| Citrus canker is a worldwide quarantine disease,and it is one of the most serious citrus diseases in China.It not only causes the loss of citrus yield and poor quality,but also threats to the environment due to the massive use of chemicals.Hence,screening resistant canker plants is the key way to solve the problem of citrus canker in the long term.In this study,6 citrus germplasms were used to evaluate the resistance by spraying high concentration and injecting with different concentrations of eGFP-labeled Xcc.For Citron C-05 and sweet orange,different inoculation methods were used,to analyze the colonization of pathogen in host and the development of disease at the cellular level,and the defense reaction of the host.Transcriptome was revealed the gene changes and regulatory mechanism of sweet orange in response to Xcc inoculation.The expression patterns of key genes of sweet orange were analyzed.The main results were described as follows:1.eGFP-labeled Xcc was uesd to spray on 6 different citrus germplasms,the results showed that there was no difference in the distribution and quantity of bacteria on the leaves at the first 3 days post inoculation(dpi).At the 6 dpi,the amount of bacteria on the leaf surface of Citron C-05 began to decrease,while the other species continued to increase.At the 10 dpi,the susceptible species showed citrus canker symptoms.At the third day after spray,we observed that the bacteria mainly concentrate in the leaf epidermis.The Xcc had invaded the mesophyll tissue at 6dpi.At the 9 dpi,bacteria began to destroy mesophyll tissue.At the 14 dpi,Xcc destroyed the mesophyll tissue of sweet orange leaves,which showed crater bump.While there was no destroy in the Citron C-05 leaf tissue after spray,and no symptoms were formed.2.6 citrus grermplasms were inoculated with different concentrations of Xcc.At the 8dpi,all the species except Citron C-05 showed early citrus canker symptoms in the area of10~6cfu/ml.At 20dpi,the varieties in 10~3 cfu/ml injected area appeared citrus canker symptoms,and more severe symptoms appeared on the higher the concentration injected area except Citron C-05.At 27 dpi,Citron C-05 still did not show typical symptoms.After injection of Xcc with a concentration of 10~4cfu/ml,the propagation and disease formation of typical symptoms in leaves were analyzed,no difference was found in six citrus germplasm of Xcc at the first 4 days after injection.After 4 days,the bacteria in the leaves of Citron C-05 grew slowly,while the bacteria in other varieties kept a rapid growth.Slices showed that,after injection the bacteria first began to accumulate in the epidermis and then expand to the mesophyll tissue,4 dpi bacteria began to destroy the mesophyll tissue,formation raised at 6 dpi.16 dpi,mesophyll tissue serious destroyed by Xcc.The bacteria grew slowly in the Citron C-05 leaves and did little damage to the normal structure.3.This paper studied the defense response of Citron C-05 and sweet orange after infection with Xcc.The results showed that Citron C-05 was induced accumulation of callose to resist the Xcc invasion,ROS accumulation and HR response were also detected.In the process of pathogen invasion,it also caused the expression of defense genes in Citron C-05.4.Transcriptome sequencing analysis was performed on samples of sweet orange inoculated with Xcc bacteria at the 2,6,8 and 12 dpi,as well as the corresponding control samples inoculated with Xoo on day 2 and 8.dpi.The results of GO enrichment analysis showed that most of the DEGs enrichment in the cell component.KEGG metabolic pathway analysis showed that the DEGs were significantly enriched in plant-pathogen interaction,plant hormone signal transduction,starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism,phenylpropanoid biosynthesis and carbon metabolism.Xcc induced the expression of a large number of cell walls related genes,such as cellulose enzyme,glycosyl hydrolase,pectin lyase,pectin lipase and expansion.5.Quantitative PCR verified the expression of the 3 genes of Pectate lyase(PL)Expansin(EXP)and Cellulase(CEL).The 3 genes were induced high expression in the presence of symptoms in sweet orange,while there was not difference in the expression of the Citron C-05.The genes ofPL,EXP and CEL were cloned.ThePL gene(CsPL,CmPL)was 1675 bp and 1657 bp respectively,with 4 exons and 3 introns,encoding 404 amino acids and the homology was 99.3%.The EXP gene(Cs EXP,Cm EXP)were both 1159bp,with 4exons and 3 introns,encoding 274 amino acids,containing 4 exons and 3 introns,encoding274 amino acids and the homology was 99.3%.The CEL gene(Cs CEL,Cm CEL)was 2278bp and 2249 bp respectively,with 7 exons and 6 introns,encoding 506 amino acids,and the homology was 99.4%.6.The PL promoter(pCsPL,p CmPL)of sweet orange and Citron C-05 were cloned,were 1807 and 1812 bp respectively,and homeology of 94.5%.Cis-acting elements related to abscisic acid response,cis-acting elements related to optical response and cis-acting elements related to anaerobic induction are common.The promoter of pCsPL also specifically contains salicylic acid response element,and the promoter of p CmPL contains methyl jasmonate response element.The EXP promoter(pCs EXP,p Cm EXP)of sweet orange and Citron C-05 were 2006 and 2032bp,respectively,with 93.9%homology.The abscisic acid response element,methyl jasmonate induction element,gibberellin induction element,hormone induction element,methyl jasmonate induction element,cell cycle regulation element,zein metabolism regulation element,MYBHv1 binding site and so on are both common.In addition,there are salicylic acid inducing elements in the pCs EXP promoter,there areα-amylase promoting elements and negative endosperm regulating elements in p Cm EXP.The CEL promoter(pCs CEL)of sweet orange and Citron C-05 were1606 and 1603 bp,respectively,and the homogeneity was 97.9%.Both including abscisic acid response element,methyl jasmonate induction element and salicylic acid induction element,and a transcription factor binding element MYBHv1 binding site,as well as anaerobic induction element,meristem expression element,endosperm expression element,and zein metabolism regulation element.7.The overexpression vectors ofCs EXP andCs CEL genes were constructed.Subcellular localization indicated that EXP and CEL proteins were located on the cell membrane.Agrobacterium-mediated transient expression assays showed that the citrus canker symptoms in the overexpression ofCs EXP andCs CEL leaves was more serious than that on blank vector control. |
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