Functional Study Of CsrbohA/D In Defense Response Against Xanthomonas Citri Ssp. Citri

Citrus is one of the most important fruit crops in terms of total output and cultivated area.Citrus canker,caused by Xanthomonas citri ssp.citri（Xcc）,is a devastating bacterial disease that infects most cultivated varieties of Rutaceae,and causes huge economic losses to the citrus industry,which seriously restricts the development of the citrus industry.Plasma membrane-bound NADPH oxidases encoded by respiratory burst oxidase homologues（rbohs）,also known as homologues of mammalian gp91^phox（phagocyte oxidase,phox）,are one of the important components of membrane redox system,which transfers electrons from cytoplasmic NADPH to O₂ to generate hydrogen peroxide（H₂O₂）.Disease resistance might be related to H₂O₂ concentration,which is mainly generated by NADPH oxidases during plant defense responses.Plant rbohs constitute a multigene family and are involved in multiple signalling pathways,including responses to abiotic and biotic stress,which have diverse and redundant functions.However,there is limited information in the literature regarding rboh in citrus and their roles in defence response.In the present work,six citrus rbohs（Csrbohs）genes were identified in the resistant genotype kumquat‘Luofu’（Fortunella margarita）and sensitive genotype grapefruit‘Duncan’（Citrus paradisi）,and their possible involvement in resistance to Xanthomonas citri ssp.citri（Xcc）was examined.Inoculation with Xcc promoted the H₂O₂ production and induced expression of the Csrbohs,especially CsrbohA and CsrbohD.Virus-induced gene silencing（VIGS）was used to verify the response of CsrbohA and CsrbohD against Xcc.Then the candidate interactors of CsRBOHA and CsRBOHD were screened and confirmed through DUALmembrane yeast two-hybrid systems and bimolecular fluorescence complementation（BiFC）.The main results were as follows:1、When citrus leaves were infiltrated with Xcc,‘Duncan’grapefruit developed water-soaked lesions 7 days after inoculation（dai）,whereas‘Luofu’kumquat exhibited almost no symptoms.The H₂O₂ content was significantly induced upon Xcc infection.When pretreated with the NADPH oxidase inhibitor DPI（diphenyleneiodonium chloride）,both kumquat and grapefruit exhibited more severe symptoms,and the H₂O₂ content decreased.It indicated that NADPH oxidase are required for the ROS burst induced by Xcc.2、Blastp searches in the C.sinensis database using Arabidopsis RBOH sequences as reference queries resulted in six Csrbohs,which was confirmed by PCR and DNA sequencing in‘Luofu’kumquat and‘Duncan’grapefruit.Thus an expanded family of at least six Csrbohs was found in citrus genome.These six Csrbohs（CsrbohA-E and CsrbohH）,have conserved NADPH oxidase,ferric reductase,FAD-and NAD-binding domains,and EF-hand motifs.The expression pattern of Csrbohs were examined following Xcc inoculation in a time course experiment at 0,1 and 6 h after inoculation（hai）.The expression analysis revealed that,CsrbohA and CsrbohD,were specifically and drastically induced at 6 hai in kumquat,JQSTJ（jinqiushatangju）,Orah and grapefruit.CsrbohD was induced at higher levels in resistant varieties.The correlation analysis showed that the expression of CsrbohD was significantly,but weakly correlated with variety resistance（r_s=0.357,P<0.05）.3、Four vectors were constructed and screened using pTRV,pFGC5941,pCLBV and pCAMBIA2301 in this study,and finally a citrus leaf blotch virus（CLBV）mediated VIGS system was established to silence CsrbohA/D in kumquat and grapefruit.The results showed that CsrbohD-silenced grapefruits displayed significantly reduced disease resistance to Xcc,while CsrbohA-silenced kumquats resulted in enhanced resistance to Xcc.The results from VIGS-based functional analysis indicate that CsrbohA and CsrbohD have opposing effects on disease development in kumquat and grapefruit.4、A DUALmembrane screen was carried out to identify the interactors of CsRBOHA and CsRBOHD.Totally,35 candidates were found and 16 candidates showed putative interaction with CsRBOHA.Among which C.sinensis leucine-rich repeat receptor-like serine/threonine/tryosine-protein kinase SOBIR1,C.sinensis putative disease resistance RPP13-like protein 1,C.sinensis probable serine/theronine-protein kinase At1g54610 and C.sinensis protein pyricularia oryzae resistance 21-like might be involved in disease resistance via bioinformatics analysis.The expression patterns of these four defense response genes against Xcc were examined in CsrbohA-silenced kumquats and grapefruits,respectively.The results showed the defense response genes were up-regulated by Xcc in both CsrbohA-silenced kumquats and grapefruits.Then one of the defense response genes‘SOBIR1’and the‘PTL3’which is the candidate functional parter of both CsRBOHA and CsRBOHD,were choosed for in planta visualization of protein interactions through BiFC.The results showed that SOBIR1 and PTL3 could interact with CsRBOHA/D.