Plant Science

Abstract Huanglongbing (HLB) primarily caused by Candidatus Liberibacter asiaticus (CLas) has been threatening citrus production globally. Under HLB conditions, an excessive accumulation of the polysaccharide callose in citrus phloem occurs, leading to phloem blockage and starch accumulation in leaves. The callose production is controlled by callose synthases (CalS), which have multiple members within plants. However, the knowledge of callose production in the citrus upon infection with CLas is limited. In this study, we firstly identified 11 CalSs in the Citrus sinensis genome through bioinformatics and found the expression pattern of CsCalS11 exhibited a positive correlation with callose deposition in CLas-infected leaves (correlation coefficient of 0.77, P ≤ 0.05). Knockdown of CsCalS11 resulted in a reduction of callose deposition and starch accumulation in CLas-infected citrus. Interestingly, we observed significantly higher concentrations of abscisic acid (ABA) in HLB-infected citrus leaves compared to uninfected ones. Furthermore, the expressions of CsABI5, CsPYR, and CsSnRK2 in the ABA pathway substantially increased in citrus leaves upon CLas infection. Additionally, the expression of CsCalS11 was significantly upregulated in citrus leaves following the application of exogenous ABA. We confirmed that CsABI5, a pivotal component of the ABA signaling pathway, regulates CsCalS11 expression by binding to its promoter using yeast one-hybrid assay, dual luciferase assay, and transient expression in citrus leaves. In conclusion, our findings strongly suggest that the CsABI5-CsCalS11 module plays a crucial role in regulating callose deposition through the ABA signaling pathway during CLas infection. The results also revealed new function of the ABA signaling pathway in plants under biotic stress.

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.

Prophages/phages are important components of the genome of ‘Candidatus Liberibacter asiaticus’ (CLas), an unculturable alphaproteobacterium associated with citrus Huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida, but different to those reported in California and China. Further studies were performed on within prophage type diversity. Analyses on Type 1 specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originated from Florida and Type 1-B, clustered with prophage P-SGCA5-1 detected in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2 specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. Results from this study provides an initial baseline information on genomic and population diversity of CLas in Texas.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas) is considered to be the main pathogen responsible for HLB. There is currently no effective practical strategy for the control of HLB. Our understanding of how pathogens cause HLB is limited because CLas has not been artificially cultured. In this study, 15 potential virulence factors were predicted from the proteome of CLas through DeepVF and PHI-base searches. One among them, FlgI, was found to inhibit yeast growth when expressed in Saccharomyces cerevisiae. The expression of the signal peptide of FlgI fused with PhoA in Escherichia coli resulted in the discovery that FlgI was a novel Sec-dependent secretory protein. We further found that the carboxyl-terminal HA-tagged FlgI was secreted via outer membrane vesicles in Sinorhizobium meliloti. Fluoresence localization of transient expression FlgI-GFP in Nicotiana benthamiana revealed that FlgI is mainly localized in the cytoplasm, cell periphery, and nuclear periphery of tobacco cells. In addition, our experimental results suggest that FlgI has a strong ability to induce callose deposition and cell necrosis in N. benthamiana. Finally, by screening a large library of compounds in a high-throughput format, we found that cyclosporin A restored the growth of FlgI-expressing yeast. These results confirm that FlgI is a novel Sec-dependent effector, enriching our understanding of CLas pathogenicity and helping to develop new and more effective strategies to manage HLB.