Long non-coding RNAs (lncRNAs) serve as crucial regulators in plant response to various diseases, while none have been systematically identified and characterized in response to citrus Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) bacteria. Here, we comprehensively investigated the transcriptional and regulatory dynamics of the lncRNAs in response to CLas. Samples were collected from leaf midribs of CLas- and mock-inoculated HLB-tolerant rough lemon (Citrus jambhiri) and HLB-sensitive sweet orange (C. sinensis) at week 0, 7, 17, and 34 following inoculation using CLas+ budwood of three biological replicates in the greenhouse. A total of 8,742 lncRNAs, including 2,529 novel lncRNAs, were identified from RNA-seq data with rRNA-removed from strand-specific libraries. Genomic variation analyses of conserved lncRNAs from 38 citrus accessions showed that 26 single nucleotide polymorphisms (SNPs) were significantly correlated with HLB. In addition, lncRNA-mRNA weighted gene co-expression network analysis (WGCNA) showed a significant module correlated with CLas-inoculation in rough lemon. Notably, the most significant LNC_28805 and multiple co-expressed genes related to plant defense in the module were targeted by miRNA5021, suggesting that LNC28805 might compete with endogenous miR5021 to maintain the homeostasis of immune gene expression levels. Candidate WRKY33 and SYP121 genes targeted by miRNA5021 were identified as two key hub genes interacting with bacteria pathogen response genes based on the prediction of protein-protein interaction (PPI) network. These two genes were also found within HLB-associated QTL in linkage group 6. Overall, our findings provide a reference for a better understanding of the role of lncRNAs involved in citrus HLB regulation.
Citrus Huanglongbing (HLB) is the most devastating disease of citrus, presumably caused by “Candidatus Liberibacter asiaticus” (CaLas). Although transcriptomic profiling of HLB-affected citrus plants has been studied extensively, the initial steps in pathogenesis have not been fully understood. In this study, RNA sequencing (RNA-seq) was used to compare very early transcriptional changes in the response of Valencia sweet orange (VAL) to CaLas after being fed by the vector, Diaphorina citri (Asian citrus psyllid, or ACP). The results suggest the existence of a delayed defense reaction against the infective vector in VAL, while the attack by the healthy vector prompted immediate and substantial transcriptomic changes that led to the rapid erection of active defenses. Moreover, in the presence of CaLas-infected psyllids, several downregulated differentially expressed genes (DEGs) were identified on the pathways, such as signaling, transcription factor, hormone, defense, and photosynthesis-related pathways at 1 day post-infestation (dpi). Surprisingly, a burst of DEGs (6,055) was detected at 5 dpi, including both upregulated and downregulated DEGs on the defense-related and secondary metabolic pathways, and severely downregulated DEGs on the photosynthesis-related pathways. Very interestingly, a significant number of those downregulated DEGs required ATP binding for the activation of phosphate as substrate; meanwhile, abundant highly upregulated DEGs were detected on the ATP biosynthetic and glycolytic pathways. These findings highlight the energy requirement of CaLas virulence processes. The emerging picture is that CaLas not only employs virulence strategies to subvert the host cell immunity, but the fast-replicating CaLas also actively rewires host cellular metabolic pathways to obtain the necessary energy and molecular building blocks to support virulence and the replication process. Taken together, the very early response of citrus to the CaLas, vectored by infective ACP, was evaluated for the first time, thus allowing the changes in gene expression relating to the primary mechanisms of susceptibility and host–pathogen interactions to be studied, and without the secondary effects caused by the development of complex whole plant symptoms.
Although there are no known sources of genetic resistance, some Citrus spp. are reportedly tolerant to huanglongbing (HLB), presumably caused by ‘Candidatus Liberibacter asiaticus’. Time-course transcriptional analysis of tolerant rough lemon (Citrus jambhiri) and susceptible sweet orange (C. sinensis) in response to ‘Ca. L. asiaticus’ infection showed more genes differentially expressed in HLB-affected rough lemon than sweet orange at early stages but substantially fewer at late time points, possibly a critical factor underlying differences in sensitivity to ‘Ca. L. asiaticus’. Pathway analysis revealed that stress responses were distinctively modulated in rough lemon and sweet orange. Although microscopic changes (e.g., callose deposition in sieve elements and phloem cell collapse) were found in both infected species, remarkably, phloem transport activity in midribs of source leaves in rough lemon was much less affected by HLB than in sweet orange. The difference in phloem cell transport activities is also implicated in the differential sensitivity to HLB between the two species. The results potentially lead to identification of key genes and the genetic mechanism in rough lemon to restrain disease development and maintain (or recover) phloem transport activity. These potential candidate genes may be used for improving citrus tolerance (or even resistance) to HLB by genetic engineering.