Regulatory Effects of Diverse DSF Family Quorum-Sensing Signals in Plant-Associated Bacteria.

Numerous bacterial species employ diffusible signal factor (DSF)-based quorum sensing (QS) as a widely conserved cell-cell signaling communication system to collectively regulate various behaviors crucial for responding to environmental changes. cis-11-Methyl-dodecenoic acid, known as DSF, was first identified as a signaling molecule in Xanthomonas campestris pv. campestris. Subsequently, many structurally related molecules have been identified in different bacterial species. This review aims to provide an overview of current understanding regarding the biosynthesis and regulatory role of DSF signals in both pathogenic bacteria and a biocontrol bacterium. Recent studies have revealed that the DSF-based QS system regulates antimicrobial factor production in a cyclic dimeric GMP-independent manner in the biocontrol bacterium Lysobacter enzymogenes. Additionally, the DSF family signals have been found to be involved in suppressing plant innate immunity. The discovery of these diverse signaling mechanisms holds significant promise for developing novel strategies to combat stubborn plant pathogens. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Unveiling the Role of Diffusible Signal Factor-Family Quorum Sensing Signals in Regulating Behavior of Xanthomonas and Lysobacter.

Diffusible signal factor (DSF) family signals represent a unique group of quorum sensing (QS) chemicals that modulate a wide range of behaviors for bacteria to adapt to different environments. However, whether DSF-mediated QS signaling acts as a public language to regulate the behavior of biocontrol and pathogenic bacteria remains unknown. In this study, we present groundbreaking evidence demonstrating that RpfFXc1 or RpfFOH11 could be a conserved DSF-family signal synthase in Xanthomonas campestris or Lysobacter enzymogenes. Interestingly, we found that both RpfFOH11 and RpfFXc1 have the ability to synthesize DSF and BDSF signaling molecules. DSF and BDSF positively regulate the biosynthesis of an antifungal factor (heat-stable antifungal factor, HSAF) in L. enzymogenes. Finally, we show that RpfFXc1 and RpfFOH11 have similar functions in regulating HSAF production in L. enzymogenes, as well as the virulence, synthesis of virulence factors, biofilm formation, and extracellular polysaccharide production in X. campestris. These findings reveal a previously uncharacterized mechanism of DSF-mediated regulation in both biocontrol and pathogenic bacteria.

Evaluating the Efficacy of Hymexazol in Controlling Globisporangium spinosum, the Newly Identified Pathogen Causing Root Rot in Houttuynia cordata.

Houttuynia cordata is a prevalent vegetable in several Asian countries and is commonly used as a traditional Chinese medicinal herb for treating various diseases in China. Unfortunately, its yield and quality are adversely affected by root rot. However, the pathogen responsible for the losses remains unidentified, and effective fungicides for its management have not been thoroughly explored. In this work, we demonstrate the first report of Globisporangium spinosum as the causative agent causing root rot of H. cordata. Moreover, we evaluated the efficacy of hymexazol to manage the disease, which displayed remarkable inhibitory effects against mycelial growth of G. spinosum in vitro, with EC50 values as low as 1.336 µg/ml. Furthermore, hymexazol completely inhibited sporangia in G. spinosum at a concentration of 0.3125 µg/ml. Specifically, we observed that hymexazol was highly efficacious in reducing the incidence of H. cordata root rot caused by G. spinosum in a greenhouse setting. These findings offer a potential management tool for utilization of hymexazol in controlling H. cordata root rot in field production.

A Glycoside Hydrolase Family 99-Like Domain-Containing Protein Modifies Outer Membrane Proteins to Maintain Xanthomonas Pathogenicity and Viability in Stressful Environments.

Protein glycosylation is an essential process that plays an important role in proteome stability, protein structure, and protein function modulation in eukaryotes. However, in bacteria, especially plant pathogenic bacteria, similar studies are lacking. Here, we investigated the relationship between protein glycosylation and pathogenicity by using Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight in rice, as a well-defined example. In our previous work, we identified a virulence-related hypothetical protein, PXO_03177, but how this protein regulates X. oryzae pv. oryzae virulence has remained unclear. BLAST analysis showed that most homologous proteins of PXO_03177 are glycoside hydrolase family 99-like domain-containing proteins. In the current study, we found that the outer membrane integrity of ΔPXO_03177 appeared to be disrupted. Extracting the outer membrane proteins (OMPs) and performing comparative proteomics and sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel staining analyses revealed that PXO_03177 deletion altered the protein levels of 13 OMPs. Western blot analyses showed that the protein level and glycosylation modification of PXO_02523, a related OmpA family-like protein, was changed in the ΔPXO_03177 mutant background strain. Additionally, the interaction between PXO_03177 and PXO_02523 was confirmed by coimmunoprecipitation. Both PXO_03177 and PXO_02523 play important roles in regulating pathogen virulence and viability in stressful environments. This work provides the first evidence that protein glycosylation is necessary for the virulence of plant pathogenic bacteria.

Two Functional Fatty Acyl Coenzyme A Ligases Affect Free Fatty Acid Metabolism To Block Biosynthesis of an Antifungal Antibiotic in Lysobacter enzymogenes.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia colifadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2 In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenesIMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

RNA-seq analysis provides insights into cold stress responses of Xanthomonas citri pv. citri.

BACKGROUND: Xanthomonas citri pv. citri (Xcc) is a citrus canker causing Gram-negative bacteria. Currently, little is known about the biological and molecular responses of Xcc to low temperatures. RESULTS: Results depicted that low temperature significantly reduced growth and increased biofilm formation and unsaturated fatty acid (UFA) ratio in Xcc. At low temperature Xcc formed branching structured motility. Global transcriptome analysis revealed that low temperature modulates multiple signaling networks and essential cellular processes such as carbon, nitrogen and fatty acid metabolism in Xcc. Differential expression of genes associated with type IV pilus system and pathogenesis are important cellular adaptive responses of Xcc to cold stress. CONCLUSIONS: Study provides clear insights into biological characteristics and genome-wide transcriptional analysis based molecular mechanism of Xcc in response to low temperature.

SstF, a novel sulforaphane-sensing transcription factor of Xanthomonas campestris, is required for sulforaphane tolerance and virulence.

Avoiding the host defence system is necessary for the survival of pathogens. However, the mechanisms by which pathogenic bacteria sense and resist host defence signals are still unknown. Sulforaphane (SFN) is a secondary metabolite of crucifers. It not only plays an important role in maintaining the local defence response but also directly inhibits the growth of some pathogens. In this study, we identified a key SFN tolerance-related gene, saxF, in Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers. More interestingly, we found that the transcription of saxF was regulated by the novel transcription factor SFN-sensing transcription factor (SstF). As a LysR family transcription factor, SstF can sense SFN and regulate the expression of saxF cluster genes to increase SFN resistance by directly binding to the promoter of saxF. In addition, we found that SstF and saxF also play an important role in positively regulating the virulence of Xcc. Collectively, our results illustrate a previously unknown mechanism by which Xcc senses the host defence signal SFN and activates the expression of SFN tolerance-related genes to increase virulence. Therefore, this study provides a remarkable result; that is, during pathogen-plant co-evolution, new functions of existing scaffolds are activated, thus improving the proficiency of the pathogenic mechanism.

Isolation, Identification, and Evaluation of the Predatory Activity of Chinese Arthrobotrys Species towards Economically Important Plant-Parasitic Nematodes.

The current investigation aimed to isolate and identify predatory fungal strains and evaluate their efficacy in mitigating the effects of plant-parasitic nematodes. We successfully isolated three distinct nematophagous fungal strains from soil samples, identified as Arthrobotrys megalosporus, A. oligospora, and A. sinensis, using conventional and molecular identification methodologies. In vitro trials illustrated the high capture efficiency of these fungi against plant-parasitic nematodes. Over an exposure period of 48 h to Aphelenchoides besseyi, Bursaphelenchus xylophilus, and Ditylenchus destructor, A. megalosporus (GUCC220044) displayed predation rates of 99.7%, 83.0%, and 21.1%, respectively. A. oligospora (GUCC220045) demonstrated predation rates of 97.3%, 97.3%, and 54.6%, and A. sinensis (GUCC220046) showed rates of 85.1%, 68.3%, and 19.0% against the same cohort of nematodes. The experimental outcomes substantiate that all three identified fungal strains demonstrate predatory activity against the tested nematodes, albeit with varying efficiencies.

Antifungal Compound from the Predatory Bacterium Lysobacter enzymogenes Inhibits a Plant Pathogenic Fungus by Targeting the AAA ATPase VpVeb1.

Heat-stable antifungal factor (HSAF) isolated from Lysobacter enzymogenes is considered a potential biocontrol agent. However, the target of HSAF in phytopathogenic fungi remains unclear. In this study, we investigated the target of HSAF in Valsa pyri that causes fatal pear Valsa canker. Thirty-one HSAF-binding proteins were captured and identified by surface plasmon resonance (SPR) and high-performance liquid chromatography-mass spectrometry (LC-MS/MS), and 11 deletion mutants were obtained. Among these mutants, only ΔVpVEB1 showed decreased sensitivity to HSAF. Additionally, ΔVpVEB1 exhibited significantly reduced virulence in V. pyri. Molecular docking and SPR results revealed that HSAF bound to threonine 569 and glycine 570 of VpVeb1, which are crucial for AAA ATPase activity. Another study showed that HSAF could decrease the ATPase activity of VpVeb1, leading to the reduced virulence of V. pyri. Taken together, this study first identified the potential target of HSAF in fungi. These findings will help us better understand the model of action of HSAF to fungi.

Sulforaphane, a secondary metabolite in crucifers, inhibits the oxidative stress adaptation and virulence of Xanthomonas by directly targeting OxyR.

Plant secondary metabolites perform numerous functions in the interactions between plants and pathogens. However, little is known about the precise mechanisms underlying their contribution to the direct inhibition of pathogen growth and virulence in planta. Here, we show that the secondary metabolite sulforaphane (SFN) in crucifers inhibits the growth, virulence, and ability of Xanthomonas species to adapt to oxidative stress, which is essential for the successful infection of host plants by phytopathogens. The transcription of oxidative stress detoxification-related genes (catalase [katA and katG] and alkylhydroperoxide-NADPH oxidoreductase subunit C [ahpC]) was substantially inhibited by SFN in Xanthomonas campestris pv. campestris (Xcc), and this phenomenon was most obvious in sax gene mutants sensitive to SFN. By performing microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA), we observed that SFN directly bound to the virulence-related redox-sensing transcription factor OxyR and weakened the ability of OxyR to bind to the promoters of oxidative stress detoxification-related genes. Collectively, these results illustrate that SFN directly targets OxyR to inhibit the bacterial adaptation to oxidative stress, thereby decreasing bacterial virulence. Interestingly, this phenomenon occurs in multiple Xanthomonas species. This study provides novel insights into the molecular mechanisms by which SFN limits Xanthomonas adaptation to oxidative stress and virulence, and the findings will facilitate future studies on the use of SFN as a biopesticide to control Xanthomonas.

The Xanthomonas citri Reverse Fitness Deficiency by Activating a Novel ß-Glucosidase Under Low Osmostress.

Bacteria can withstand various types of environmental osmostress. A sudden rise in osmostress affects bacterial cell growth that is countered by activating special genes. The change of osmostress is generally a slow process under the natural environment. However, the collective response of bacteria to low osmostress remains unknown. This study revealed that the deletion of phoP (ΔphoP) from X. citri significantly compromised the growth and virulence as compared to the wild-type strain. Interestingly, low osmostress reversed physiological deficiencies of X. citri phoP mutant related to bacterial growth and virulence. The results also provided biochemical and genetic evidence that the physiological deficiency of phoP mutant can be reversed by low osmostress induced ß-glucosidase (BglS) expression. Based on the data, this study proposes a novel regulatory mechanism of a novel ß-glucosidase activation in X. citri through low osmostress to reverse the fitness deficiency.

Proteomic and Transcriptomic Analyses Provide Novel Insights into the Crucial Roles of Host-Induced Carbohydrate Metabolism Enzymes in Xanthomonas oryzae pv. oryzae Virulence and Rice-Xoo Interaction.

BACKGROUND: Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, a devastating rice disease. The Xoo-rice interaction, wherein wide ranging host- and pathogen-derived proteins and genes wage molecular arms race, is a research hotspot. Hence, the identification of novel rice-induced Xoo virulence factors and characterization of their roles affecting rice global gene expression profiles will provide an integrated and better understanding of Xoo-rice interactions from the molecular perspective. RESULTS: Using comparative proteomics and an in vitro interaction system, we revealed that 5 protein spots from Xoo exhibited significantly different expression patterns (|fold change| > 1.5) at 3, 6, 12 h after susceptible rice leaf extract (RLX) treatment. MALDI-TOF MS analysis and pathogenicity tests showed that 4 host-induced proteins, including phosphohexose mutase, inositol monophosphatase, arginase and septum site-determining protein, affected Xoo virulence. Among them, mutants of two host-induced carbohydrate metabolism enzyme-encoding genes, ΔxanA and Δimp, elicited enhanced defense responses and nearly abolished Xoo virulence in rice. To decipher rice differentially expressed genes (DEGs) associated with xanA and imp, transcriptomic responses of ΔxanA-treated and Δimp-treated susceptible rice were compared to those in rice treated with PXO99A at 1 and 3 dpi. A total of 1521 and 227 DEGs were identified for PXO99A vs Δimp at 1 and 3 dpi, while for PXO99A vs ΔxanA, there were 131 and 106 DEGs, respectively. GO, KEGG and MapMan analyses revealed that the DEGs for PXO99A vs Δimp were mainly involved in photosynthesis, signal transduction, transcription, oxidation-reduction, hydrogen peroxide catabolism, ion transport, phenylpropanoid biosynthesis and metabolism of carbohydrates, lipids, amino acids, secondary metabolites, hormones, and nucleotides, while the DEGs from PXO99A vs ΔxanA were predominantly associated with photosynthesis, signal transduction, oxidation-reduction, phenylpropanoid biosynthesis, cytochrome P450 and metabolism of carbohydrates, lipids, amino acids, secondary metabolites and hormones. Although most pathways were associated with both the Δimp and ΔxanA treatments, the underlying genes were not the same. CONCLUSION: Our study identified two novel host-induced virulence factors XanA and Imp in Xoo, and revealed their roles in global gene expression in susceptible rice. These results provide valuable insights into the molecular mechanisms of pathogen infection strategies and plant immunity.

The predatory soil bacterium Lysobacter reprograms quorum sensing system to regulate antifungal antibiotic production in a cyclic-di-GMP-independent manner.

Soil bacteria often harbour various toxins to against eukaryotic or prokaryotic. Diffusible signal factors (DSFs) represent a unique group of quorum sensing (QS) chemicals that modulate interspecies competition in bacteria that do not produce antibiotic-like molecules. However, the molecular mechanism by which DSF-mediated QS systems regulate antibiotic production for interspecies competition remains largely unknown in soil biocontrol bacteria. In this study, we find that the necessary QS system component protein RpfG from Lysobacter, in addition to being a cyclic dimeric GMP (c-di-GMP) phosphodiesterase (PDE), regulates the biosynthesis of an antifungal factor (heat-stable antifungal factor, HSAF), which does not appear to depend on the enzymatic activity. Interestingly, we show that RpfG interacts with three hybrid two-component system (HyTCS) proteins, HtsH1, HtsH2, and HtsH3, to regulate HSAF production in Lysobacter. In vitro studies show that each of these proteins interacted with RpfG, which reduced the PDE activity of RpfG. Finally, we show that the cytoplasmic proportions of these proteins depended on their phosphorylation activity and binding to the promoter controlling the genes implicated in HSAF synthesis. These findings reveal a previously uncharacterized mechanism of DSF signalling in antibiotic production in soil bacteria.

RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris.

Homologous regulatory factors are widely present in bacteria, but whether homologous regulators synergistically or differentially regulate different biological functions remains mostly unknown. Here, we report that the homologous regulators RpoN1 and RpoN2 of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) play different regulatory roles with respect to virulence traits, flagellar biosynthesis, and basal metabolism. RpoN2 directly regulated Xcc fliC and fliQ to modulate flagellar synthesis in X. campestris, thus affecting the swimming motility of X. campestris. Mutation of rpoN2 resulted in reduced production of biofilms and extracellular polysaccharides in Xcc. These defects may together cause reduced virulence of the rpoN2 mutant against the host plant. Moreover, we demonstrated that RpoN1 could regulate branched-chain fatty acid production and modulate the synthesis of diffusible signal factor family quorum sensing signals. Although RpoN1 and RpoN2 are homologues, the regulatory roles and biological functions of these proteins were not interchangeable. Overall, our report provides new insights into the two different molecular roles that form the basis for the transcriptional specialization of RpoN homologues.

Biological Functions of ilvC in Branched-Chain Fatty Acid Synthesis and Diffusible Signal Factor Family Production in Xanthomonas campestris.

In bacteria, the metabolism of branched-chain amino acids (BCAAs) is tightly associated with branched-chain fatty acids (BCFAs) synthetic pathways. Although previous studies have reported on BCFAs biosynthesis, more detailed associations between BCAAs metabolism and BCFAs biosynthesis remain to be addressed. In this study, we deleted the ilvC gene, which encodes ketol-acid reductoisomerase in the BCAAs synthetic pathway, from the Xanthomonas campestris pv. campestris (Xcc) genome. We characterized gene functions in BCFAs biosynthesis and production of the diffusible signal factor (DSF) family signals. Disruption of ilvC caused Xcc to become auxotrophic for valine and isoleucine, and lose the ability to synthesize BCFAs via carbohydrate metabolism. Furthermore, ilvC mutant reduced the ability to produce DSF-family signals, especially branched-chain DSF-family signals, which might be the main reason for Xcc reduction of pathogenesis toward host plants. In this report, we confirmed that BCFAs do not have major functions in acclimatizing Xcc cells to low temperatures.