Screening And Functional Analysis Of WRKY Transcription Factor And Pathogenesis-related Protein Genes Associated With Citrus Canker

Citrus canker is a bacterial disease caused by Xanthomonas axonopodis pv.citri(Xac)which affects most cultivated varieties,and can result in huge economic losses to citrus industry.Breeding resistance varieties is a fundamental way to solve the problem.Transforming the resistance genes through transgenic technology to citrus is an effective method to battle the citrus disease.WRKY transcription factors and Pathogenesis-related proteins(PRs)play important roles in interaction between pathogen and plants.Based on the transcriptome database of the susceptible variety Newhall navel orange and the resistant variety Calamondin infected by Xac,twenty-four WRKY and four PR genes,which showed significantly different expression in the two varieties during Xac inoculation,were selected to be evaluated by q PCR(quantitative PCR)in this study.Finally,roles of four(Cs WRKY22,Cs WRKY50,Cs WRKY72-1 and Cs WRKY72-2)Cs WRKY and two(Cs PR1-1 and Cs PR1-2)Cs PR-1 from these genes in citrus canker development were further explored.The detailed results as follows:1.Bioinformatics analysis and cloning of the candidate genes.The c DNAs of Cs WRKY22,Cs WRKY50,Cs WRKY72-1,Cs WRKY72-2,Cs PR1-1,and Cs PR1-2 were cloned from Newhall navel orange and their ORFs were 921 bp,480bp,1809 bp,1767bp,501 bp and 480 bp,respectively.The results of phylogenetic analysis showed that the six genes had a close relationship with Tc WRKY29,Zj WRKY50,Tc WRKY72,Tc PR-1-1 and Dl PR-1 family from Theobroma cacao,Ziziphus Jujube,and Dimocarpus longan,respectively.2.Subcellular localization analysis of the Cs WRKY proteins.Using GFP as reporter genes,Subcellular localization characteristics of the four Cs WRKY in onion epidermal cells was investigated using Agrabacteria-mediated method.Fluorescence microscopy observation showed that all the Cs WRKY proteins were located in the nucleus,indicating they are transcription factors.3.Expression charateristics of Cs WRKY genes induced by citrus canker.The expression levels of four Cs WRKY genes in susceptible variety Newhall navel orange and resistant variety Calamondin were analyzed in real time fluorescent quantitative PCR(q PCR)after inoculation of citrus canker.The results showed that the inducible expression of Cs WRKY22 in Newhall navel orange was significantly higher than that in the Calamondin,indicating that it could be involved in the susceptible response of the host,while inducible expression of Cs WRKY50 in Newhall navel orange was obviously lower than that in in the Calamondin,suggesting Cs WRKY50 may be involved in the immune response of Xac resistance.Expression of Cs WRKY72-1was repressed by Xac in both of Newhall navel orange and Calamondin while Expression of Cs WRKY72-2 was repressed by Xac only in Newhall navel orange.This indicated that Xac may inhibit the WRKY72-1 and WRKY72-2-mediated host basal immunity.4.expression charateristics of Cs WRKY genes induced by salicylic acid(SA)and methyl jasmonate(Me JA).To determine whether the Cs WRKY genes is included with SA or JA hormone signal pathway,Expression level of the four Cs WRKY genes in Calamondin was investigated after SA and Me JA treatment.q PCR analysis showed after SA induction,expressions of all the Cs WRKY in Calamondin decreased,but increased in Newhall navel orange though it was not significant.The results showed that SA inhibit the WRKYs' expression in Calamondin,but activated their expression in Newhall.The similar results were observed after treating with MeJA.5.Production of transgenic plants overexpressing candidate genesIn order to evaluate the role of candidate genes in citrus canker development,a seris of p LGNe plant expression vectors containing candidate genes were constructed.All the transgenic vectors were transformed into citrus genome by Agrobacterium tumefaciens.GUS histochemical staining and PCR were used to screen transgenic plants.Finally,nine Cs WRKY22 transgenic plants,five Cs WRKY50 transgenic plants,ten Cs WRKY72-1 transgenic plants,five Cs WRKY72-2 transgenic plants,two Cs PR1-1 transgenic plants and nine Cs PR1-2 transgenic plants were obtained.6.Evaluation of canker resistance of transgenic plants.In vitro citrus canker disease resistance evaluation showed that 3 Cs WRKY22 transgenic plants were significantly susceptible than that of control wild-type orange,while 2 Cs WRKY72-1 transgenic plants,2 Cs PR-1-1 and 1 Cs PR-1-2 transgenic plants were significantly resistant than that of the control plants.The rest of transgenic plants had no significant resistance change compared with the control plants.7.Observation of transgenic plant phenotype.Analysis of the phenotype of transgenic plants showed that transgenic plants overexpressing Cs WRKY22 showed phenotypic difference with other transgenic plants and the control wild-type orange.The transgenic plants overexpressing Cs WRKY22 were short and their leaves were narrow and thick,and were bent to the backside.Among them,the phenotypic changes of the three transgenic plants were the most significant.Our results showed the disease susceptibility of transgenic plants overexpressing Cs WRKY22 were correlated with the phenotypic changes.