Preliminary Study On The Effect Of Salicylic Acid And High Temperature On RNA Silencing

RNA silencing is a kind of sequence specific post-transcriptional gene inactivation.High temperature could weaken viral symptoms and reduce the virus titer in plants, andsalicylic acid (SA) mediated disease defense also was reported. In our study, wedemonstrated the molecular mechanisms of GFP co-infiltration induced RNA silencing andvirus mediated RNA silencing was influenced by high temperature and SA using wild typeand NahG transgenic tobacco.The results are as follows.(1) Agrobacterium mediated transient infiltration assay showed that high temperatureand SA could promote RNA silencing. When the cultivated temperature was30℃, wild typetabacco showed no GFP expression at infiltration area at5dpi. While when the cultivatedtemperature was24℃, wild type tabacco still displayed GFP expression at infiltration area.Compared with wild type tabacco, SA-deficient NahG transgenic tobacco plants displayedweak GFP fluorescence when cultivated at30℃, and even stronger GFP expression couldbe detected in24℃NahG transgenic tobacco plants infiltration area than in24℃wild typetabacco. NahG was salicylic acid hydroxylase gene, catalysing SA into catechins, whichmade SA amount decline to a lower level than wild type tabacco. So, the weak degree ofRNA silencing might be related to the decline of SA. Moreover, the assay of spraying SAalso proved that SA could enhance RNA silencing.In order to study the influence of hightemperature and SA on RNA silencing and their relationf, we detected five genes, AOX1,RDR1, PR1a,PR1b and PR2, related to SA induced disease resistance. The results showedthat, all of the five genes had the highest expression in30℃wild type tabacco showingstrongest RNA silencing, which illustrated that both RNA silencing and SA mediated SARdefense response could be stimulated by SA and high temperature, the two kinds ofdefense responses had a cross interaction. Besides, it had been proved that high temperaturecould raise the SA level of plants. All the results obtained in the present study confirmed thathigh temperature enhanced RNA silencing by increasing SA level.(2) PVX inoculation assay showed that high temperature and SA could promote RNA silencing induced by viruses. When the cultivated temperature was30℃, wild type tabaccoshowed no PVX symptoms in the leaves above the inoculated leaves at14dpi. While whenthe cultivated temperature was24℃, wild type tabacco displayed obvious symptoms.SA-deficient NahG transgenic tobacco plants displayed obvious symptoms when cultivatedat30℃, and even stronger symptoms could be observed in24℃NahG transgenic tobaccoplants than in24℃wild type tobacco plants. The PVX titer detected by ELISA wascorresponded to the above observation results. This illustrated that high temperature couldenhance RNA silencing induced by virus, and when we used NahG transgenic tobaccoplants to decline the SA level, high temperature could not activated virus induced RNAsilencing, so SA might play a critical role in RNA silencing pathway. In order to study theinfluence of high temperature and SA on RNA silencing, we detected five genes related withdisease resistance and could be induced by SA. The results showed that AOX1, PR1a, PR1band PR2had the high expression level in24℃NahG transgenic tobacco plants showingstrongest PVX symptoms, than in30℃NahG transgenic tobacco plants, and the expressionof these four genes was higher in24℃WT than in30℃WT. which illustrated that AOX1,PR1a, PR1b and PR2could be directly induced by PVX virus and had nothing to do withthe degree of RNA silencing.(3) In order to study the genes related to the enhanced RNA silencing in plants, hightemperature and SA treated Nicotiana benthamiana plants were analysed by transcriptomesequencing. Solexa sequencing analysis showed that, compared with control, differentiallyexpressed（DE）genes in high temperatue and SA treatment were601and14respectively.Among these DE genes,5genes were shared in high temperatue and SA treatment. Moreover,596and9genes were specific respectively. In high temperatue and SA treatment,363and7genes were up-regulated respectively,besides,238and7genes were down-regulatedrespectively.We detected the expression level of four up-regulated genes of30℃treatmentand four up-regulated genes of SA treatment by qRT-PCR, they werecomp42463_c0,comp40795_c0,comp44509_c0,comp46958_c0,comp62975_c0,comp30068_c0,comp61011_c0and comp40205_c0, the log2change fold were3.5851,2.1887,2.4862,2.3758,2.8046,1.9292,1.1253and1.0992respectively. The resultswere basically identical with the RNA sequencing results, which shows that the transcriptome sequencing results are reliable. Among these genes, comp46958_c0encodedproline rich protein, which accumulated largely under stress condition and play a importantrole in stabling biological macromolecular structure, reducing acid cell and removing toxicammonia. comp62975_c0was speculated nonspecific lipid transfer protein,which wasconsidered play a part in the synthesis and transportation of wax, disease resistance,reproductive development and other important physiological processes. In addition, hightemperature treated materials enriched a lot of genes in photosynthesis. So we speculatedthat RNA silencing might affect photosynthetic pathway.