Frontiers in Plant Science

The tomato-potato psyllid, Bactericera cockerelli (Šulc), belonging to the Hemiptera order, is an insect pest of solanaceous crops and vectors a fastidious bacterium, Candidatus Liberibacter solanacearum (CLso), the presumptive causal agent of zebra chip and vein greening diseases in potatoes and tomatoes, respectively. The genome of B. cockerelli has been sequenced recently, providing new avenues to elucidate mechanistic insights into pathogenesis in vegetable crops. In this study, we performed RNA-sequencing of the critical psyllid organs (salivary glands and ovaries) involved in CLso pathology and transmission to host plants. Transcriptome analysis revealed differentially expressed genes and organ-specific enrichment of gene ontology (GO) terms related to metabolic processes, response to stress/stimulus, phagocytosis, proteolysis, endocytosis, and provided candidate genes encoding transcription factors (TFs). To examine gene regulatory networks across the psyllid organs under CLso(-) and CLso(+) conditions, we performed weighted gene co-expression network analysis (WGCNA), and unique modules differentiating the psyllid organs were identified. A comparative GO analysis of the unique gene modules revealed functional terms enriched in response to stress, gene regulation, and cell division processes in the ovaries. In contrast, respiration, transport, and neuronal transmission-related GO terms were enriched in the salivary glands. Altogether, this study reveals new insights into tissue-specific expression of the psyllid organs in the absence or presence of CLso bacterium. This knowledge can be leveraged to develop new pest and disease management strategies by delineating the regulatory networks involved in the psyllid-CLso interaction.

IntroductionHuanglongbing (HLB), a disease that’s ubiquitous worldwide, wreaks havoc on the citrus industry. The primary culprit of HLB is the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas) that infects the phloem, but its damaging mechanism is yet to be fully understood.Methods and resultsIn this study, a multitude of tools including weighted correlation network analysis (WGCNA), protein-protein interaction (PPI) network analysis and gene expression profiling are employed to unravel the intricacies of its pathogenesis. The investigation pinpoints various central genes, such as the ethylene-responsive transcription factor 9 (ERF9) and thioredoxin reductase 1 (TrxR1), that are associated with CLas invasion and resultant disturbances in numerous biological operations. Additionally, the study uncovers a range of responses through the detection of differential expressed genes (DEGs) across different experiments. The discovery of core DEGs leads to the identification of pivotal genes such as the sieve element occlusion (SEO) and the wall-associated receptor kinase-like 15 (WAKL15). PPI network analysis highlights potential vital proteins, while GO and KEGG pathway enrichment analysis illustrate a significant impact on multiple defensive and metabolic pathways. Gene set enrichment analysis (GSEA) indicates significant alterations in biological processes such as leaf senescence and response to biotic stimuli.DiscussionThis all-encompassing approach extends valuable understanding into the pathogenesis of CLas, potentially aiding future research and therapeutic strategies for HLB.

IntroductionAsian citrus psyllid (ACP, Diaphorina citri) is an important transmission vector of “Candidatus Liberibacter asiaticus” (CLas), the causal agent of Huanglongbing (HLB), the most destructive citrus disease in the world. As there are currently no HLB-resistant rootstocks or varieties, the control of ACP is an important way to prevent HLB. Some viruses of insect vectors can be used as genetically engineered materials to control insect vectors.MethodsTo gain knowledge on viruses in ACP in China, the prevalence of five RNA and DNA viruses was successfully determined by optimizing reverse transcription polymerase chain reaction (RT-PCR) in individual adult ACPs. The five ACP-associated viruses were identified as follows: diaphorina citri bunyavirus 2, which was newly identified by high-throughput sequencing in our lab, diaphorina citri reovirus (DcRV), diaphorina citri picorna-like virus (DcPLV), diaphorina citri bunyavirus (DcBV), and diaphorina citri densovirus-like virus (DcDV).ResultsDcPLV was the most prevalent and widespread ACP-associated virus, followed by DcBV, and it was detected in more than 50% of all samples tested. DcPLV was also demonstrated to propagate vertically and found more in salivary glands among different tissues. Approximately 60% of all adult insect samples were co-infected with more than one insect pathogen, including the five ACP-associated viruses and CLas.DiscussionThis is the first time these viruses, including the newly identified ACP-associated virus, have been detected in individual adult ACPs from natural populations in China’s five major citrus-producing provinces. These results provide valuable information about the prevalence of ACP-associated viruses in China, some of which have the potential to be used as biocontrol agents. In addition, analysis of the change in prevalence of pathogens in a single insect vector is the basis for understanding the interactions between CLas, ACP, and insect viruses.

IntroductionCandidatus Liberibacter solanacearum (CLso) is a regulated plant pathogen in European and some Asian countries, associated with severe diseases in economically important Apiaceous and Solanaceous crops, including potato, tomato, and carrot. Eleven haplotypes of CLso have been identified based on the difference in rRNA and conserved genes and host and pathogenicity. Although it is pathogenic to a wide range of plants, the mechanisms of plant response and functional decline of host plants are not well defined. This study aims to describe the underlying mechanism of the functional decline of tomato plants infected by CLso by analyzing the transcriptomic response of tomato plants to CLso haplotypes A and B.MethodsNext-generation sequencing (NGS) data were generated from total RNA of tomato plants infected by CLso haplotypes A and B, and uninfected tomato plants, while qPCR analysis was used to validate the in-silico expression analysis. Gene Ontology and KEGG pathways were enriched using differentially expressed genes.ResultsPlants infected with CLso haplotype B saw 229 genes upregulated when compared to uninfected plants, while 1,135 were downregulated. Healthy tomato plants and plants infected by haplotype A had similar expression levels, which is consistent with the fact that CLso haplotype A does not show apparent symptoms in tomato plants. Photosynthesis and starch biosynthesis were impaired while starch amylolysis was promoted in plants infected by CLso haplotype B compared with uninfected plants. The changes in pathway gene expression suggest that carbohydrate consumption in infected plants was more extensive than accumulation. In addition, cell-wall-related genes, including steroid biosynthesis pathways, were downregulated in plants infected with CLso haplotype B suggesting a reduction in membrane fluidity, cell signaling, and defense against bacteria. In addition, genes in phenylpropanoid metabolism and DNA replication were generally suppressed by CLso infection, affecting plant growth and defense.DiscussionThis study provides insights into plants’ defense and functional decline due to pathogenic CLso using whole transcriptome sequencing and qPCR validation. Our results show how tomato plants react in metabolic pathways during the deterioration caused by pathogenic CLso. Understanding the underlying mechanisms can enhance disease control and create opportunities for breeding resistant or tolerant varieties.

The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including ‘Candidiatus Phytoplasma solani’ are unknown. Six putative pathogenicity factors/effectors from six different strains of ‘Ca. P. solani’ were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate–glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate–glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.

Citrus Huanglongbing (HLB) is the most destructive citrus disease worldwide, mainly caused by ‘Candidatus Liberibacter asiaticus’ (CLas). It encodes a large number of Sec-dependent effectors that contribute to HLB progression. In this study, an elicitor triggering ROS burst and cell death in Nicotiana benthamiana, CLIBASIA_04425 (CLas4425), was identified. Of particular interest, its cell death-inducing activity is associated with its subcellular localization and the cytoplasmic receptor Botrytis-induced kinase 1 (BIK1). Compared with CLas infected psyllids, CLas4425 showed higher expression level in planta. The transient expression of CLas4425 in N. benthamiana and its overexpression in Citrus sinensis enhanced plant susceptibility to Pseudomonas syringae pv. tomato DC3000 ΔhopQ1-1 and CLas, respectively. Furthermore, the salicylic acid (SA) level along with the expression of genes NPR1/EDS1/NDR1/PRs in SA signal transduction was repressed in CLas4425 transgenic citrus plants. Taken together, CLas4425 is a virulence factor that promotes CLas proliferation, likely by interfering with SA-mediated plant immunity. The results obtained facilitate our understanding of CLas pathogenesis.

Psyllid yellows, vein-greening (VG), and zebra chip (ZC) diseases, which are primarily transmitted by potato psyllid (PoP) carrying Candidatus Liberibacter solanacearum (CLso), have caused significant losses in solanaceous crop production worldwide. Pathogens interact with their vectors at the organic and cellular levels, while the potential changes that may occur at the biochemical level are less well reported. In this study, the impact of CLso on the metabolism of PoP and the identification of biomarkers from infected psyllids were examined. Using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis, metabolomic changes in CLso-infected psyllids were compared to uninfected ones. A total of 34 metabolites were identified as potential biomarkers of CLso infection, which were primarily related to amino acid, carbohydrate, and lipid metabolism. The significant increase in glycerophospholipids is thought to be associated with CLso evading the insect vector’s immune defense. Matrix-assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) was used to map the spatial distribution of these biomarkers, revealing that 15-keto-Prostaglandin E2 and alpha-D-Glucose were highly expressed in the abdomen of uninfected psyllids but down-regulated in infected psyllids. It is speculated that this down-regulation may be due to CLso evading surveillance by immune suppression in the PoP midgut. Overall, valuable biochemical information was provided, a theoretical basis for a better understanding of psyllid-pathogen interactions was offered, and the findings may aid in breaking the transmission cycle of these diseases.

Citrus Huanglongbing (HLB), caused by “Candidatus Liberibacter asiaticus” (CLas), is the most destructive disease threatening global citrus industry. Most commercial cultivars were susceptible to HLB, although some showed tolerant to HLB phenotypically. Identifying tolerant citrus genotypes and understanding the mechanism correlated with tolerance to HLB is essential for breeding citrus variety tolerance/resistance to HLB. In this study, the graft assay with CLas-infected bud were performed in four citrus genotypes, including Citrus reticulata Blanco, C. sinensis, C. limon, and C. maxima. HLB tolerance was observed in C. limon and C. maxima, while C. Blanco and C. sinensis were susceptible to HLB. The time-course transcriptomic analysis revealed a significant variation in differentially expressed genes (DEGs) related to HLB between susceptible and tolerant cultivar group at early and late infection stage. Functional analysis of DEGs indicated that the activation of genes involved in SA-mediated defense response, PTI, cell wall associated immunity, endochitinase, phenylpropanoid and alpha-linolenic/linoleic lipid metabolism played an important in the tolerance of C. limon and C. maxima to HLB at early infection stage. In addition, the overactive plant defense combined with the stronger antibacterial activity (antibacterial secondary and lipid metabolism) and the suppression of pectinesterase were contributed to the long-term tolerance to HLB in C. limon and C. maxima at late infection stage. Particularly, the activation of ROS scavenging genes (catalases and ascorbate peroxidases) could help to reduce HLB symptoms in tolerant cultivars. In contrast, the overexpression of genes involved in oxidative burst and ethylene metabolism, as well as the late inducing of defense related genes could lead to the early HLB symptom development in susceptible cultivars at early infection stage. The weak defense response and antibacterial secondary metabolism, and the induce of pectinesterase were responsible for sensitivity to HLB in C. reticulata Blanco and C. sinensis at late infection stage. This study provided new insights into the tolerance/sensitivity mechanism against HLB and valuable guidance for breeding of HLB-tolerant/resistant cultivars.

Huanglongbing (HLB) is a devastating disease that affects all commercial citrus species worldwide. The disease is associated with bacteria of three species of the genus ‘Candidatus Liberibacter’ transmitted by psyllid vectors. To date, HLB has no cure, so preventing its introduction into HLB-free areas is the best strategy to control its spread. For that, the use of accurate, sensitive, specific, and reliable detection methods is critical for good integrated management of this serious disease. This study presents a new real-time recombinase polymerase amplification (RPA) protocol able to detect the three ‘Ca. Liberibacter’ species associated with HLB in both plant and insect samples, validated according to European and Mediterranean Plant Protection Organization (EPPO) guidelines and tested on 365 samples from nine different geographic origins. This new protocol does not require nucleic acid purification or specialized equipment, making it ideal to be used under field conditions. It is based on specific primers and probe targeting a region of fusA gene, which shows a specificity of 94%–100%, both in silico and in vitro, for the ‘Ca. Liberibacter’ species associated with HLB. The analytical sensitivity of the new protocol is excellent, with a reliable detection limit in the order of 101 copies per microliter in HLB-infected plant and insect material. The repeatability and reproducibility of the new methods showed consistent results. Diagnostic parameters of the new RPA protocol were calculated and compared with the gold standard technique, a quantitative real-time PCR, in both crude extracts of citrus plants and insect vectors. The agreement between the two techniques was almost perfect according to the estimated Cohen’s kappa index, with a diagnostic sensitivity and specificity of 83.89% and 100%, respectively, and a relative accuracy of 91.59%. Moreover, the results are obtained in less than 35 min. All these results indicate the potential of this new RPA protocol to be implemented as a reliable on-site detection kit for HLB due to its simplicity, speed, and portability.

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.