Transcriptomic Analysis Of Nicotiana Benthamiana That Showed More Severe Symptom When Infected By Beet Black Scorch Virus Along With Its Satellite RNAs

Beet black scorch virus(BBSV)is able to systematically infect Nicotiana benthamiana.Interestingly,it was reported that the systemic symptom caused by BBSV became more severe when BBSV satellite RNA(sat-RNA)was envolved in the infection,especially under a low temperature(18?).To know the mechanism through which the BBSV sat-RNA facilitates the infection of BBSV in N.benthamiana,in this study,the transcriptome of N.benthamiana infected by BBSV and its satellite RNA were analyzed by using microarray and high throughput RNA sequencing(RNA-seq)techniques.Hopefully,this research will provide valuable data for understanding of the infection processes and pathogenicity of BBSV associated with its sat-RNA.At the early days after inoculation,microarray and RNA-seq techniques were applied for comparative study the gene expression profiles in the local leaves of N.benthamiana infected by BBSV alone or together with its sat-RNA.In gene chip analysis,4503 differentially expressed genes were obtained by comparisons of the gene expression levels of N.benthamiana at different time points after infection by BBSV with or without its satellite RNA,using mock-inoculated plants as control.Among those activated genes,besides many are involved in important biological processes such as ribosome,proteasome metabolisms and photosynthesis pathway,remarkably the genes that are involved in the biosynthesis of plant hormones and the plant-pathogen interaction were differentially expressed and up-regulated rapidly after infection by BBSV,but which was attenuated when sat-RNAs were added.In further high throughput RNA-seq experiments were carried out to complement the deficiency of microarray in exploring unknown genes that may be involved in BBSV infection process.The results generated by RNA-seq were consistent with those by microarray,both demonstrating that resistant genes/pathways were significantly changed and this trend is compromised in the presence of satellite RNA.Further comparative analysis of the transcriptomic data of N.benthamiana infected by BBSV or BBSV plus sat-RNA showed that the enhanced pathogenicity by satellite RNA were possibly related to the gene silencing pathway and the genes that were differentially expressed,such as plant resistance genes(35 differentially expression genes,DEGs),transcription factors(47 DEGs),receptor proteins(49 DEGs),ubiquitination pathways(34 DEGs)and auxin pathways(17 DEGs).However,the up-regulation level in these pathways could be suppressed by sat-RNA to attenuate the defensive response of N.benthamiana to BBSV infection.In order to understand and verify the role of sat-RNA in the infective processes,the genes related to gene silencing pathway were deeply analyzed.Results showed that the expression of RDR6(RNA-directed RNA polymerase 6),DCL2(Dicer-like protein 2),AGOl(argonaute protein 1)and AG02(argonaute protein 2)were up regulated after BBSV infection,suggesting that the antiviral ability of N.benthamiana may be increased by higher level expression of these genes.In contrast to BBSV infection alone,the addition of satellite RNA inhibited the up-regulation of AGO expression,suggesting that the decreased resistance by sat-RNA may due to reduction of the expression level of AGO genes.Comparing with the untreated healthy plants,it was shown that the expression of AG02 was up-regulated while the expression of miR403 was down-regulated.In light of the fact that AGCO2 mRNA contains the target sequence of miR403 to aggravate AGO1 for degradation of AG02,it is presumed that the up-regulation of AGO2 is regulated by inhibiting the expression of miR403 when the plant was infected by BBSV alone,but the decrease of AGO2 accumulation while miR403 singnificantly declining suggested that there were other paths responsible for AGO2 regulation at the addition of sat-RNA.Since the reliable candidate genes obtained from microarray or RNA-seq have to be verified by quantitative real-time PCR(qPCR)detection,the screening of internal reference genes suitable for the analysis of transcriptional levels in virus-infected plants were carried out.According to the sequences in data base and previou reports,26 candidate genes were selected and tested by qPCR for their expression level in the leaves of N.benthamiana infected by the viruses.The qPCR results were analyzied by geNorm,NormFinder and BestKeeper and it was shown that NbUP7 and GBP were the most stable internal reference genes suit for qPCR detection in transcriptomic analysis of N.benthamiana infected by Tobacco necrosis virus A,BBSV,Beet necrotzic yellow vein virus,Barley stripe mosaic virus or Potato virus X,respectively.Intergatively,by these results,it is speculated that the defensive response of N.benthamiana to the BBSV infection could be attenuated byits sat-RNA through inhibiting differential expressions of the genes envolved in plant resistant pathway,causing the deficient defense and more severe symptoms of N.benthamiana infected by BBSV.