Functional Study Of CsRBOH5 Sites Ser148 And Ser334 Phosphorylation On Citrus Bacterial Canker Resistance

Citrus bacterial canker（CBC）is a bacterial disease caused by Xanthomonas citri subsp.Citri（Xcc）,which has caused serious damage to citrus production worldwide.The screening of citrus disease resistance and susceptibility genes for molecular breeding is an effective measure to prevent and control CBC at this stage.Prior research has demonstrated that the overexpression of Cs LYK6 in sweet orange resulted in the augmentation of the phosphorylation of CsRBOH5 at Ser148 and Ser334,an elevation in RBOH enzyme activity and ROS content,and an enhancement in disease resistance.Consequently,it was postulated that the levels of phosphorylation at Ser148 and Ser334 of CsRBOH5 were associated with ROS synthesis,which in turn was linked to the degree of canker resistance.This study aimed to demonstrate this speculation to probe the function of phosphorylation of Ser148 and Ser334 at CsRBOH5 in resistance to CBC.The key results as following.1.Bioinformatics analysis of CsRBOH5.Bioinformatics analysis of CsRBOH5 revealed that CsRBOH5 contains the NADPH structural domain.The Ser148 site of CsRBOH5 was located in the NADPH domain,and Ser334 followed with two EF chiral structural domains at the N terminus.2.Phosphorylation at Ser148 and Ser334 of CsRBOH5 positively regulates CBC resistance.The area of the phosphorylation-mimicking gene at CsRBOH5 was 48.4%lower than the transgenic plants of the unmutated gene;Similarly,the area of the phosphorylation-mimicking gene at the Ser334 site at CsRBOH5was 51.4%lower compared to the transgenic plants of the unmutated gene.The simultaneous phosphorylation of Ser148 and Ser334 of CsRBOH5 caused a more pronounced effect against CBC.Transgenic plants’phosphorylation-mimicking genes had significantly reduced sickness index relative to transgenic plants with unmutated genes.3.The phosphorylation sites Ser148/Ser334 of CsRBOH5 resist CBC by-regulating oxygen species burst.Wanjincheng with transient expression vector including Ser148 or Ser334 sites of CsRBOH5 showed an increase in RBOH enzyme activity and ROS content in the phosphorylation-mimicking group,with the opposite trend in the dephosphorylation-mimicking.In addition,the effects were enhanced when two sites were non-phosphorylated or phosphorylated simultaneously.Ser148 or Ser334 sites of CsRBOH5 with phosphorylation-mimicking overexpression vectors were transformed to Wanjincheng.Phosphorylation-mimicking transgenic plants enhanced RBOH enzyme activity、ROS content and disease resistance to CBC.The results demonstrated the sustained activation of the CsRBOH5 phosphorylation site positively regulates CBC resistance by regulating H₂O₂ homeostasis.4.CsRBOH5 interacts with Cs MAPK6 protein.The interaction between of CsRBOH5 and Cs MAPK6was identified by yeast two-hybrid and bimolecular fluorescence complementation methods.Cs MAPK6 in citrus contains the Serine/Threonine protein kinase domain.Cs MAPK6 was differentially expressed in the disease susceptible variety Wanjincheng and the disease-resistant variety Kumquat.Cs MAPK6 may be a CBC-related gene by regulating the phosphorylation of Ser148/Ser334 of CsRBOH5.In summary,this study confirmed that the phosphorylation of Ser148 and Ser334 at CsRBOH5 has an important function in CBC resistance.This study explored the mechanism of CsRBOH5-mediating resistance to CBC and provided target genes and target site for molecular breeding against CBC.