Biological And Transcriptional Analyses Of Wild Citrus Responses To 'Candidatus Liberibacter Asiaticus' Infection

Citrus Huanglongbing(HLB),is one of the most devastating citrus diseases in the world.It can affect almost all commercial citrus cultivars and no effective chemicals are currently available towards the pathogen inside trees.Currently,three species of‘Candidatus Liberibacter',‘Ca.Liberibacter asiaticus'(Las),‘Ca.Liberibacter africanus'(Laf),and ‘Ca.Liberibacter americanus'(Lam),are associated with HLB based on 16 S rDNA sequence.Among these three species,Las is widely distributed around the world.Information about resistance genes against HLB is currently limited,inhibiting the progress of HLB resistance breeding.Previous studies showed that Citrus hystrix,a wild citrus variety,was tolerant to HLB.However,its defense mechanism remains to be determined.As an important original center of citrus,China is rich in resources of wild citrus species,but lack of systematic studies focused on HLB susceptibility or resistance of these wild citrus.In this study,biological indexing,Las titer monitoring and RNA-seq analysis of three wild citrus species,C.hystrix,C.ichangensis and C.daoxianensis,and susceptible C.sinensis were performed to identitfy the sources of HLB resistance in wild citrus and decipher their potential resistance mechanism.This study provided new insights into the host response of HLB-tolerant wild citrus species to Las.It is useful for HLB-tolerant/resistant citrus breeding in the future.The main results are as follows:1.Three wild citrus species and a sweet orange addressed in this thesis were confirmed to be infected with Las at ca.90 dpi(days post inoculation).The titer of Las in C.sinensis was much higher than those in C.ichangensis and C.daoxianensis since ca.120 dpi,which indicated that Las reproduced faster in C.sinensis than in wildcitrus species.No typical symptom was observed on C.hystrix and C.ichangensis,whereas mild yellowing and severe yellowing symptoms were found from C.daoxianensis and C.sinensis,respectively.It suggested that C.hystrix and C.ichangensis were tolerant to HLB.2.Gene expression profiles of HLB-tolerant C.hystrix and HLB-susceptible C.sinensis infected with Las were examined using RNA-seq.A total of 252 and 604 genes were differentially regulated by HLB infection in C.hystrix and C.sinensis,respectively.Among them,179 genes were up-expressed and 73 were down-expressed in C.hystrix,254 genes were up-expressed and 350 were down-expressed in C.sinensis,respectively.Of the 4651 genes differentially expressed in C.hystrix compared to C.sinensis,2137 genes were up-expressed and 2514 were down-expressed.Biological pathways including carbohydrates metabolism,cell wall metabolism,secondary metabolism,plant hormone signaling,pathogenesis related(PR)-proteins,and redox state were significantly changed by Las infection.Notably,two genes encoding starch synthase(glgA)were significantly up-regulated,four times higher in C.sinensis than in C.hystrix.The beta-amylase(BAM)gene associated with starch degradation was down-regulated in C.sinensis,whereas the gene was not differentially expressed in C.hystrix.Several genes involved in light reaction of photosynthesis were down-regulated in C.sinensis but not differentially expressed in C.hystrix.Most of the differentially expressed genes(DEGs)involved in secondary metabolism were up-regulated in C.hystrix,while most of them were down-regulated in C.sinensis.In addition,several PR-proteins related genes were induced in the two species,and some genes related to disease resistance proteins exhibited higher expression level in C.hystrix.Of the selected 20 DEGs invloved in different pathways,the expression profiles of 18 genes tested by RT-qPCR were consistent with the RNA-Seq data,demonstrating the reliability of RNA-seq analysis.3.Compatative transcriptome approach was applied to analyze the response of C.ichangensis,C.daoxianensis and C.sinensis to Las at late stage(ca.240 dpi).The total of 592,987,and 2844 DEGs were indetified in C.ichangensis,C.daoxianensis and C.sinensis,respectively.The number of DEGs in the three citrus species reflected the the severe degree of disease symptoms caused by the pathogen.In the late stage of Las infection,genes related to starch synthesis and PP2 proteins were up-regulated in C.daoxianensis and C.sinensis,and no significant change in C.ichangensis.It may explain the no typical symptom of C.ichangensis infected with Las.Besides,secondary metabolism pathways were activited in all three species.Genes related to cellulose synthesis were up-regulated in C.ichangensis and C.daoxianensis,while those genes were down-regulated in C.sinensis.A large number of PR-proteins related genes were repressed in C.sinensis,but not in C.ichangensis and C.daoxianensis.Some genes related to disease resistance proteins exhibited higher expression level in the two wild citrus species than those in C.sinensis.4.Different wild citrus species,especially C.hystrix and C.ichangensis,showed somewhat similarities in response to Las.Most of DEGs involved in pathways related to plant defense were up-regulated in all four citrus species.However,some genes within plant defence pathways exhibited higher expression level in three wild citrus species than those in C.sinensis.In addition,genes related to starch synthesis and PP2 proteins were up-regulated in C.daoxianensis and C.sinensis,whereas these genes were not induced in C.hystrix and C.ichangensis.Overall,biological indexing and transcriptome analsis partially explained the HLB-tolerant mechanism of the two wild citrus species(C.hystrix and C.ichangensis).