbe-miR159,and p CVA mediated systemic overexpression of nbe-miR159,the expression levels of Niben8009 and Niben7027 were still negatively correlated to those of nbe-miR159.By TRV-mediated expression of Niben8009 or Niben7027 in N.benthamiana,the accumulation level of TbCSV and TbCSB was detected at 7 dpi.The results showed that after Niben8009 or Niben7027 were silenced,the accumulation of TbCSV and TbCSB decreased in both inoculated and system leaves.Similarly,PVX was used to mediate the overexpression of Niben8009 and Niben7027 in N.benthamiana,and the accumulation of TbCSV and TbCSB was detected at 7 dpi,too.The results showed that after Niben8009 or Niben7027 were overexpressed,the accumulation of TbCSV and TbCSB increased in both inoculated and systemic leaves.These results indicate that nbe-miR159 responds to TbCSV/TbCSB infection by regulating its downstream target genes(Niben8009 and Niben7027),and the identified target genes Niben8009 and Niben7027 have positive regulatory effects on the accumulation of TbCSV and TbCSB.4.Responses of GA pathway-related genes upon TbCSV/TbCSB infectionIn order to determine the response of the GA pathway to virus infection,we selected 3 key genes in the GA synthesis pathway and used q RT-PCR to detect their expression in N.benthamiana infected by TbCSV/TbCSB at 5 dpi 10 dpi,15 dpi and 20 dpi.The results showed that the key genes GA20,GA-3?,KS of GA synthesis pathway were down-regulated after TbCSV/TbCSB infection.At the same time,the GA content in N.benthamiana infected by TbCSV/TbCSB at 5 dpi,10 dpi,15 dpi,and 20 dpi was lower than that in uninfected N.benthamiana at the same period.The study showed that,when the exogenous GA was sprayed on the N.benthamiana which inoculated with TbCSV/TbCSB,the accumulation of TbCSV and TbCSB were lower than that of spraying with water at 5 dpi,10 dpi,15 dpi,and 20 dpi,respectively.Meanwhile,the expression of nbe-miR159 in N.benthamiana treated with GA and inoculated with TbCSV/TbCSB was significantly higher than that of water treatment group.This indicated that GA treatment can not only increase the expression of nbe-miR159 in N.benthamiana,but also reduce the accumulation of TbCSV and TbCSB.This study showed that TbCSV/TbCSB infection in N.benthamiana can affect a large number of endogenous miRNAs expression,among which,the expression level of nbe-miR159 not only affected the phenotype of N.benthamiana,but also was closely related to the virus infection.Besieds,the GA pathway participated in the resistance of N.benthamiana to TbCSV/TbCSB and involved in regulating the accumulation of TbCSV and TbCSB.These results lay a foundation for further analysis of the interaction mechanism between the host and TbCSV/TbCSB.