Overexpressing Early Auxin-responsive Gene CsGH3 Enhances Canker Resistance In Citrus

Citrus canker,caused by Xanthomonas axonopodis pv.citri(Xac),is a quarantine disease found worldwide.The disease causes leaf,stem,and fruit lesions and fruit drop.This leads to a decreased citrus fruit production and substantial economic losses.Auxin,an essential plant hormone in regulating plant development,is involved in plant disease susceptibility.Our studying shows that pathogens can manipulate the auxin signal transduction pathway to improve their pathogenicity.Based on the laboratory's transcriptome analysis of susceptible ‘Newhall' Navel Orange(Citrus sinensis Osbeck)and resistant Calamondin(C.madurensis)after infection citrus canker,we found that auxin early-response genes,GH3.1,GH3.6 and GH3.1L,showed significantly increased expression,especially five days after Xac inoculation.Expression levels of the three genes in ‘Newhall' navel orange was significantly higher than that in the Calamondin,indicating that GH3.1,GH3.6 and GH3.1L were closely related to citrus canker resistance.GH3 protein has adenylate-forming enzyme activity.These enzymes catalyze the transfer of IAA into acyl-IAA,which inactivates the free IAA.These datas suggested that GH3.1,GH3.6 and GH3.1L have important role in citrus canker development.In order to study roles of auxin early-response genes CsGH3.1,CsGH3.6 and CsGH3.1 L in citrus canker development,we cloned these genes from ‘Newhall' Navel Orange and analyzed thier expression of characteristics in ‘Newhall' Navel Orange and Calamondin after Xac infection.Moreover,the overexpression and RNAi vectors of the three genes was constructed and delivered into Wangjincheng orange(C.sinensis Osbeck)genome by argarbacteria-mediated transformation and roles of the three genes in citrus canker resistance was further evaluated in transgenic plants:(1)Sequence analysis showed that the length of CsGH3.1,CsGH3.6 and CsGH3.1 L genes in ‘Newhall' Navel Orange is 1797 bp,1887 bp and 1836 bp respectively,and coded 599,629 and 612 amino acids respectively.Homology analysis showed that CsGH3.1,CsGH3.6 and CsGH3.1L in ‘Newhall' Navel Orange shared 100% with that in Clementine(C.clementine).Evolutionary tree analysis showed that CsGH3.1,CsGH3.6,CsGH3.1L protein is closer with homologous protein,AtGH3.1,OsGH3.6 and AtGH3.1 protein,respectively.And their conservative structures has the same domain.GH3.1 and GH3.6 promoter sequences from ‘Newhall' Navel Orange and Calamondin have more than 90% homology,and both of GH3.1 promoters in two varieties have auxin-response element TGA–box.(2)Expression levels of CsGH3.1,CsGH3.6 and CsGH3.1L in ‘Newhall' Navel Orange and Calamondin leaves after inoculation were investigated by real-time fluorescent quantitative PCR(qPCR)method.Results showed that,compared with the induction of 0 days,CsGH3.1,CsGH3.6 and CsGH3.1L in ‘Newhall' Navel Orange are significantly induced by Xac,especially the expression levels in susceptible ‘Newhall' Navel Orange is higher than that in resistant Calamondin varities.The results suggest that CsGH3.1,CsGH3.6 and CsGH3.1L may play an important role in citrus cankerdevelopment.(3)‘Newhall' Navel Orange leaves ere treated with different hormones and inhibitors(10 ?mol· L-1)and then were inoculated with Xac.Disease spot areas in leaves were valuated at 2,3,5 days after infection.Results showed that IAA,IBA and NAA improved canker development,but not significant,while NPA,SA,PP333 significantly suppressed disease development.The results suggest that inhibition of auxin signaling can enhance the resistance of citrus canker.(4)Expression levels of CsGH3.1,CsGH3.6 and CsGH3.1L in ‘Newhall' Navel Orange leaves were detected at 0,6,24,and 72 hours after different hormones and inhibitors(10 ?mol · L-1)treatment.The results showed that CsGH3.1 and CsGH3.1L were up-regulated by IAA,IBA and NAA,but not induced by SA,PP333 and NPA.CsGH3.6 wsa not induced by these hormones and inhibitors before or after treatment.These results suggest that CsGH3.1 and CsGH3.1L may participate in citrus auxin signaling pathways regulation.(5)Transgenic plants overexpressing CsGH3.1,CsGH3.6 or CsGH3.1L displayed changed phenotypes,such as leaf curl upwards,smaller,shallow color,droop and plants withered.Meanwhile,no changed phenotypes were obsreved in RNAi transgenic plants repressing CsGH3.1,CsGH3.6 or CsGH3.1L.(6)Resistance evaluation showed that overexpression of CsGH3.1,CsGH3.6 and CsGH3.1L significantly enhanced plant resistance to citrus canker compared with wild type,while repression of CsGH3.1,CsGH3.6 or CsGH3.1L has no obvious effect on plant resistance to citrus canker.The above data indicated that CsGH3.1,CsGH3.6 and CsGH3.1L is involved in citrus canker development,and overexpressing CsGH3.1,CsGH3.6 and CsGH3.1L enhances plant resistance to citrus canker through repressing auxin accumulation during pathogen infection.