Salicylic Acid (SA) is a pivotal phytohormone in plant innate immunity enhancement of triggered by various pathogens, such as Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). WRKY is a plant specific transcription factor (TF) family, which plays crucial roles in plant response to biotic stresses. So far, the evolutionary history, functions, and expression patterns under SA treatment and CLas infection of WRKY family are poorly understood in Citrus, despite the release of the genome of several Citrus species. A comprehensive genomic and expressional analysis is worth to conduct for this family.
Here, a genome-wide identification of WRKY TFs was performed in two Citrus species: Citrus sinensis (HLB-sensitive) and Poncirus trifoliata (HLB-tolerant). In total, 52 CsWRKYs and 51 PtrWRKYs were identified, whose physical and chemical properties, chromosome locations, phylogenetic relationships and structural characteristics were comparatively analyzed. Especially, expression patterns of these WRKY genes before and after SA treatment and CLas infection were compared. Based on this result, seven pairs of orthologous WRKY genes showing opposite expression patterns in two Citrus species were screened out. Moreover, two pairs of orthologous WRKY genes with significant differences in the number or type of stress-responsive cis-elements in the promoter regions were discovered. Subcellular localization and transcriptional activation activity assays revealed that these two pairs of orthologous genes are classic WRKY TFs localize in the nucleus and could function as transcriptional activators.
In this study, we systematically analyzed the genomic characterization of WRKY family in two Citrus species, together with the analyses of expression patterns under SA signaling and CLas infection. Our study laid a foundation for further study on the function of WRKY TFs in HLB response and SA signaling of Citrus.
Candidatus Liberibacter asiaticus (CLas) is the causative agent of Huanglongbing (HLB), which has caused great economic losses to the citrus industry. The molecular mechanism of the host response to CLas in wild citrus germplasm has been reported less. Eighteen weeks after inoculation via grafting, all the CLas-inoculated Chongyi wild mandarin (Citrus reticulata) were positive and showed severe anatomical aberrations, suggesting its susceptibility to HLB. Transcriptomics and metabolomics analyses of leaves, barks, and roots from mock-inoculated (control) and CLas-inoculated seedlings were performed. Comparative transcriptomics identified 3,628, 3,770, and 1,716 differentially expressed genes (DEGs) between CLas-infected and healthy tissues in the leaves, barks, and roots, respectively. The CLas-infected tissues had higher transcripts per kilobase per million values and more genes that reached their maximal expression, suggesting that HLB might cause an overall increase in transcript accumulation. However, HLB-triggered transcriptional alteration showed tissue specificity. In the CLas-infected leaves, many DEGs encoding immune receptors were downregulated. In the CLas-infected barks, nearly all the DEGs involved in signaling and plant-pathogen interaction were upregulated. In the CLas-infected roots, DEGs encoding enzymes or transporters involved in carotenoid biosynthesis and nitrogen metabolism were downregulated. Metabolomics identified 71, 62, and 50 differentially accumulated metabolites (DAMs) in the CLas-infected leaves, barks and roots, respectively. By associating DEGs with DAMs, nitrogen metabolism was the only pathway shared by the three infected tissues and was depressed in the CLas-infected roots. In addition, 26 genes were determined as putative markers of CLas infection, and a hypothesized model for the HLB susceptibility mechanism in Chongyi was proposed. Our study may shed light on investigating the molecular mechanism of the host response to CLas infection in wild citrus germplasm.