The Mechanism Of Citrus Disease Resistance Against Huanglongbing Induced By Plant Immunity-inducing Agent

Huanglongbing(HLB)is the most destructive citrus pathosystem worldwide,and it restricts the development of citrus industry seriously.HLB is caused by the phloem-restricted bacterium,Candidatus liberibacter asiaticus and is transmitted by the citrus psyllid Diaphorina citri.Measures to prevent and control Citrus HLB include control of the citrus psyllid,cultivation of HLB-free nursery trees and removal of infected trees,however,the disease is still difficult to get effective control.That induce plant resistance is an important measure to reduce the occurrence and development of diseases.The study focused on the mechanism of plant immunity inducing agent ATaiLing inducing citrus disease resistance against HLB.The major results are as follows:1.The plant immunity inducer ATaiLing could induce Citrus disease resistance.The results showed that ATaiLing-treated plants displayed a higher level of chlorophyll content compared to control plants.And ATaiLing could promote plant root development,improve root vigor and fruit quality,at the meantime,significantly improve the prevention and control of HLB.The quantity of CLas in infected plants was reduced to a certain extent after treatment with ATaiLing.Scanning electron microscopy observation showed that the sieve tissue in treated plant was more clear than the control.In addition,qPCR analysis indicated that ATaiLing was able to induce an increase of expression level of Citrus resistance-related genes CsNPR1,CsPR1,CsPR5 and CsLOX2.1.After spraying treatment with ATaiLing,the CsNPR1 gene related to salicylic acid(SA)pathway was up-regulated by about 4 fold,and the SA downstream genes of CsPR1 and CsPR5,which are two markers of systemic acquired resistance(SAR),were also up-regulated by about 10 and 7 fold,respectively.ATaiLing also caused an up-regulation of CsLOX2.1,a key gene for the biosynthesis of jasmonic acid(JA),to about 5-fold.2.Proteomics and transcriptomics studies confirmed that ATaiLing significantly regulated expression level of Citrus resistance-related proteins and genes.The proteomics data showed that ATaiLing caused 447 differentially expressed proteins(DEPs)in citrus plants,Gene Ontology analysis showed that most of DEPs were involved in the plant responses to the stimulus.KEGG pathway enrichment indicated that a lot of DEPs were involved in SA,JA and phenylpropanoid biosynthesis pathways that were related to disease resistance.Furthermore,the transcriptomic data showed that most of differentially expressed genes(DEGs)involved in SA,JA and phenylpropanoid synthesis pathways were also elevated by ATaiLing treatment.Interestingly,ATaiLing also induced up-regulated expression of chlorophyll synthesis-related proteins and genes in plants.3.To further validate the proteomics and transcriptional data,12 DEPs and 12 DEGs were confirmed using the qPCR.The results indicated that transcriptional level of the selected proteins and genes was consistent with the proteome and transcriptome data.Comprehensive analysis was performed combining the proteome and transcriptome data,2 target proteins,Salicylate carboxymethyltransferase(CsSAMT)and Linoleate 13S-lipoxygenase 2-1(Cs LOX2.1),were further chosen to prepare their antibodies to confirm their function.CsSAMT and CsLOX2.1 were first expressed by E.coli with pET-28 a and pET-32 a,respectively,and purified via His-tag.Purified proteins were used as antigens to infect rabbit for antibodies preparation.CsSAMT is a salicylic acid carboxymethyltransferase that catalyzes the methylation of the free carboxyl-terminus of the phytohormone SA,thereby converting SA into SA methyl ester(MeSA).Lipoxygenase CsLOX2.1 is involved in the biosynthesis of JA and plays a very important role in plant defense responses.Western blot analysis confirmed that ATaiLing could induce the up-regulation of expression level of CsSAMT and CsLOX2.1,while ATaiLing caused the accumulation of endogenous SA and JA in Citrus plants.