Functional Study Of CsLYK6-a Receptor Kinase Involved In Citrus Canker Resistance

Citrus bacterial canker（CBC）is a bacterial disease caused by Xanthomonas citri subsp.citri（Xcc）,which is one of most serious diseases causing huge losses to the citrus industry.Molecular breeding using CBC resistance genes was one of the main methods to prevent CBC.Based on the previous transcriptome data and q RT-PCR analysis in resistant and susceptible citrus varieties induced by citrus bacterial canker,we found that CsLYK6 might be a resistant protein of citrus in response to citrus canker disease.In this study,we carried out the citrus functional verification combining the molecular mechanism analysis with transcriptome,proteome,physiological and biochemical analysis of CsLYK6 overexpression plants.The main findings are as follows:（1）CsLYK6 is a putative Xcc-influenced CBC resistance geneCsLYK6 was not significantly induced by Xcc infextion in CBC susceptible variety--Wanjincheng,but was significantly up-regulated in CBC resistant variety--Calamondin,indicating that CsLYK6 may be a resistance gene associated with CBC.CsLYK6 is located on chromosome 6of citrus with an open reading frame of 2 037 bp,encoding 678 aa,containing a Lys M functional domain,a kinase domain,a transmembrane domain and a signal peptide region.In citrus,CsLYK6protein was localized on the cell membrane detected by onion cell transient expression.（2）Overexpression of CsLYK6 confers CBC resistanceIn this study,3 CsLYK6 overexpression transgenic plants were obtained using Agrobacterium-mediated gene transformation.With respect to their phenotypes,the three transgenic plants exhibited a normal growth rate compared to the WT plants.q RT-PCR further confirmed that these three overexpression plants expressed high levels of CsLYK6（49-fold to61-fold over WT respectively）.The lesion size and disease index of the overexpression plants were significantly lower than the WT.The growth of the pathogen was significantly inhibited by the overexpression of CsLYK6.We finally concluded from the Xcc assay that overexpression of CsLYK6 confers CBC resistance.（3）CsLYK6 regulates CsRBOH5 mediated CBC resistance by CsWRKY33The phosphorylated proteome and transcriptome of CsLYK6 OE plants were used to identify key downstream genes.We found that CsLYK6 overexpression led to a significant increase of the phosphorylation level of CsRBOH5,CsCPK13 and CsMAPK6.The relative expression of CsRBOH5 related to reactive oxygen species（ROS）was also significantly up-regulated,indicating that CsRBOH5 plays an important role in the downstream regulation of CsLYK6.There should be an upstream transcriptional regulator of CsRBOH5.To detect the transcription factor,the promoter of CsRBOH5 was taken as a bait to screen the citrus Y1H.Finally,transcription factor CsWRKY33was obtained.Further Y1H verification found that CsWRKY33 can bind the CsRBOH5 promoter region and activate reporter gene expression,indicating the interaction between CsWRKY33 and CsRBOH5.We concluded that CsLYK6 may participate in the plant immune process by regulating the expression of CsWRKY33 and activating CsRBOH5.（4）CsLYK6 positively regulates CsRBOH5 mediated ROS homeostasisDetermining the bio-chemical index of CsLYK6 OE plants found that RBOH enzyme activity,H₂O₂ and O₂^·-contents in CsLYK6 overexpression plants were higher significantly compared with the WT.Based on these findings,we conclude that CsLYK6 positively regulates CBC resistance through CsRBOH5 mediated ROS homeostasis.These results further highlight the importance of this kinase family in plant pathogen resistance.