Jain, Mukesh

Phosphatidylcholine (PtdCho) is an unusual membrane phospholipid present in some endosymbiotic and intracellular pathogenic prokaryotes. ‘ Candidatus Liberibacter asiaticus’ (CLas) is a phloem-limited, uncultured, fastidious α-Proteobacterium associated with the devastating citrus “greening” disease (huanglongbing). Phylogenetically related but nonpathogenic Liberibacter crescens (Lcr) was used as a culturable surrogate to examine PtdCho biosynthesis in pathogenic CLas. Genes encoding key enzymes for two alternative PtdCho biosynthetic routes are present in the Lcr genome: the one-step cytidine diphosphate (CDP)-choline ( pcs-encoding phosphatidylcholine synthase) and the three-step methyl-transferase pathway ( pmt-encoding phospholipid N-methyltransferase). However, only the CDP-choline pathway genes for incorporating exogenous Cho were identified in the CLas genome. Exogenous Cho enhanced growth and alleviated osmotic stress in wild-type Lcr and in the pmt insertion mutant strains when cultured in a sugar-rich medium. Quantitative RT-PCR analyses confirmed active uptake and condensation of nutritional Cho into PtdCho by CLas in both its plant host and psyllid vector. CLas-infected grapefruit leaves showed transcriptional activation of Cho biosynthesis genes and 2.8-fold higher levels of Cho. In plant cells, the compatible osmolyte glycine-betaine (GlyBet) is also derived from Cho. Expression of GlyBet biosynthesis genes and the GlyBet content were similar in both CLas-infected and healthy leaf tissue. The data presented here suggest that CLas likely exploits the Cho biosynthetic pathway in citrus hosts to expand the nutritional Cho pool. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Abstract Objective ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus ‘greening’ disease. All attempts to achieve axenic growth and complete Koch’s postulates with CLas have failed to date, at best yielding complex cocultures with very low CLas titers detectable only by PCR. Reductive genome evolution has rendered all pathogenic ‘Ca. Liberibacter’ spp. deficient in multiple key biosynthetic, metabolic and structural pathways that are highly unlikely to be rescued in vitro by media supplementation alone. By contrast, Liberibacter crescens (Lcr) is axenically cultured and its genome is both syntenic and highly similar to CLas. Our objective is to achieve replicative axenic growth of CLas via addition of missing culturability-related Lcr genes. Results Bioinformatic analyses identified 405 unique ORFs in Lcr but missing (or truncated) in all 24 sequenced CLas strains. Site-directed mutagenesis confirmed and extended published EZ-Tn5 mutagenesis data, allowing elimination of 310 of these 405 genes as nonessential, leaving 95 experimentally validated Lcr genes as essential for CLas growth in axenic culture. Experimental conditions for conjugation of large GFP-expressing plasmids from Escherichia coli to Lcr were successfully established for the first time, providing a practical method for transfer of large groups of ‘essential’ Lcr genes to CLas.

The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic ‘Candidatus Liberibacter’ spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of ‘Ca. L. asiaticus’ bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that ‘Ca. L. asiaticus’ BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. ‘Ca. L. asiaticus’ BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of ‘Ca. L. asiaticus’ acquisition and transmission by fecund psyllids throughout the limited flush period in citrus. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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 β-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.

Liberibacter crescens is a bacterium that is closely related to plant pathogens that have caused billions of dollars in crop losses in recent years. Particularly devastating are citrus losses due to citrus greening disease, also known as Huanglongbing, which is caused by “ Candidatus Liberibacter asiaticus” and carried by the Asian citrus psyllid. L. crescens is the only close relative of “ Ca . Liberibacter asiaticus” that can currently be grown in culture, and it therefore serves as an important model organism for the growth, genetic manipulation, and biological control of the pathogenic species. Here, we show that one of the greatest limitations to L. crescens growth is the sharp increase in alkaline conditions it produces as a consequence of consumption of its preferred nutrient source. In addition to new information about L. crescens growth and metabolism, we provide new guidelines for culture conditions that improve the survival and yield of L. crescens .

‘Candidatus Liberibacter’ spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. ‘Ca. L. asiaticus’, causal agent of Huanglongbing or citrus “greening,” and ‘Ca. L. solanacearum’, causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

In recent decades, ‘Candidatus Liberibacter spp.’ have emerged as a versatile group of psyllid-vectored plant pathogens and endophytes capable of infecting a wide range of economically important plant hosts. The most notable example is ‘Candidatus Liberibacter asiaticus’ (CLas) associated with Huanglongbing (HLB) in several major citrus-producing areas of the world. CLas is a phloem-limited α-proteobacterium that is primarily vectored and transmitted among citrus species by the Asian citrus psyllid (ACP) Diaphorina citri. HLB was first detected in North America in Florida (USA) in 2005, following introduction of the ACP to the State in 1998. HLB rapidly spread to all citrus growing regions of Florida within three years, with severe economic consequences to growers and considerable expense to taxpayers of the state and nation. Inability to establish CLas in culture (except transiently) remains a significant scientific challenge toward effective HLB management. Lack of axenic cultures has restricted functional genomic analyses, transfer of CLas to either insect or plant hosts for fulfillment of Koch’s postulates, characterization of host-pathogen interactions and effective screening of antibacterial compounds. In the last decade, substantial progress has been made toward CLas culturing: (i) three reports of transient CLas cultures were published, (ii) a new species of Liberibacter was identified and axenically cultured from diseased mountain papaya (Liberibacter crescens strain BT-1), (iii) psyllid hemolymph and citrus phloem sap were biochemically characterized, (iv) CLas phages were identified and lytic genes possibly affecting CLas growth were described, and (v) genomic sequences of 15 CLas strains were made available. In addition, development of L. crescens as a surrogate host for functional analyses of CLas genes, has provided valuable insights into CLas pathogenesis and its physiological dependence on the host cell. In this review we summarize the conclusions from these important studies.

The oxidative (H2O2) burst is a seminal feature of the basal plant defense response to attempted pathogen invasions. In ‘Candidatus Liberibacter asiaticus’ UF506, expression of the SC2 prophage-encoded secreted peroxidase (F489_gp15) increases bacterial fitness and delays symptom progression in citrus. Two chromosomal 1-Cys peroxiredoxin genes, CLIBASIA_RS00940 (Lasprx5) and CLIBASIA_RS00445 (Lasbcp), are conserved among all sequenced ‘Ca. L. asiaticus’ strains, including those lacking prophages. Both LasBCP and LasdPrx5 have only a single conserved peroxidatic Cys (CP/SH) and lack the resolving Cys (CR/SH). Lasprx5 appeared to be a housekeeping gene with similar moderate transcript abundance in both ‘Ca. L. asiaticus’–infected psyllids and citrus. By contrast, Lasbcp was expressed only in planta, similar to the expression of the SC2 peroxidase. Since ‘Ca. L. asiaticus’ is uncultured, Lasbcp and Lasprx5 were functionally validated in a cultured surrogate species, Liberibacter crescens, and both genes significantly increased oxidative stress tolerance and cell viability in culture. LasBCP was nonclassically secreted and, in L. crescens, conferred 214-fold more resistance to tert-butyl hydroperoxide (tBOOH) than wild type. Transient overexpression of Lasbcp in tobacco suppressed H2O2-mediated transcriptional activation of RbohB, the key gatekeeper of the systemic plant defense signaling cascade. Lasbcp expression did not interfere with the perception of ‘Ca. L. asiaticus’ flagellin (flg22Las) but interrupted the downstream activation of RbohB and stereotypical deposition of callose in tobacco. Critically, LasBCP also protected against tBOOH-induced peroxidative degradation of lipid membranes in planta, preventing subsequent accumulation of antimicrobial oxylipins that can also trigger the localized hypersensitive cell death response.