Effect Of Calcium And Calcium Channel Protein Cngc1/2 On Citrus Canker Disease

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp.citri（Xcc）,which endangers almost all the main citrus cultivars and affects the development of citrus industry.Therefore,it is of great significance to study the measures for controlling citrus canker.Previous investigation on 39 citrus orchards in Hunan Province showed that soil acidification and exchangeable calcium deficiency were serious in citrus orchards,and calcium deficiency existed in leaves.As one of the large quantities of elements required by plants,calcium deficiency will cause imbalance of plant nutrition,decline of growth potential and weakening of immune level.However,the role of calcium in Xcc infection of citrus leaves remains unclear.In this study,the rootstock Poncirus trifoliata（L.）Raf was used as the experimental material to analyze the tolerance level of Poncirus trifoliata to calcium and the effect of calcium on the growth and development of Poncirus trifoliata.The calcium ion(Ca²⁺)concentration suitable for the growth of Poncirus trifoliata was clarified,and the role of calcium in leaf response to Xcc infection was preliminarily analyzed.（Cyclic Nucleotide-Gated Channels,CNGC）genes with high expression induced by Xcc in resistant germplasm Citron C-05（Citrus medica L.）and susceptible germplasm Citrus sinensis were screened based on transcriptome level,and the role of CNGC channel protein CNGC1/2 in response to Xcc infection in citrus was analyzed in depth,which provided a theoretical basis for scientific fertilization in citrus orchards and formulation of ulcer disease control strategies.The main research results are as follows:（1）Poncirus trifoliata seedlings were treated with 0,0.75,3,30 mmol·L^-1Ca²⁺by sand culture method,and 0 mmol·L^-1Ca²⁺was used as control.The promotion effects of different treatments on plant height,main root length and biomass ofPoncirus trifoliata were different.When the Ca²⁺concentration was 3 mmol·L^-1,the aboveground and underground development were the best.It showed that calcium deficiency and excessive calcium would affect the normal growth and development of Poncirus trifoliata.（2）The pathogenicity of different Ca²⁺treatments on Xcc was observed after inoculation with Xcc（10⁵cfu/m L）.The results showed that with the increase of Ca²⁺concentration,leaf symptoms gradually reduced,but there was no significant difference in the growth of Xcc.The cell wall synthesis related genes Pt CESA4,Pt PME and Pt FLA were down-regulated by Xcc under low calcium stress,and up-regulated by Xcc under high calcium stress.The expression levels of immune pathway-related genes Pt GSL,Pt GST1 and Pt WRKY22 induced by Xcc under high calcium stress were higher than those under calcium deficiency stress.The results showed that the expression of genes related to cell wall synthesis was induced by calcium,and the increase of Ca²⁺concentration may promote the thickening of leaf cell wall and inhibit the formation of typical symptoms of Xcc breaking through leaf epidermis.（3）39 CNGC channel genes were screened from transcriptome data,of which Cm CNGC1 and Cs CNGC2 were highly expressed in Citron C-05 and Bingtang Sweet Orange leaves induced by Xcc,respectively.Real-time fluorescent quantitative PCR（q RT-PCR）was used to verify the expression characteristics of these two genes induced by Xcc in the leaves of Bingtang Sweet Orange and Citron C-05.The expression of Cm CNGC1 was up-regulated by Xcc in Citron C-05 leaves,but there was no significant change in Bingtang Sweet Orange.The expression of Cs CNGC2was up-regulated in Bingtang Sweet Orange leaves induced by Xcc,but there was no significant change in Citron C-05 leaves.The amino acid sequences encoded by CNGC1/2 homologous genes in different citrus germplasms were analyzed,and it was found that the amino acid sequences encoded by these two genes in different citrus germplasms were quite different.（4）The function of CNGC1/2 was verified by Tobacco transient transfection,Citrus transient expression system and Arabidopsis genetic transformation.The subcellular localization results showed that Cm CNGC1 was localized in the nucleus,and Cs CNGC2 was localized in the plasma membrane.The results of transient over-expression of Bingtang Sweet Orange leaves showed that the over-expression Cm CNGC1 region had milder susceptibility than the empty vector（control）,and the Xcc content was lower than that of the control.The susceptibility of the over-expression Cs CNGC2 region was more severe than that of the control,and the Xcccontent was higher than that of the control.It indicated that CNGC1 and CNGC2might be regulated during the typical symptom stage of citrus leaf canker induced by Xcc.Cm CNGC1 and Cs CNGC2 overexpression transgenic Arabidopsis T1 lines were obtained,and wild-type Arabidopsis（Columbia-0,Col-0）was used as control,and（Pseudomonas syringae tomato DC3000,Pst DC3000）was inoculated.The results showed that the growth and post-inoculation symptoms of Pst DC3000 in Cm CNGC1overexpression plants were not obvious compared with those of wild type.Compared with the wild type,the overexpression plants of Cs CNGC2 had higher bacterial content and more severe symptoms.