Mandadi, Kranthi K.

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.

Citrus greening, also known as Huanglongbing (HLB), is caused by the unculturable bacterium Candidatus Liberibacter spp. (e.g., CLas), and has caused a devastating decline in citrus production in many areas of the world. As of yet, there are no definitive treatments for controlling the disease. Antimicrobial peptides (AMPs) that have the potential to block secretion-dependent effector proteins at the outer-membrane domains were screened in silico. Predictions of drug-receptor interactions were built using multiple in silico techniques, including molecular docking analysis, molecular dynamics, molecular mechanics generalized Born surface area analysis, and principal component analysis. The efflux pump TolC of the Type 1 secretion system interacted with natural bacteriocin plantaricin JLA-9, blocking the β barrel. The trajectory-based principal component analysis revealed the possible binding mechanism of the peptides. Furthermore, in vitro assays using two closely related culturable surrogates of CLas (Liberibacter crescens and Rhizobium spp.) showed that Plantaricin JLA-9 and two other screened AMPs inhibited bacterial growth and caused mortality. The findings contribute to designing effective therapies to manage plant diseases associated with Candidatus Liberibacter spp.

AbstractA major bottleneck in identifying therapies to control citrus greening and other devastating plant diseases caused by fastidious pathogens is our inability to culture the pathogens in defined media or axenic cultures. As such, conventional approaches for antimicrobial evaluation (genetic or chemical) rely on time-consuming, low-throughput and inherently variable whole-plant assays. Here, we report that plant hairy roots support the growth of fastidious pathogens like Candidatus Liberibacter spp., the presumptive causal agents of citrus greening, potato zebra chip and tomato vein greening diseases. Importantly, we leverage the microbial hairy roots for rapid, reproducible efficacy screening of multiple therapies. We identify six antimicrobial peptides, two plant immune regulators and eight chemicals which inhibit Candidatus Liberibacter spp. in plant tissues. The antimicrobials, either singly or in combination, can be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.

AbstractCandidatus Liberibacter asiaticus (CLas) has been associated with Huanglongbing, a lethal vector-borne disease affecting citrus crops worldwide. While comparative genomics has provided preliminary insights into the metabolic capabilities of this uncultured microorganism, a comprehensive functional characterization is currently lacking. Here, we reconstructed and manually curated genome-scale metabolic models for the six CLas strains A4, FL17, gxpsy, Ishi-1, psy62, and YCPsy, in addition to a model of the closest related culturable microorganism, L. crescens BT-1. Predictions about nutrient requirements and changes in growth phenotypes of CLas were confirmed using in vitro hairy root-based assays, while the L. crescens BT-1 model was validated using cultivation assays. Host-dependent metabolic phenotypes were revealed using expression data obtained from CLas-infected citrus trees and from the CLas-harboring psyllid Diaphorina citri Kuwayama. These results identified conserved and unique metabolic traits, as well as strain-specific interactions between CLas and its hosts, laying the foundation for the development of model-driven Huanglongbing management strategies.