Study On The Mechanism Of Azelaic Acid Induced Tobacco Disease Resistance

Tobacco diseases including tobacco black shank,brown spot and bacterial wilt often decrease the yield and quality of tobacco.Azelaic acid(Az A)is a long-range transduction signal that elicits local and systemic immunity in plants and induces systemic acquired resistance(SAR)via the NO-ROS-Az A-G3P(glycerol3-phosphate)signaling pathway.The protein kinase GCN2(general control nonderepressible-2)responds to various biotic and abiotic stresses and can be activated by azelaic acid.Previous study showed that azelaic acid spraying could induce plant disease resistance.However,the mechanism of azelaic acid induced disease resistance is still unclear.In this study,we analyzed the expression patterns of antioxidant indicators and disease-resistance-related genes in tobacco after spraying with different concentrations of azelaic acid.We used RNA-seq to analyze the molecular mechanism of tobacco immunity induced by azelaic acid in response to tobacco.The mechanism of induction of disease resistance and NtGCN2-mediated disease resistance in tobacco after spraying with different concentrations of azelaic acid was also analyzed.The results of the study were listed as following:1.The results of the analysis of antioxidant indexes and expression patterns of disease-resistance-related genes in tobacco K326 treated with 0.25 m M,0.5 m M,1 m M and 2 m M of azelaic acid showed that the spraying of azelaic acid increased antioxidant enzyme activity,antioxidant-related gene and disease-resistance-related gene expression,and decreased superoxide anion accumulation and MDA content.This suggests that spraying azelaic acid may have enhanced the disease resistance of tobacco by increasing the antioxidant capacity and activating the expression of disease-resistant genes.It was found that the expression levels of antioxidant and disease resistance related genes in tobacco were higher 48 hours after spraying with 0.5 m M,1 m M,and2 m M 的 Az A.2.RNA-seq was used to analyse tobacco K326 at 48 h after 0 m M,0.5 m M,1m M and 2 m M azelaic acid spraying.The results showed that there were 620 differentially expressed genes at 48 h after the three concentrations of azelaic acid spraying compared to 0 m M,of which 457 genes were up-regulated and 153 genes were down-regulated.In comparison.The other 10 genes were inconsistently expressed in the three differential groups.GO enrichment analysis revealed that these differentially expressed genes involve biological processes such as ethylene-activated signaling pathway,response to salicylic acid,regulation of protein serine/threonine,etc.KEGG enrichment analysis showed that these differentially expressed genes involve plant hormone signal transduction,MAPK signaling pathway-plant,plant-pathogen interaction and phenylpropanoid biosynthesis.Screening these differentially expressed genes resulted in eight disease-resistant genes and five antioxidant-related genes whose expression was up-regulated by azelaic acid spraying.Eight disease-resistant genes included the calcium-binding protein-related genes CML39,the LRR receptor-like serine/threonine protein kinase-related gene FLS2,and the WRKY transcription factorrelated gene WRKY3.The five antioxidant-related genes included the peroxidaserelated genes PER15,PER51,PER15 and pox N1.3.Tobacco K326 was inoculated with Alternaria alternata,Ralstonia solanacearum and Phytophthora nicotianae 48 h after spraying with different concentrations of azelaic acid.The results showed that azelaic acid reduced the spot diameter of tobacco inoculated with Alternaria alternata and the spot diameter of tobacco isolated from leaves inoculated with Phytophthora nicotianae,with a greater reduction in spot diameter with 0.5 m M azelaic acid.Azelaic acid reduced the disease index and improved the efficacy of azelaic acid in tobacco inoculated with Ralstonia solanacearum,with 2 m M azelaic acid increasing the efficacy.Analysis of antioxidant indicators showed that azelaic acid spraying increased SOD activity and reduced superoxide anion accumulation and MDA content in pathogen-inoculated tobacco.In addition,azelaic acid spraying increased POD and CAT activity in isolated leaves of tobacco inoculated with Alternaria alternata.Analysis of the expression pattern of disease-resistance-related genes showed that spraying 2 m M and 0.5 m M azelaic acid respectively increased the expression of Nt NPR1,Nt PAL and Nt COMT diseaseresistance-related genes in isolated leaves of tobacco inoculated with Ralstonia solanacearum and Phytophthora nicotianae.4.NtGCN2 overexpressing tobacco was sprayed with different concentrations of azelaic acid.The results of the antioxidant analysis showed that the spraying of azelaic acid increased the antioxidant enzyme activity and reduced the MDA content in NtGCN2 overexpressing tobacco.This indicated that spraying azelaic acid improved the antioxidant capacity of NtGCN2 overexpression tobacco,with the spraying of 0.5m M azelaic acid being more effective.At the same time,NtGCN2 overexpressed tobacco increased antioxidant enzyme activity and reduced superoxide anion accumulation and MDA content compared to K326,indicating that NtGCN2 overexpression improved the antioxidant capacity of tobacco.The statistical results of the disease incidence of NtGCN2 overexpressed tobacco inoculated with Alternaria alternata at 48 h after azelaic acid spraying showed that azelaic acid spraying reduced the spot diameter of NtGCN2 overexpressed tobacco,NtGCN2 overexpression also reduces spot diameter.The results of DAB and NBT staining showed that azelaic acid spraying improved the antioxidant capacity in NtGCN2 overexpressing tobacco.These results indicated that azelaic acid can enhance the resistance of NtGCN2 overexpressing plants to Alternaria alternata.