Lectins and polysaccharide EPS I have flow-responsive roles in the attachment and biofilm mechanics of plant pathogenic Ralstonia.

Bacterial biofilm formation and attachment to hosts are mediated by carbohydrate-binding lectins, exopolysaccharides, and their interactions in the extracellular matrix (ECM). During tomato infection Ralstonia pseudosolanacearum (Rps) GMI1000 highly expresses three lectins: LecM, LecF, and LecX. The latter two are uncharacterized. We evaluated the roles in bacterial wilt disease of LecF, a fucose-binding lectin, LecX, a xylose-binding lectin, and the Rps exopolysaccharide EPS I. Interestingly, single and double lectin mutants attached to tomato roots better and formed more biofilm under static conditions in vitro. Consistent with this finding, static bacterial aggregation was suppressed by heterologous expression of lecFGMI1000 and lecXGMI1000 in other Ralstonia strains that naturally lack these lectins. Crude ECM from a ΔlecF/X double mutant was more adhesive than the wild-type ECM, and LecF and LecX increased Rps attachment to ECM. The enhanced adhesiveness of the ΔlecF/X ECM could explain the double mutant's hyper-attachment in static conditions. Unexpectedly, mutating lectins decreased Rps attachment and biofilm viscosity under shear stress, which this pathogen experiences in plant xylem. LecF, LecX, and EPS I were all essential for biofilm development in xylem fluid flowing through cellulose-coated microfluidic channels. These results suggest that under shear stress, LecF and LecX increase Rps attachment by interacting with the ECM and plant cell wall components like cellulose. In static conditions such as on root surfaces and in clogged xylem vessels, the same lectins suppress attachment to facilitate pathogen dispersal. Thus, Rps lectins have a dual biological function that depends on the physical environment.

Nitric Oxide Regulates the Ralstonia solanacearum Type III Secretion System.

Ralstonia solancearum causes bacterial wilt disease on diverse plant hosts. R. solanacearum cells enter a host from soil or infested water through the roots, then multiply and spread in the water-transporting xylem vessels. Despite the low nutrient content of xylem sap, R. solanacearum grows very well inside the host, using denitrification to respire in this hypoxic environment. R. solanacearum growth in planta also depends on the successful deployment of protein effectors into host cells via a type III secretion system (T3SS). The T3SS is absolutely required for R. solanacearum virulence, but it is metabolically costly and can trigger host defenses. Thus, the pathogen's success depends on optimized regulation of the T3SS. We found that a byproduct of denitrification, the toxic free-radical nitric oxide (NO), positively regulates the R. solanacearum T3SS both in vitro and in planta. Using chemical treatments and R. solanacearum mutants with altered NO levels, we show that the expression of a key T3SS regulator, hrpB, is induced by NO in culture. Analyzing the transcriptome of R. solanacearum responding to varying levels of NO both in culture and in planta revealed that the T3SS and effectors were broadly upregulated with increasing levels of NO. This regulation was specific to the T3SS and was not shared by other stressors. Our results suggest that R. solanacearum may experience an NO-rich environment in the plant host and that this NO contributes to the activation of the T3SS during infection. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Molecular signatures between citrus and Candidatus Liberibacter asiaticus.

Citrus Huanglongbing (HLB), also known as citrus greening, is one of the most devastating citrus diseases worldwide. Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain associated with HLB, which is yet to be cultured in vitro. None of the commercial citrus cultivars are resistant to HLB. The pathosystem of Ca. Liberibacter is complex and remains a mystery. In this review, we focus on the recent progress in genomic research on the pathogen, the interaction of host and CLas, and the influence of CLas infection on the transcripts, proteins, and metabolism of the host. We have also focused on the identification of candidate genes for CLas pathogenicity or the improvements of HLB tolerance in citrus. In the end, we propose potentially promising areas for mechanistic studies of CLas pathogenicity, defense regulators, and genetic improvement for HLB tolerance/resistance in the future.

Tomato deploys defence and growth simultaneously to resist bacterial wilt disease.

Plant disease limits crop production, and host genetic resistance is a major means of control. Plant pathogenic Ralstonia causes bacterial wilt disease and is best controlled with resistant varieties. Tomato wilt resistance is multigenic, yet the mechanisms of resistance remain largely unknown. We combined metaRNAseq analysis and functional experiments to identify core Ralstonia-responsive genes and the corresponding biological mechanisms in wilt-resistant and wilt-susceptible tomatoes. While trade-offs between growth and defence are common in plants, wilt-resistant plants activated both defence responses and growth processes. Measurements of innate immunity and growth, including reactive oxygen species production and root system growth, respectively, validated that resistant plants executed defence-related processes at the same time they increased root growth. In contrast, in wilt-susceptible plants roots senesced and root surface area declined following Ralstonia inoculation. Wilt-resistant plants repressed genes predicted to negatively regulate water stress tolerance, while susceptible plants repressed genes predicted to promote water stress tolerance. Our results suggest that wilt-resistant plants can simultaneously promote growth and defence by investing in resources that act in both processes. Infected susceptible plants activate defences, but fail to grow and so succumb to Ralstonia, likely because they cannot tolerate the water stress induced by vascular wilt.

Seasonal Transcriptome Profiling of Susceptible and Tolerant Citrus Cultivars to Citrus Huanglongbing.

Citrus huanglongbing (HLB) caused by 'Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. Most commercial citrus cultivars are susceptible to HLB, with a few more tolerant exceptions such as 'LB8-9' Sugar Belle mandarin. Transcriptomic analyses have been widely used to investigate the potential mechanisms for disease susceptibility, resistance, or tolerance. Previous transcriptomic studies related to HLB mostly focused on single time point data collection. We hypothesize that changes in day length and temperature throughout the seasons have profound effects on citrus-CLas interactions. Here, we conducted RNA-seq analyses on HLB-susceptible Valencia sweet orange and HLB-tolerant mandarin 'LB8-9' in winter, spring, summer, and fall. Significant variations in differentially expressed genes (DEGs) related to HLB were observed among the four seasons. For both cultivars, the highest number of DEGs were found in the spring. CLas infection stimulates the expression of immune-related genes such as NBS-LRR, RLK, RLCK, CDPK, MAPK pathway, reactive oxygen species (ROS), and PR genes in both cultivars, consistent with the model that HLB is a pathogen-triggered immune disease. HLB-positive mandarin 'LB8-9' trees contained higher concentrations of maltose and sucrose, which are known to scavenge ROS. In addition, mandarin 'LB8-9' showed higher expression of genes involved in phloem regeneration, which might contribute to its HLB tolerance. This study shed light on the pathogenicity mechanism of the HLB pathosystem and the tolerance mechanism against HLB, providing valuable insights into HLB management.

Candidatus Liberibacter: From Movement, Host Responses, to Symptom Development of Citrus Huanglongbing.

Candidatus Liberibacter spp. are fastidious α-proteobacteria that cause multiple diseases on plant hosts of economic importance, including the most devastating citrus disease: Huanglongbing (HLB). HLB was reported in Asia a century ago but has since spread worldwide. Understanding the pathogenesis of Candidatus Liberibacter spp. remains challenging as they are yet to be cultured in artificial media and infect the phloem, a sophisticated environment that is difficult to manipulate. Despite those challenges, tremendous progress has been made on Ca. Liberibacter pathosystems. Here, we first reviewed recent studies on genetic information of flagellar and type IV pili biosynthesis, their expression profiles, and movement of Ca. Liberibacter spp. inside the plant and insect hosts. Next, we reviewed the transcriptomic, proteomic, and metabolomic studies of susceptible and tolerant plant genotypes to Ca. Liberibacter spp. infection and how Ca. Liberibacter spp. adapt in plants. Analyses of the interactions between plants and Ca. Liberibacter spp. imply the involvement of immune response in the Ca. Liberibacter pathosystems. Lastly, we reviewed how Ca. Liberibacter spp. movement inside and interactions with plants lead to symptom development.

Ralstonia solanacearum Depends on Catabolism of Myo-Inositol, Sucrose, and Trehalose for Virulence in an Infection Stage-Dependent Manner.

The soilborne pathogen Ralstonia solanacearum causes a lethal bacterial wilt disease of tomato and many other crops by infecting host roots, then colonizing the water-transporting xylem vessels. Tomato xylem sap is nutritionally limiting but it does contain some carbon sources, including sucrose, trehalose, and myo-inositol. Transcriptomic analyses revealed that R. solanacearum expresses distinct catabolic pathways at low cell density (LCD) and high cell density (HCD). To investigate the links between bacterial catabolism, infection stage, and virulence, we measured in planta fitness of bacterial mutants lacking specific carbon catabolic pathways expressed at either LCD or HCD. We hypothesized that early in disease, during root infection, the bacterium depends on carbon sources catabolized at LCD, while HCD carbon sources are only required later in disease during stem colonization. A R. solanacearum ΔiolG mutant unable to use the LCD-catabolized nutrient myo-inositol was defective in tomato root colonization, but after it reached the stem this strain colonized and caused symptoms as well as wild type. In contrast, R. solanacearum mutants unable to use the HCD-catabolized nutrients sucrose (ΔscrA), trehalose (ΔtreA), or both (ΔscrA/treA), infected roots as well as wild-type R. solanacearum but were defective in colonization and competitive fitness in midstems and had reduced virulence. Further, xylem sap from tomato plants colonized by ΔscrA, ΔtreA, or ΔscrA/treA R. solanacearum mutants contained twice as much sucrose as sap from plants colonized by wild-type R. solanacearum. Together, these findings suggest that quorum sensing specifically adapts R. solanacearum metabolism for success in the different nutritional environments of plant roots and xylem sap.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Metabolomics of tomato xylem sap during bacterial wilt reveals Ralstonia solanacearum produces abundant putrescine, a metabolite that accelerates wilt disease.

Ralstonia solanacearum thrives in plant xylem vessels and causes bacterial wilt disease despite the low nutrient content of xylem sap. We found that R. solanacearum manipulates its host to increase nutrients in tomato xylem sap, enabling it to grow better in sap from infected plants than in sap from healthy plants. Untargeted GC/MS metabolomics identified 22 metabolites enriched in R. solanacearum-infected sap. Eight of these could serve as sole carbon or nitrogen sources for R. solanacearum. Putrescine, a polyamine that is not a sole carbon or nitrogen source for R. solanacearum, was enriched 76-fold to 37 µM in R. solanacearum-infected sap. R. solanacearum synthesized putrescine via a SpeC ornithine decarboxylase. A ΔspeC mutant required ≥ 15 µM exogenous putrescine to grow and could not grow alone in xylem even when plants were treated with putrescine. However, co-inoculation with wildtype rescued ΔspeC growth, indicating R. solanacearum produced and exported putrescine to xylem sap. Intriguingly, treating plants with putrescine before inoculation accelerated wilt symptom development and R. solanacearum growth and systemic spread. Xylem putrescine concentration was unchanged in putrescine-treated plants, so the exogenous putrescine likely accelerated disease indirectly by affecting host physiology. These results indicate that putrescine is a pathogen-produced virulence metabolite.

Generating new prions by targeted mutation or segment duplication.

Yeasts contain various protein-based genetic elements, termed prions, that result from the structural conversion of proteins into self-propagating amyloid forms. Most yeast prion proteins contain glutamine/asparagine (Q/N)-rich prion domains that drive prion activity. Here, we explore two mechanisms by which new prion domains could evolve. First, it has been proposed that mutation and natural selection will tend to result in proteins with aggregation propensities just low enough to function under physiological conditions and thus that a small number of mutations are often sufficient to cause aggregation. We hypothesized that if the ability to form prion aggregates was a sufficiently generic feature of Q/N-rich domains, many nonprion Q/N-rich domains might similarly have aggregation propensities on the edge of prion formation. Indeed, we tested four yeast Q/N-rich domains that had no detectable aggregation activity; in each case, a small number of rationally designed mutations were sufficient to cause the proteins to aggregate and, for two of the domains, to create prion activity. Second, oligopeptide repeats are found in multiple prion proteins, and expansion of these repeats increases prion activity. However, it is unclear whether the effects of repeat expansion are unique to these specific sequences or are a generic result of adding additional aggregation-prone segments into a protein domain. We found that within nonprion Q/N-rich domains, repeating aggregation-prone segments in tandem was sufficient to create prion activity. Duplication of DNA elements is a common source of genetic variation and may provide a simple mechanism to rapidly evolve prion activity.

NorA, HmpX, and NorB Cooperate to Reduce NO Toxicity during Denitrification and Plant Pathogenesis in Ralstonia solanacearum.

Ralstonia solanacearum, which causes bacterial wilt disease of many crops, requires denitrifying respiration to survive in its plant host. In the hypoxic environment of plant xylem vessels, this pathogen confronts toxic oxidative radicals like nitric oxide (NO), which is generated by both bacterial denitrification and host defenses. R. solanacearum has multiple distinct mechanisms that could mitigate this stress, including putative NO-binding protein (NorA), nitric oxide reductase (NorB), and flavohaemoglobin (HmpX). During denitrification and tomato pathogenesis and in response to exogenous NO, R. solanacearum upregulated norA, norB, and hmpX. Single mutants lacking ΔnorB, ΔnorA, or ΔhmpX increased expression of many iron and sulfur metabolism genes, suggesting that the loss of even one NO detoxification system demands metabolic compensation. Single mutants suffered only moderate fitness reductions in host plants, possibly because they upregulated their remaining protective genes. However, ΔnorA/norB, ΔnorB/hmpX, and ΔnorA/hmpX double mutants grew poorly in denitrifying culture and in planta. It is likely that the loss of norA, norB, and hmpX is lethal, since the methods used to construct the double mutants could not generate a triple mutant. Functional aconitase activity assays showed that NorA, HmpX, and especially NorB are important for maintaining iron-sulfur cluster proteins. Additionally, plant defense genes were upregulated in tomatoes infected with the NO-overproducing ΔnorB mutant, suggesting that bacterial detoxification of NO reduces the ability of the plant host to perceive the presence of the pathogen. Thus, R. solanacearum's three NO detoxification systems each contribute to and are collectively essential for overcoming metabolic nitrosative stress during denitrification, for virulence and growth in the tomato, and for evading host plant defenses. IMPORTANCE The soilborne plant pathogen Ralstonia solanacearum (Rs) causes bacterial wilt, a serious and widespread threat to global food security. Rs is metabolically adapted to low-oxygen conditions, using denitrifying respiration to survive in the host and cause disease. However, bacterial denitrification and host defenses generate nitric oxide (NO), which is toxic and also alters signaling pathways in both the pathogen and its plant hosts. Rs mitigates NO with a trio of mechanistically distinct proteins: NO-reductase (NorB), predicted iron-binding (NorA), and oxidoreductase (HmpX). This redundancy, together with analysis of mutants and in-planta dual transcriptomes, indicates that maintaining low NO levels is integral to Rs fitness in tomatoes (because NO damages iron-cluster proteins) and to evading host recognition (because bacterially produced NO can trigger plant defenses).

In through the Out Door: A Functional Virulence Factor Secretion System Is Necessary for Phage Infection in Ralstonia solanacearum.

Bacteriophages put intense selective pressure on microbes, which must evolve diverse resistance mechanisms to survive continuous phage attacks. We used a library of spontaneous Bacteriophage Insensitive Mutants (BIMs) to learn how the plant pathogen Ralstonia solanacearum resists the virulent lytic podophage phiAP1. Phenotypic and genetic characterization of many BIMs suggested that the R. solanacearum Type II Secretion System (T2SS) plays a key role in phiAP1 infection. Using precision engineered mutations that permit T2SS assembly but either inactivate the T2SS GspE ATPase or sterically block the secretion portal, we demonstrated that phiAP1 needs a functional T2SS to infect R. solanacearum. This distinction between the static presence of T2SS components, which is necessary but not sufficient for phage sensitivity, and the energized and functional T2SS, which is sufficient, implies that binding interactions alone cannot explain the role of the T2SS in phiAP1 infection. Rather, our results imply that some aspect of the resetting of the T2SS, such as disassembly of the pseudopilus, is required. Because R. solanacearum secretes multiple virulence factors via the T2SS, acquiring resistance to phiAP1 also dramatically reduced R. solanacearum virulence on tomato plants. This acute fitness trade-off suggests this group of phages may be a sustainable control strategy for an important crop disease. IMPORTANCE Ralstonia solanacearum is a destructive plant pathogen that causes lethal bacterial wilt disease in hundreds of diverse plant hosts, including many economically important crops. Phages that kill R. solanacearum could offer effective and environmentally friendly wilt disease control, but only if the bacterium cannot easily evolve resistance. Encouragingly, most R. solanacearum mutants resistant to the virulent lytic phage phiAP1 no longer secreted multiple virulence factors and had much reduced fitness and virulence on tomato plants. Further analysis revealed that phage phiAP1 needs a functional type II secretion system to infect R. solanacearum, suggesting this podophage uses a novel infection mechanism.

Citrus Huanglongbing is a pathogen-triggered immune disease that can be mitigated with antioxidants and gibberellin.

Huanglongbing (HLB) is a devastating disease of citrus, caused by the phloem-colonizing bacterium Candidatus Liberibacter asiaticus (CLas). Here, we present evidence that HLB is an immune-mediated disease. We show that CLas infection of Citrus sinensis stimulates systemic and chronic immune responses in phloem tissue, including callose deposition, production of reactive oxygen species (ROS) such as H2O2, and induction of immunity-related genes. The infection also upregulates genes encoding ROS-producing NADPH oxidases, and downregulates antioxidant enzyme genes, supporting that CLas causes oxidative stress. CLas-triggered ROS production localizes in phloem-enriched bark tissue and is followed by systemic cell death of companion and sieve element cells. Inhibition of ROS levels in CLas-positive stems by NADPH oxidase inhibitor diphenyleneiodonium (DPI) indicates that NADPH oxidases contribute to CLas-triggered ROS production. To investigate potential treatments, we show that addition of the growth hormone gibberellin (known to have immunoregulatory activities) upregulates genes encoding H2O2-scavenging enzymes and downregulates NADPH oxidases. Furthermore, foliar spray of HLB-affected citrus with gibberellin or antioxidants (uric acid, rutin) reduces H2O2 concentrations and cell death in phloem tissues and reduces HLB symptoms. Thus, our results indicate that HLB is an immune-mediated disease that can be mitigated with antioxidants and gibberellin.

The Immune Receptor Roq1 Confers Resistance to the Bacterial Pathogens Xanthomonas, Pseudomonas syringae, and Ralstonia in Tomato.

Xanthomonas species, Pseudomonas syringae and Ralstonia species are bacterial plant pathogens that cause significant yield loss in many crop species. Generating disease-resistant crop varieties can provide a more sustainable solution to control yield loss compared to chemical methods. Plant immune receptors encoded by nucleotide-binding, leucine-rich repeat (NLR) genes typically confer resistance to pathogens that produce a cognate elicitor, often an effector protein secreted by the pathogen to promote virulence. The diverse sequence and presence/absence variation of pathogen effector proteins within and between pathogen species usually limits the utility of a single NLR gene to protecting a plant from a single pathogen species or particular strains. The NLR protein Recognition of XopQ 1 (Roq1) was recently identified from the plant Nicotiana benthamiana and mediates perception of the effector proteins XopQ and HopQ1 from Xanthomonas and P. syringae respectively. Unlike most recognized effectors, alleles of XopQ/HopQ1 are highly conserved and present in most plant pathogenic strains of Xanthomonas and P. syringae. A homolog of XopQ/HopQ1, named RipB, is present in most Ralstonia strains. We found that Roq1 confers immunity to Xanthomonas, P. syringae, and Ralstonia when expressed in tomato. Strong resistance to Xanthomonas perforans was observed in three seasons of field trials with both natural and artificial inoculation. The Roq1 gene can therefore be used to provide safe, economical, and effective control of these pathogens in tomato and other crop species and reduce or eliminate the need for traditional chemical controls.