| Rice stripe virus(RSV)is an important virus in rice production,which seriously affects the yield and quality of rice in China,but there is no effective anti-RSV agent in production yet.Previous studies in our laboratory have found that the plant-derived active substances ailanthone has a very good effect in inducing anti-RSV effects in Nicotiana benthamiana.In order to investigate the mechanism of action of ailanthone to induce host plants to enhance their resistance to the virus,this paper was conducted on the host plant Nicotiana benthamiana,which can be frictionally inoculated to transmit RSV,the molecular mechanism of RSV resistance induced by ailanthone in the host plant Nicotiana benthamiana was investigated by transcriptome sequencing and virus-induced gene silencing(VIGS),and the main findings are summarized below.(1)The induction of RSV resistance in the host plant Nicotiana benthamiana at different concentrations of ailanthone was examined by RT-qPCR using BTH,a common induction agent used in production,as a control agent.The results showed that the inhibition of RSV-CP expression in Nicotiana benthamiana was 13.2%,41.3%,59.9%,82.0% and 94.4%when the concentrations of ailanthone were 0.25 μM,0.5 μM,0.75 μM,1.0μM and 1.25 μM,respectively;The control agent BTH at concentrations of 250 μM,500 μM,750 μM,1000 μM and 1250 μM resulted in 5.2%,46.5%,70.8%,82.0% and 89.9% of RSV-CP expression in Nicotiana benthamiana,respectively.It is suggested that ailanthone has the effect of inducing anti-RSV in the host plant Nicotiana benthamiana,and its effect increases with increasing concentration and is significantly better than the control agent BTH.(2)The expression of RSV-CP was determined in Nicotiana benthamiana at different times after induction by 0.75 μM ailanthone.The results showed that the relative expression of RSV-CP in leaves of Nicotiana benthamiana inoculated with RSV after spraying 0.75 μM ailanthone decreased gradually with the time after inoculation compared with the expression of the control at the corresponding time points,and the relative expression of RSV-CP decreased by 45.8%,68.1%,86.1% and 96.6%at 3,5,7 and 9 d after inoculation with RSV,respectively.Phenotypic observations revealed that at 15 d of RSV inoculation,the control group showed obvious symptoms of disease in the upper new leaves of Nicotiana benthamiana,while those treated with 0.75 μM ailanthone showed no obvious symptoms,further confirming that ailanthone has a good effect in inducing RSV resistance in Nicotiana benthamiana.(3)Quantitative analysis by transcriptome sequencing yielded 255.11 G of Clean bases,a set of 20 differentially expressed genes.GO functional enrichment and KEGG pathway enrichment analysis of differentially expressed genes showed that ailanthone has immunomodulatory activity in host plant Nicotiana benthamiana and RSV interactions,mainly involving metabolic pathways such as plant hormone signal transduction,photosynthesis,phenylpropanoid biosynthesis,starch and sucrose metabolism.The induction of resistance to RSV in Nicotiana benthamiana by ailanthone is predicted to be mainly related to plant hormone signal transduction pathway.Eight genes from the plant hormone signal transduction pathway that were more differentially expressed between the ailanthone treated group and the sailanthone treated group inoculated with RSV were selected for RT-qPCR validation,and the results of the assay were consistent with the transcriptome sequencing results.(4)To further confirm that the induction of the anti-RSV effect of Nicotiana benthamiana by ailanthone is related to the plant hormone signal transduction pathway.Two genes,SAUR20,a growth hormone response gene,and ERF7,an ethylene response factor,were randomly selected from among eight genes in the plant hormone signal transduction pathway.The VIGS technique was used to silence the endogenous genes SAUR20 and ERF7,respectively,and it was found that those with SAUR20 and ERF7 silenced were more susceptible to the disease than those without.And 0.75μM ailanthone still induced up-regulated expression of SAUR20 and ERF7 genes in silenced Nicotiana benthamiana and had some inhibitory effect on RSV-CP expression in its leaves,(the expression of SAUR20 was significantly lower and the expression of ERF7 was significantly higher in silenced Nicotiana benthamiana than in unsilenced Nicotiana benthamiana).The expression of RSV-CP was significantly higher in silenced Nicotiana benthamiana than in silenced by 0.75 μM ailanthone and unsilenced Nicotiana benthamiana,which is consistent with the results of western blot for the accumulation of RSV-CP in silenced Nicotiana benthamiana.It is suggested that the growth hormone response gene SAUR20 and the ethylene response factor ERF7 play an important role in the induction of RSV resistance in Nicotiana benthamiana by ailanthone,and are the two key genes in the induction of RSV resistance in Nicotiana benthamiana by ailanthone.In summary,the plant-derived active substance ailanthone has an induced anti-RSV effect on Nicotiana benthamiana,and its inducing effect increases with the duration of ailanthone treatment within a certain period of time;The induction of RSV resistance in Nicotiana benthamiana by ailanthone may be mediated through the plant hormone signal transduction pathway in Nicotiana benthamiana,and the growth hormone response gene SAUR20 and ethylene response factor ERF7 play an important role in this. |
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