Transcriptomic And Proteomic Analysis Of Citrus Hystrix Responses To ’Candidatus Liberibacter Asiaticus’ In Early And Late Stage Of Infection

Huanglongbing(HLB)is a century-old disease,spreads rapidly and severely impedes worldwide citrus production.There is no effective treatment so far.The causal agents of HLB are phloem-limited and fastidious α-proteobacteria,including ‘Candidatus Liberibacter asiaticus’(CLas),‘Ca.L.africanus’(CLaf),and ‘Ca.L.americanus’(CLam).The pathogens are transmitted by grafting and insect vector,the citrus psyllid(Diaphorina citri,Trioza erytreae and Cacopsylla citrisuga).All known commercial citrus species and cultivars are susceptible to HLB.In previous study,biological indexing,qPCR monitoring and transcriptome analysis showed that Kaffir lime(Citrus hystrix)is tolerant to HLB.To further decipher the potential tolerance mechanism,cytopathology,transcriptomic and proteomic analysis were performed on HLB-infected C.hystrix in early stages(inoculated after 4 months)and in late stages(inoculated after 14 months).The main results are as follows:1.In order to improve the detection sensitivity,a droplet digital PCR(ddPCR)detection method for CLas was established.This method is accurate,sensitive,and capable of absolute quantification.Its detection sensitivity is 100 times higher than real-time quantitative PCR.After grafting the HLB-infected scion on C.hystrix,it was almost asymptomatic and its microstructure was not significantly different from that of healthy plants on the early or late stage of infection,indicating that C.hystrix has the potential resistance or tolerance to HLB.2.Transcriptome sequencing from HLB-infected C.hystrix in early and late stages showed that there are 181 genes were differently expressed in the early stage and 1384 genes were differentially expressed in the late stage.Of the genes differently expressed in early infection,32 were up regulated and 149 were down regulated.Of the genes differently expressed in late infection,386 were up regulated and 998 were down regulated.Expression trend analysis showed two kinds of significant trends,decrease then increase,and increase then decrease,were enriched.The KEGG enrichment analysis indicated that genes in the phenylpropanoid biosynthesis,monoterpene biosynthesis,starch and sucrose metabolism,plant hormone signal transduction and plant-pathogen interaction pathways were found in significantly enrichment express trends,suggesting that C.hystrix may produce physical or chemical barriers by regulating the level of hormone metabolism,synthesizing secondary metabolites(such as flavonoids,lignin,phenolic compounds,etc.)and disease-resistance related proteins to limit the colonization and further expansion of CLas.In the early stage of infection,the starch synthesis and degradation-related genes were down-regulated synergistically and PP2 protein-related genes were not differently expressed.In the late stage of infection,the genes encoding callose synthase were slightly up regulated and PP2 protein-related genes were still not differently expressed.The expression profiles of 22 genes tested by RT-qPCR are consistent with RNA-seq sequencing date,demonstrating the reliability and accuracy of the sequencing results.3.iTRAQ-based proteomic analysis showed that 378 proteins were differently expressed in the early stage of CLas infection and 488 proteins were differently expressed in the late stage of CLas infection.Among these different expressed proteins,232 were up regulated and 146 were down regulated in early infection,172 were up regulated and 316 were down regulated in late infection.Based on the KEGG pathway enrichment analysis on differently expressed proteins,the major enrichment pathways in the early stage were plant-pathogen interaction,sulfur metabolism,sesquiterpenoid and triterpene biosynthesis,and diterpene biosynthesis.Significantly enriched pathways in the late stage were ubiquitin-mediated proteolysis,phosphatidylinositol signaling system,and phenylpropanoid biosynthesis.The differentially expressed proteins in the early stage were less than those in the late stage.Overall,different expressed proteins showed upregulated trend in early infection and showed downregulated trend in the late stage.There are 56 differential proteins associated with the transcriptome,in which 45 were identical to the gene expression trends.Most of the differential proteins participated in the response of plant resistance,such as phenylalanine ammonia-lyase,glutathione S-transferase,miraculin-like,GDSL esterase,peroxidase,peroxygenase,cellulose synthase,germacrene D synthase,extensin,lectin and so on.