| Citrus bacterial canker disease(CBCD),caused by Xanthomonas axonopodis pv.citri(Xcc),is a worldwide quarantine disease,which results significantly influences to citrus industry,and it is difficult to prevent and control.Therefore,the circulation of citrus materials carrying Xcc from epizootie region to non-epizootie region must be strictly prohibited at all time and the detection of Xcc must be performed before entering the non-epizootie region.However,those strategies could not control the expansion of disease forever.Hence,to fundamentally solve this problem,the most effective and economical approach relies on the selection or breeding of resistant cultivars and the observation of infection and colonization of the pathogen in citrus is indispensable to the research of interaction between citrus and Xcc as well as the susceptibility and resistance of citrus.To better understand the pathogen in the invasion process of pathogen colonization of the host,and the analysis of the development of a more direct infection after the host condition,confocal laser scanning microscopy was used in the observation of the invasion process to the plant tissue in real time by Xcc that marked by eGFP,which provides the technical method for the study of intuitive citrus canker pathogen infection.In this study,Xcc labeled by eGFP was used to inoculated citrus canker disease resistant germplasm citron C-05 and susceptible germplasm ‘Bingtang’ sweet orange for comparing the difference of invasive progress.As we knew,the expression of the resistance related gene,flagellin sensitive gene FLS2 showed a declining trend in susceptible plant,wheras enhanced expression in resistant plant;therefore,in order to investigate the functional difference of FLS2 in susceptible and resistant varieties,we cloned and analyzed the FLS2 gene and its promoter,and explored the resistance mechanism of FLS2 gene in Citron C-05.The followings are main results:(1)Observation the symptoms in different age citrus leaves after inoculation by Xcc,the results showed that after 25 days inoculation,young leaves and leaf only mild shows the most serious symptoms,while the adult leaves shows no lesion,so as that fully expandedleaf at 1/2 maximum leaf area of citrus leaves is the most suitable for the observation of inoculated material.(2)By using laser confocal microscopy,the invasion process by inoculated Xcc with eGFP labeled to citrus leaf tissue was observed,there are the results: after 48 h inoculation,bacteria have invaded in to the leaf tissue,and in healthy tissue the bacteria multiply against the blade causing disease susceptible materials after 60 h inoculation in ‘Bingtang’ sweet orange which cost 72 h in citron C-05;comparison of the onset of symptoms after the inoculation,a volcano like lesions appeared in ‘Bingtang’ sweet orange,and then tissue ulceration,although the C-05 would generate citric lesion symptom,the lesion and lesion surrounding leaf tissue showed a browning,atrophy and dry symptoms;slice observation of citrus leaves after inoculation was took to indicated that Xcc had branched the whole vaccination area(including leaf stomata and the leaf tissues around the stomata)there are no special laws,so that bacteria for invasion and no self selective pores,invasion of leaf tissue is random;Z-stack tomography was used to scan the citrus leaves after inoculation,showed that the Xcc mainly concentrated in mesophyll cells and there are no had obvious evidences proof that Xcc exist in the cell,which infered the colonization of Xcc mainly in the intercellular space;the amount of formation on ‘Bingtang’ sweet orange leaves is significantly redundant than its in citron C-05.The above results show that there’s a possible reaction mechanism of citrus Leaves was triggered in citron C-05,which is inhibited in‘Bingtang’ sweet orange.(3)TA cloning of FLS2-1 and FLS2-2 gene were amplified in ‘Bingtang’ sweet orange and citron C-05 and the structure of them were analyzed by bioinformatics.The cDNA fragment length of FLS2-1 and FLS2-2 genes of sweet orange were 3132 bp and 3129bp;The cDNA fragment length of FLS2-1 and FLS2-2 genes of citron C-05 were 3129 bp and3126 bp,which encoding 1043 、 1042 and 1042 、 1041 amino acids respectively.The homology of amino acid sequence between FLS2-1 and FLS2-2 in ‘Bingtang’ sweet orange and citron C-05 was 97.03% and 99.62% respectively,which indicated that the difference of the amino acid sequences of two alleles between the same citrus germplasm are very small.The homology of FLS2-1 amino acid sequence between ‘Bingtang’ sweet orange and citron C-05 was 92.91% while the homology of FLS2-2 amino acid sequence between ‘Bingtang’sweet orange and citron C-05 was 94.53%.The different protein domains and two or threelevel structure analysis between resistant and susceptible materials of two FLS2 gene showed that the protein structure of two gene were consistent with the analytical results of the gene structure that reported in Arabidopsis and rice,which contains an extracellular domain,transmembrane structure and intracellular kinase domain.But citron C-05 contains 17 extracellular LRR domains,which is lost in ‘Bingtang’ sweet orange;beyond that,there was no significant difference of other protein functional domains and two or three level structure between the two material,which suggesting that there was no necessarylly relation between the difference of two gene structure of FLS2 and expression levels of FLS2 gene in two genotypes.(4)The successful cloning of promoter in ‘Bingtang’ sweet orange,citron C-05.The length of FLS2 gene promoter sequences in ‘Bingtang’ sweet orange exhibited different sequence size,which is 1579 bp and 1662 bp respectively.By the comparison of promoter sequences between ‘Bingtang’ sweet orange and citron C-05,the homology of them is95.89 %.The comparison of three promoter sequences of cis acting elements showed that citron C-05 obsesses one more Box-W1 element than the other as the sequence of Box-W1 element were the same to the reported.It is indicated that W-box may play an important role in the regulation of the resistance to Xcc in citron C-05.‘Bingtang’ sweet orange possess high levels of transcripts exist in orange(5UTR Py-rich stretch)and excitation response element(ELI-box3),which does not exist in citron C-05.Excitation response element(ELI-box3)is unique components in ‘Bingtang’ sweet orange 1579 bp promoter sequence.Promoter deletion part(792 bp~817 bp)of ‘Bingtang’ sweet orange in 1579 bp size may be part of the citrus DEXH box.TGACG-motif was missing in ‘Bingtang’ sweet orangeNCBI sequence alignment showed that deletions may be part of the citrus DEXH box,the loss of these two elements may be related to the high sensitivity to Xcc of ‘Bingtang’sweet orange.The absence of these two components may be related to the high sensitivity of sugar orange. |
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