Structure of Lipopolysaccharide from Liberibacter crescens Is Low Molecular Weight and Offers Insight into Candidatus Liberibacter Biology.

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium (Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a ß-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

HPE1, an Effector from Zebra Chip Pathogen Interacts with Tomato Proteins and Perturbs Ubiquitinated Protein Accumulation.

The gram-negative bacterial genus Liberibacter includes economically important pathogens, such as 'Candidatus Liberibacter asiaticus' that cause citrus greening disease (or Huanglongbing, HLB) and 'Ca. Liberibacter solanacearum' (Lso) that cause zebra chip disease in potato. Liberibacter pathogens are fastidious bacteria transmitted by psyllids. Pathogen manipulation of the host' and vector's immune system for successful colonization is hypothesized to be achieved by Sec translocon-dependent effectors (SDE). In previous work, we identified hypothetical protein effector 1 (HPE1), an SDE from Lso, that acts as a suppressor of the plant's effector-triggered immunity (ETI)-like response. In this study, using a yeast two-hybrid system, we identify binding interactions between tomato RAD23 proteins and HPE1. We further show that HPE1 interacts with RAD23 in both nuclear and cytoplasmic compartments in planta. Immunoblot assays show that HPE1 is not ubiquitinated in the plant cell, but rather the expression of HPE1 induced the accumulation of other ubiquitinated proteins. A similar accumulation of ubiquitinated proteins is also observed in Lso infected tomato plants. Finally, earlier colonization and symptom development following Lso haplotype B infection are observed in HPE1 overexpressing plants compared to wild-type plants. Overall, our results suggest that HPE1 plays a role in virulence in Lso pathogenesis, possibly by perturbing the ubiquitin-proteasome system via direct interaction with the ubiquitin-like domain of RAD23 proteins.

Discovery of novel SecA inhibitors against "Candidatus Liberibacter asiaticus" through virtual screening and biological evaluation.

"Candidatus Liberibacter asiaticus" (Ca. L. asiaticus) is the causal agent of Huanglongbing disease of citrus and current study focuses on the discovery of novel small-molecule inhibitors against SecA protein of Ca. L. asiaticus. In this study, homologous modeling was used to construct the three-dimensional structure of SecA. Then, molecular docking-based virtual screening and two rounds of in vitro bacteriostatic experiments were utilized to identify novel small-molecule inhibitors of SecA. Encouragingly, 93 compounds were obtained and two of them (P684-2850, P684-3808) showed strong antimicrobial activities against Liberibacter crescens BT-1 in bacteriostatic experiments. Finally, molecular dynamics simulations were employed to explore the binding modes of the receptor-ligand complexes. Results in MD simulations showed that compound P684-3808 was relatively stable during simulation, while compound P684-2850 left the binding pocket. Compound P684-3808 might be suitable as a lead compound for further development of antimicrobial compounds against SecA of Ca. L. asiaticus.

'Candidatus Liberibacter Asiaticus' SDE1 Effector Induces Huanglongbing Chlorosis by Downregulating Host DDX3 Gene.

'Candidatus Liberibacter asiaticus' (CLas) is the pathogenic bacterium that causes the disease Huanglongbing (HLB) in citrus and some model plants, such as Nicotiana benthamiana. After infection, CLas releases a set of effectors to modulate host responses. One of these critical effectors is Sec-delivered effector 1 (SDE1), which induces chlorosis and cell death in N. benthamiana. In this study, we revealed the DEAD-box RNA helicase (DDX3) interacts with SDE1. Gene silencing study revealed that knockdown of the NbDDX3 gene triggers leaf chlorosis, mimicking the primary symptom of CLas infection in N. benthamiana. The interactions between SDE1 and NbDDX3 were localized in the cell membrane. Overexpression of SDE1 resulted in suppression of NbDDX3 gene expression in N. benthamiana, which suggests a critical role of SDE1 in modulating NbDDX3 expression. Furthermore, we verified the interaction of SDE1 with citrus DDX3 (CsDDX3), and demonstrated that the expression of the CsDDX3 gene was significantly reduced in HLB-affected yellowing and mottled leaves of citrus. Thus, we provide molecular evidence that the downregulation of the host DDX3 gene is a crucial mechanism of leaf chlorosis in HLB-affected plants. The identification of CsDDX3 as a critical target of SDE1 and its association with HLB symptom development indicates that the DDX3 gene is an important target for gene editing, to interrupt the interaction between DDX3 and SDE1, and therefore interfere host susceptibility.

Sec-Delivered Effector 1 (SDE1) of 'Candidatus Liberibacter asiaticus' Promotes Citrus Huanglongbing.

Sec-delivered effector 1 (SDE1) from the huanglongbing (HLB)-associated bacterium 'Candidatus Liberibacter asiaticus' was previously characterized as an inhibitor of defense-related, papain-like cysteine proteases in vitro and in planta. Here, we investigated the contributions of SDE1 to HLB progression. We found that SDE1 expression in the model plant Arabidopsis thaliana caused severe yellowing in mature leaves, reminiscent of both 'Ca. L. asiaticus' infection symptoms and accelerated leaf senescence. Induction of senescence signatures was also observed in the SDE1-expressing A. thaliana lines. These signatures were apparent in older leaves but not in seedlings, suggesting an age-associated effect. Furthermore, independent lines of transgenic Citrus paradisi (L.) Macfadyen (Duncan grapefruit) that express SDE1 exhibited hypersusceptibility to 'Ca. L. asiaticus'. Similar to A. thaliana, transgenic citrus expressing SDE1 showed altered expression of senescence-associated genes, but only after infection with 'Ca. L. asiaticus'. These findings suggest that SDE1 is a virulence factor that contributes to HLB progression, likely by inducing premature or accelerated senescence in citrus. This work provides new insight into HLB pathogenesis.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Linking metabolic phenotypes to pathogenic traits among "Candidatus Liberibacter asiaticus" and its hosts.

Candidatus 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.

Characterization of the heavy metal binding properties of periplasmic metal uptake protein CLas-ZnuA2.

Candidatus Liberibacter asiaticus (CLas), a phloem-limited unculturable Gram-negative bacterium, causes citrus greening disease. The proteome analysis of CLas showed the presence of a heavy metal permease and Co/Zn/Cd cation exporter system. However, there is no designated metal uptake protein specific for the heavy metal permease in CLas. One of the metal uptake proteins, designated as CLas-ZnuA2, in our previous studies, showed a lower metal-binding affinity for Mn2+ and Zn2+ and was postulated to bind and transport metals rather non-specifically. The present study focused on the characterization of the heavy metal binding properties of CLas-ZnuA2 using SPR, CD, DSC and crystallographic studies. The crystal structure analysis of Cd2+ bound CLas-ZnuA2 showed octahedral geometry for Cd2+ binding as compared to a non-preferred square-pyramidal geometry for Mn2+ and Zn2+ binding in earlier reported crystal structures. In SPR analysis, the binding affinities of 4.7 × 10-6 M, 7.2 × 10-6 M, 5.3 × 10-5 M and 4.3 × 10-5 M for Hg2+, Cd2+, Ba2+ and Co2+ respectively were higher as compared to earlier reported values for Mn2+ and Zn2+. Likewise, CD and DSC analysis showed relatively higher thermal stability for CLas-ZnuA2 on heavy metal binding. Taken together with the expression of the permease and exporter system for heavy metals, our results indicate that CLas-ZnuA2 may be involved in sequestering and transport of various transition divalent metals in environmentally stressed conditions.

Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells.

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.