Structural insights into Xanthomonas campestris pv. campestris NAD+ biosynthesis via the NAM salvage pathway.

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) via the nicotinamide (NAM) salvage pathway. While the structural biochemistry of eukaryote NAMPT has been well studied, the catalysis mechanism of prokaryote NAMPT at the molecular level remains largely unclear. Here, we demonstrated the NAMPT-mediated salvage pathway is functional in the Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) for the synthesis of NAD+, and the enzyme activity of NAMPT in this bacterium is significantly higher than that of human NAMPT in vitro. Our structural analyses of Xcc NAMPT, both in isolation and in complex with either the substrate NAM or the product nicotinamide mononucleotide (NMN), uncovered significant details of substrate recognition. Specifically, we revealed the presence of a NAM binding tunnel that connects the active site, and this tunnel is essential for both catalysis and inhibitor binding. We further demonstrated that NAM binding in the tunnel has a positive cooperative effect with NAM binding in the catalytic site. Additionally, we discovered that phosphorylation of the His residue at position 229 enhances the substrate binding affinity of Xcc NAMPT and is important for its catalytic activity. This work reveals the importance of NAMPT in bacterial NAD+ synthesis and provides insights into the substrate recognition and the catalytic mechanism of bacterial type II phosphoribosyltransferases.

Early transcriptional changes of heavy metal resistance and multiple efflux genes in Xanthomonas campestris pv. campestris under copper and heavy metal ion stress.

BACKGROUND: Copper-induced gene expression in Xanthomonas campestris pv. campestris (Xcc) is typically evaluated using targeted approaches involving qPCR. The global response to copper stress in Xcc and resistance to metal induced damage is not well understood. However, homologs of heavy metal efflux genes from the related Stenotrophomonas genus are found in Xanthomonas which suggests that metal related efflux may also be present. METHODS AND RESULTS: Gene expression in Xcc strain BrA1 exposed to 0.8 mM CuSO4.5H2O for 15 minutes was captured using RNA-seq analysis. Changes in expression was noted for genes related to general stress responses and oxidoreductases, biofilm formation, protein folding chaperones, heat-shock proteins, membrane lipid profile, multiple drug and efflux (MDR) transporters, and DNA repair were documented. At this timepoint only the cohL (copper homeostasis/tolerance) gene was upregulated as well as a chromosomal czcCBA efflux operon. An additional screen up to 4 hrs using qPCR was conducted using a wider range of heavy metals. Target genes included a cop-containing heavy metal resistance island and putative metal efflux genes. Several efflux pumps, including a copper resistance associated homolog from S. maltophilia, were upregulated under toxic copper stress. However, these pumps were also upregulated in response to other toxic heavy metals. Additionally, the temporal expression of the coh and cop operons was also observed, demonstrating co-expression of tolerance responses and later activation of part of the cop operon. CONCLUSIONS: Overall, initial transcriptional responses focused on combating oxidative stress, mitigating protein damage and potentially increasing resistance to heavy metals and other biocides. A putative copper responsive efflux gene and others which might play a role in broader heavy metal resistance were also identified. Furthermore, the expression patterns of the cop operon in conjunction with other copper responsive genes allowed for a better understanding of the fate of copper ions in Xanthomonas. This work provides useful evidence for further evaluating MDR and other efflux pumps in metal-specific homeostasis and tolerance phenotypes in the Xanthomonas genus. Furthermore, non-canonical copper tolerance and resistance efflux pumps were potentially identified. These findings have implications for interpreting MIC differences among strains with homologous copLAB resistance genes, understanding survival under copper stress, and resistance in disease management.

Identification of the ERF gene family of Mangifera indica and the defense response of MiERF4 to Xanthomonas campestris pv. mangiferaeindicae.

An important regulatory role for ethylene-responsive transcription factors (ERFs) is in plant growth and development, stress response, and hormone signaling. However, AP2/ERF family genes in mango have not been systematically studied. In this study, a total of 113 AP2/ERF family genes were identified from the mango genome and phylogenetically classified into five subfamilies: AP2 (28 genes), DREB (42 genes), ERF (33 genes), RAV (6 genes), and Soloist (4 genes). Of these, the ERF family, in conjunction with Arabidopsis and rice, forms a phylogenetic tree divided into seven groups, five of which have MiERF members. Analysis of gene structure and cis-elements showed that each MiERF gene contains only one AP2 structural domain, and that MiERF genes contain a large number of cis-elements associated with hormone signaling and stress response. Collinearity tests revealed a high degree of homology between MiERFs and CsERFs. Tissue-specific and stress-responsive expression profiling revealed that MiERF genes are primarily involved in the regulation of reproductive growth and are differentially and positively expressed in response to external hormones and pathogenic bacteria. Physiological results from a gain-of-function analysis of MiERF4 transiently overexpressed in tobacco and mango showed that transient expression of MiERF4 resulted in decreased colony count and callose deposition, as well as varying degrees of response to hormonal signals such as ETH, JA, and SA. Thus, MiERF4 may be involved in the JA/ETH signaling pathway to enhance plant defense against pathogenic bacteria. This study provides a basis for further research on the function and regulation of MiERF genes and lays a foundation for the selection of disease-resistant genes in mango.

Functional characterization and transcriptional analysis of degQ of Xanthomonas campestris pathovar campestris.

High-temperature-requirement protein A (HtrA) family proteins play important roles in controlling protein quality and are recognized as virulence factors in numerous animal and human bacterial pathogens. The role of HtrA family proteins in plant pathogens remains largely unexplored. Here, we investigated the HtrA family protein, DegQ, in the crucifer black rot pathogen Xanthomonas campestris pathovar campestris (Xcc). DegQ is essential for bacterial attachment and full virulence of Xcc. Moreover, the degQ mutant strain showed increased sensitivity to heat treatment and sodium dodecyl sulfate. Expressing the intact degQ gene in trans in the degQ mutant could reverse the observed phenotypic changes. In addition, we demonstrated that the DegQ protein exhibited chaperone-like activity. Transcriptional analysis displayed that degQ expression was induced under heat treatment. Our results contribute to understanding the function and expression of DegQ of Xcc for the first time and provide a novel perspective about HtrA family proteins in plant pathogen.

A conserved microtubule-binding region in Xanthomonas XopL is indispensable for induced plant cell death reactions.

Pathogenic Xanthomonas bacteria cause disease on more than 400 plant species. These Gram-negative bacteria utilize the type III secretion system to inject type III effector proteins (T3Es) directly into the plant cell cytosol where they can manipulate plant pathways to promote virulence. The host range of a given Xanthomonas species is limited, and T3E repertoires are specialized during interactions with specific plant species. Some effectors, however, are retained across most strains, such as Xanthomonas Outer Protein L (XopL). As an 'ancestral' effector, XopL contributes to the virulence of multiple xanthomonads, infecting diverse plant species. XopL homologs harbor a combination of a leucine-rich-repeat (LRR) domain and an XL-box which has E3 ligase activity. Despite similar domain structure there is evidence to suggest that XopL function has diverged, exemplified by the finding that XopLs expressed in plants often display bacterial species-dependent differences in their sub-cellular localization and plant cell death reactions. We found that XopL from X. euvesicatoria (XopLXe) directly associates with plant microtubules (MTs) and causes strong cell death in agroinfection assays in N. benthamiana. Localization of XopLXe homologs from three additional Xanthomonas species, of diverse infection strategy and plant host, revealed that the distantly related X. campestris pv. campestris harbors a XopL (XopLXcc) that fails to localize to MTs and to cause plant cell death. Comparative sequence analyses of MT-binding XopLs and XopLXcc identified a proline-rich-region (PRR)/α-helical region important for MT localization. Functional analyses of XopLXe truncations and amino acid exchanges within the PRR suggest that MT-localized XopL activity is required for plant cell death reactions. This study exemplifies how the study of a T3E within the context of a genus rather than a single species can shed light on how effector localization is linked to biochemical activity.

Sources and methods of manufacturing xanthan by fermentation of various carbon sources.

Xanthan gum, an anionic polysaccharide with an exceptionally high molecular weight, is produced by the bacterium Xanthomonas sp. It is a versatile compound that has been utilized in various industries for decades. Xanthan gum was the second exopolysaccharide to be commercially produced, following dextran. In 1969, the US Food and Drug Administration (FDA) approved xanthan gum for use in the food and pharmaceutical industries. The food industry values xanthan gum for its exceptional rheological properties, which make it a popular thickening agent in many products. Meanwhile, the cosmetics industry capitalizes on xanthan gum's ability to form stable emulsions. The industrial production process of xanthan gum involves fermenting Xanthomonas in a medium that contains glucose, sucrose, starch, etc. as a substrate and other necessary nutrients to facilitate growth. This is achieved through batch fermentation under optimal conditions. However, the increasing costs of glucose in recent years have made the production of xanthan economically unviable. Therefore, many researchers have investigated alternative, cost-effective substrates for xanthan production, using various modified and unmodified raw materials. The objective of this analysis is to investigate how utilizing different raw materials can improve the cost-efficient production of xanthan gum.

Diffusible signal factor primes plant immunity against Xanthomonas campestris pv. campestris (Xcc) via JA signaling in Arabidopsis and Brassica oleracea.

Background: Many Gram-negative bacteria use quorum sensing (QS) signal molecules to monitor their local population density and to coordinate their collective behaviors. The diffusible signal factor (DSF) family represents an intriguing type of QS signal to mediate intraspecies and interspecies communication. Recently, accumulating evidence demonstrates the role of DSF in mediating inter-kingdom communication between DSF-producing bacteria and plants. However, the regulatory mechanism of DSF during the Xanthomonas-plant interactions remain unclear. Methods: Plants were pretreated with different concentration of DSF and subsequent inoculated with pathogen Xanthomonas campestris pv. campestris (Xcc). Pathogenicity, phynotypic analysis, transcriptome combined with metabolome analysis, genetic analysis and gene expression analysis were used to evaluate the priming effects of DSF on plant disease resistance. Results: We found that the low concentration of DSF could prime plant immunity against Xcc in both Brassica oleracea and Arabidopsis thaliana. Pretreatment with DSF and subsequent pathogen invasion triggered an augmented burst of ROS by DCFH-DA and DAB staining. CAT application could attenuate the level of ROS induced by DSF. The expression of RBOHD and RBOHF were up-regulated and the activities of antioxidases POD increased after DSF treatment followed by Xcc inoculation. Transcriptome combined with metabolome analysis showed that plant hormone jasmonic acid (JA) signaling involved in DSF-primed resistance to Xcc in Arabidopsis. The expression of JA synthesis genes (AOC2, AOS, LOX2, OPR3 and JAR1), transportor gene (JAT1), regulator genes (JAZ1 and MYC2) and responsive genes (VSP2, PDF1.2 and Thi2.1) were up-regulated significantly by DSF upon Xcc challenge. The primed effects were not observed in JA relevant mutant coi1-1 and jar1-1. Conclusion: These results indicated that DSF-primed resistance against Xcc was dependent on the JA pathway. Our findings advanced the understanding of QS signal-mediated communication and provide a new strategy for the control of black rot in Brassica oleracea.

The functional and structural characterization of Xanthomonas campestris pv. campestris core effector XopP revealed a new kinase activity.

Exo70B1 is a protein subunit of the exocyst complex with a crucial role in a variety of cell mechanisms, including immune responses against pathogens. The calcium-dependent kinase 5 (CPK5) of Arabidopsis thaliana (hereafter Arabidopsis), phosphorylates AtExo70B1 upon functional disruption. We previously reported that, the Xanthomonas campestris pv. campestris effector XopP compromises AtExo70B1, while bypassing the host's hypersensitive response, in a way that is still unclear. Herein we designed an experimental approach, which includes biophysical, biochemical, and molecular assays and is based on structural and functional predictions, utilizing AplhaFold and DALI online servers, respectively, in order to characterize the in vivo XccXopP function. The interaction between AtExo70B1 and XccXopP was found very stable in high temperatures, while AtExo70B1 appeared to be phosphorylated at XccXopP-expressing transgenic Arabidopsis. XccXopP revealed similarities with known mammalian kinases and phosphorylated AtExo70B1 at Ser107, Ser111, Ser248, Thr309, and Thr364. Moreover, XccXopP protected AtExo70B1 from AtCPK5 phosphorylation. Together these findings show that XccXopP is an effector, which not only functions as a novel serine/threonine kinase upon its host target AtExo70B1 but also protects the latter from the innate AtCPK5 phosphorylation, in order to bypass the host's immune responses. Data are available via ProteomeXchange with the identifier PXD041405.

RNase D Is Involved in 5S rRNA Degradation and Exopolysaccharide Production in Xanthomonas campestris pv. campestris.

Ribonucleases (RNases) play critical roles in RNA metabolism and are collectively essential for cell viability. However, most knowledge about bacterial RNases comes from the studies on Escherichia coli; very little is known about the RNases in plant pathogens. The crucifer black rot pathogen Xanthomonas campestris pv. campestris (Xcc) encodes 15 RNases, but none of them has been functionally characterized. Here, we report the physiological function of the exoribonuclease RNase D in Xcc and provide evidence demonstrating that the Xcc RNase D is involved in 5S rRNA degradation and exopolysaccharide (EPS) production. Our work shows that the growth and virulence of Xcc were not affected by deletion of RNase D but were severely attenuated by RNase D overexpression. However, deletion of RNase D in Xcc resulted in a significant reduction in EPS production. In addition, either deletion or overexpression of RNase D in Xcc did not influence the tRNAs tested, inconsistent with the finding in E. coli that the primary function of RNase D is to participate in tRNA maturation and its overexpression degrades tRNAs. More importantly, deletion, overexpression, and in vitro enzymatic analyses revealed that the Xcc RNase D degrades 5S rRNA but not 16S and 23S rRNAs that share an operon with 5S rRNA. Our results suggest that Xcc employs RNase D to realize specific modulation of the cellular 5S rRNA content after transcription and maturation whenever necessary. The finding expands our knowledge about the function of RNase D in bacteria.

Novel pyrimidin-4-one derivatives as potential T3SS inhibitors against Xanthomonas campestris pv. campestris.

BACKGROUND: Cruciferous black rot is caused by Xanthomonas campestris pv. campestris (Xcc) infection and is a widespread disease worldwide. Excessive and repeated use of bactericide is an important cause of the development of bacterial resistance. It is imperative to take new approaches to screening compounds that target virulence factors rather than kill bacterial pathogens. The type III secretion system (T3SS) invades a variety of cells by transporting virulence effector factors into the cytoplasm and is an attractive antitoxic target. Toward the search of new T3SS inhibitors, an alternative series of novel pyrimidin-4-one derivatives were designed and synthesized and assessed for their effect in blocking the virulence. RESULTS: All of the target compounds were characterized by proton (1 H) nuclear magnetic resonance (NMR), carbon-13 (13 C) NMR, fluorine-19 (19 F) NMR and high-resolution mass spectrometry (HRMS). All compounds were evaluated using high-throughput screening systems against Xcc. The results of the biological activity test revealed that the compound SPF-9 could highly inhibit the activity of xopN gene promoter and the hypersensitivity (HR) of tobacco without affecting bacterial growth. Moreover, messenger RNA (mRNA) level measurements showed that compound SPF-9 inhibited the expression of some representative genes (hrp/hrc genes). Compound SPF-9 weakened the pathogenicity of Xcc to Raphanus sativus L. CONCLUSION: Compound SPF-9 has good potential for further development as a novel T3SS inhibitor against Xcc. © 2023 Society of Chemical Industry.

Unleashing the potential of xanthan: a comprehensive exploration of biosynthesis, production, and diverse applications.

Employing aerobic fermentation, Gram-negative bacteria belonging to the genus Xanthomonas produce the high molecular weight natural heteropolysaccharide known as xanthan. It has various amounts of O-acetyl and pyruvyl residues together with D-glucosyl, D-mannosyl, and D-glucuronyl acid residues in a molar ratio of 2:2:1. The unique structure of xanthan allowed its various applications in a wide range of industries such as the food industry, pharmacology, cosmetics and enhanced oil recovery primarily in petroleum. The cultivation medium used in the manufacture of this biopolymer is critical. Many attempts have been undertaken to generate xanthan gum from agro-based and food industry wastes since producing xanthan gum from synthetic media is expensive. Optimal composition and processing parameters must also be considered to achieve an economically viable manufacturing process. There have been several attempts to adjust the nutrient content and feeding method, temperature, pH, agitation and the use of antifoam in xanthan fermentations. Various modifications in technological approaches have been applied to enhance its physicochemical properties which showed significant improvement in the area studied. This review describes the biosynthesis production of xanthan with an emphasis on the importance of the upstream processes involving medium, processing parameters, and other factors that significantly contributed to the final application of this precious polysaccharide.

The FabA-FabB Pathway Is Not Essential for Unsaturated Fatty Acid Synthesis but Modulates Diffusible Signal Factor Synthesis in Xanthomonas campestris pv. campestris.

Most bacteria use type II fatty acid synthesis (FAS) systems for synthesizing fatty acids, of which the conserved FabA-FabB pathway is considered to be crucial for unsaturated fatty acid (UFA) synthesis in gram-negative bacteria. Xanthomonas campestris pv. campestris, the phytopathogen of black rot disease in crucifers, produces higher quantities of UFAs under low-temperature conditions for increasing membrane fluidity. The fabA and fabB genes were identified in the X. campestris pv. campestris genome by BLAST analysis; however, the growth of the X. campestris pv. campestris fabA and fabB deletion mutants was comparable to that of the wild-type strain in nutrient and minimal media. The X. campestris pv. campestris ΔfabA and ΔfabB strains produced large quantities of UFAs and, altogether, these results indicated that the FabA-FabB pathway is not essential for growth or UFA synthesis in X. campestris pv. campestris. We also observed that the expression of X. campestris pv. campestris fabA and fabB restored the growth of the temperature-sensitive Escherichia coli fabA and fabB mutants CL104 and CY242, respectively, under non-permissive conditions. The in-vitro assays demonstrated that the FabA and FabB proteins of X. campestris pv. campestris catalyzed FAS. Our study also demonstrated that the production of diffusible signal factor family signals that mediate quorum sensing was higher in the X. campestris pv. campestris ΔfabA and ΔfabB strains and greatly reduced in the complementary strains, which exhibited reduced swimming motility and attenuated host-plant pathogenicity. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

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.

BDSF Is a Degradation-Prone Quorum-Sensing Signal Detected by the Histidine Kinase RpfC of Xanthomonas campestris pv. campestris.

Diffusible signal factors (DSFs) are medium-chain fatty acids that induce bacterial quorum sensing. Among these compounds, BDSF is a structural analog of DSF that is commonly detected in bacterial species, and it is the predominant in planta quorum-sensing signal in Xanthomonas campestris. How BDSF is sensed in Xanthomonas spp. and the functional diversity between BDSF and DSF remain unclear. In this study, we generated genetic and biochemical evidence that BDSF is a low-active regulator of X. campestris pv. campestris quorum sensing, whereas trans-BDSF does not seem to be a signaling compound. BDSF is detected by the sensor histidine kinase RpfC. Although BDSF has relatively low physiological activities, it binds to the RpfC sensor with a high affinity and activates RpfC autophosphorylation to a level that is similar to that induced by DSF in vitro. The inconsistency in the physiological and biochemical activities of BDSF is not due to RpfC-RpfG phosphorylation or RpfG hydrolase. Neither BDSF nor DSF controls the phosphotransferase and phosphatase activities of RpfC or the ability of RpfG hydrolase activity to degrade the bacterial second messenger cyclic di-GMP. We demonstrated that BDSF is prone to degradation by RpfB, a critical fatty acyl coenzyme A ligase involved in the turnover of DSF-family signals. rpfB mutations lead to substantial increases in BDSF-induced quorum sensing. Although DSF and BDSF are similarly detected by RpfC, our data suggest that their differential degradation in cells is the major factor responsible for the diversity in their physiological effects. IMPORTANCE The diffusible signal factor (DSF) family consists of quorum-sensing signals employed by Gram-negative bacteria. These signals are a group of cis-2-unsaturated fatty acids, such as DSF, BDSF, IDSF, CDSF, and SDSF. However, the functional divergence of various DSF signals remains unclear. The present study demonstrates that though BDSF is a low active quorum-sensing signal, it binds histidine kinase RpfC with a higher affinity and activates RpfC autophosphorylation to the similar level as DSF. Rather than regulation of enzymatic activities of RpfC and its cognate response regulator RpfG encoding a c-di-GMP hydrolase, BDSF is prone to degradation in bacterial cells by RpfB, which effectively avoided the inhibition of bacterial growth by accumulating high concentrations of BDSF. Therefore, our study sheds new light on the functional differences of quorum-sensing signals and shows that bacteria balance quorum sensing and growth by fine-tuning concentrations of signaling chemicals.

Improving the Transglycosylation Activity of α-Glucosidase from Xanthomonas campestris Through Semi-rational Design for the Synthesis of Ethyl Vanillin-α-Glucoside.

The α-glucosidase (EC 3.2.1.20) XgtA produced by Xanthomonas campestris shows high α-glucosyl transfer activity toward alcoholic and phenolic hydroxyl groups. Ethyl vanillin-α-glucoside, a precursor-aroma compound with improved water solubility and thermal stability, can be synthesized through the transglycosylation of ethyl vanillin by XgtA. However, its low ethyl vanillin-α-glucoside yield and ability to hydrolyze ethyl vanillin-α-glucoside limits for industrial applications. Rational design and site-directed mutagenesis were employed to generate three variants of X. campestris α-glucosidase, L145I, S272T, and L145I/S272T, with improved transglycosylation activity toward EV. The highest yield is up to 52.41% by L145I/S272T, which also displayed remarkably lower hydrolysis activity toward the glycoside product EVG compared to XgtA. These results also showed that the mutation in sugar-binding subsite + 1 is more effective than subsite -1 for enhancing the ratio of transglycosylation/hydrolysis for the α-glucosidase XgtA.

Expression and function of clpS and clpA in Xanthomonas campestris pv. campestris.

ATP-dependent proteases (FtsH, Lon, and Clp family proteins) are ubiquitous in bacteria and play essential roles in numerous regulatory cell processes. Xanthomonas campestris pv. campestris is a Gram-negative pathogen that can cause black rot diseases in crucifers. The genome of X. campestris pv. campestris has several clp genes, namely, clpS, clpA, clpX, clpP, clpQ, and clpY. Among these genes, only clpX and clpP is known to be required for pathogenicity. Here, we focused on two uncharacterized clp genes (clpS and clpA) that encode the adaptor (ClpS) and ATPase subunit (ClpA) of the ClpAP protease complex. Transcriptional analysis revealed that the expression of clpS and clpA was growth phase-dependent and affected by the growth temperature. The inactivation of clpA, but not of clpS, resulted in susceptibility to high temperature and attenuated virulence in the host plant. The altered phenotypes of the clpA mutant could be complemented in trans. Site-directed mutagenesis revealed that K223 and K504 were the amino acid residues critical for ClpA function in heat tolerance. The protein expression profile shown by the clpA mutant in response to heat stress was different from that exhibited by the wild type. In summary, we characterized two clp genes (clpS and clpA) by examining their expression profiles and functions in different processes, including stress tolerance and pathogenicity. We demonstrated that clpS and clpA were expressed in a temperature-dependent manner and that clpA was required for the survival at high temperature and full virulence of X. campestris pv. campestris. This work represents the first time that clpS and clpA were characterized in Xanthomonas.

HetI-Like Phosphopantetheinyl Transferase Posttranslationally Modifies Acyl Carrier Proteins in Xanthomonas spp.

In Xanthomonas spp., the biosynthesis of the yellow pigment xanthomonadin and fatty acids originates in the type II polyketide synthase (PKS II) and fatty acid synthase (FAS) pathways, respectively. The acyl carrier protein (ACP) is the central component of PKS II and FAS and requires posttranslational phosphopantetheinylation to initiate these pathways. In this study, for the first time, we demonstrate that the posttranslational modification of ACPs in X. campestris pv. campestris is performed by an essential 4'-phosphopantetheinyl transferase (PPTase), XcHetI (encoded by Xc_4132). X. campestris pv. campestris strain XchetI could not be deleted from the X. campestris pv. campestris genome unless another PPTase-encoding gene such as Escherichia coli acpS or Pseudomonas aeruginosa pcpS was present. Compared with wild-type strain X. campestris pv. campestris 8004 and mutant XchetI::PapcpS, strain XchetI::EcacpS failed to generate xanthomonadin pigments and displayed reduced pathogenicity for the host plant, Brassica oleracea. Further experiments showed that the expression of XchetI restored the growth of E. coli acpS mutant HT253 and, when a plasmid bearing XchetI was introduced into P. aeruginosa, pcpS, which encodes the sole PPTase in P. aeruginosa, could be deleted. In in vitro enzymatic assays, XcHetI catalyzed the transformation of 4'-phosphopantetheine from coenzyme A to two X. campestris pv. campestris apo-acyl carrier proteins, XcAcpP and XcAcpC. All of these findings indicate that XcHetI is a surfactin PPTase-like PPTase with a broad substrate preference. Moreover, the HetI-like PPTase is ubiquitously conserved in Xanthomonas spp., making it a potential new drug target for the prevention of plant diseases caused by Xanthomonas.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The Xanthomonas type-III effector XopS stabilizes CaWRKY40a to regulate defense responses and stomatal immunity in pepper (Capsicum annuum).

As a critical part of plant immunity, cells that are attacked by pathogens undergo rapid transcriptional reprogramming to minimize virulence. Many bacterial phytopathogens use type III effector (T3E) proteins to interfere with plant defense responses, including this transcriptional reprogramming. Here, we show that Xanthomonas outer protein S (XopS), a T3E of Xanthomonas campestris pv. vesicatoria (Xcv), interacts with and inhibits proteasomal degradation of WRKY40, a transcriptional regulator of defense gene expression. Virus-induced gene silencing of WRKY40 in pepper (Capsicum annuum) enhanced plant tolerance to Xcv infection, indicating that WRKY40 represses immunity. Stabilization of WRKY40 by XopS reduces the expression of its targets, which include salicylic acid-responsive genes and the jasmonic acid signaling repressor JAZ8. Xcv bacteria lacking XopS display significantly reduced virulence when surface inoculated onto susceptible pepper leaves. XopS delivery by Xcv, as well as ectopic expression of XopS in Arabidopsis thaliana or Nicotiana benthamiana, prevented stomatal closure in response to bacteria and biotic elicitors. Silencing WRKY40 in pepper or N. benthamiana abolished XopS's ability to prevent stomatal closure. This suggests that XopS interferes with both preinvasion and apoplastic defense by manipulating WRKY40 stability and downstream gene expression, eventually altering phytohormone crosstalk to promote pathogen proliferation.

The lolB gene in Xanthomonas campestris pv. campestris is required for bacterial attachment, stress tolerance, and virulence.

BACKGROUND: Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium that can cause black rot disease in crucifers. The lipoprotein outer membrane localization (Lol) system is involved in the lipoprotein sorting to the outer membrane. Although Xcc has a set of annotated lol genes, there is still little known about the physiological role in this phytopathogen. In this study, we aimed to characterize the role of LolB of Xcc in bacterial attachment, stress tolerance, and virulence. RESULTS: To characterize the role of LolB, lolB mutant was constructed and phenotypic evaluation was performed. The lolB mutant revealed reductions in bacterial attachment, extracellular enzyme production, and virulence. Mutation of lolB also resulted in reduced tolerance to a myriad of stresses, including heat and a range of membrane-perturbing agents. Trans-complementation of lolB mutant with intact lolB gene reverted these altered phenotypes to the wild-type levels. From subsequent reporter assay and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, the expression of genes that encode the major extracellular enzymes and the stress-related proteins was reduced after lolB mutation. CONCLUSIONS: The results in this work contribute to the functional understanding of lolB in Xanthomonas for the first time, and provide new insights into the function of lolB in bacteria.

Analysis of transcriptional regulators HpaR1 and Clp regulating the expression of glycoside hydrolase-encoding gene in the Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that causes black rot in host. It is an important model strain for studying the interaction between the phytopathogen and plants. In Xcc, global transcription regulator HpaR1 that belongs to the GntR family regulates many cellular processes such as the movement and synthesis of extracellular polysaccharides and extracellular enzymes, and is associated with hypersensitive response (HR) and pathogenicity. On the other hand, the global transcriptional regulator Clp regulates the secretion and synthesis of extracellular enzymes and extracellular polysaccharides, and is associated with the pathogenicity of Xanthomonas. Previous studies have shown that both HpaR1 and Clp bind to the promoter region of the glycoside hydrolase encoding gene (named ghy gene). This study investigates the molecular mechanism of the co-regulation of HpaR1 and Clp on the expression of ghy gene. Through electrophoresis mobility shift assay (EMSA), we found that both HpaR1 and Clp bind to the promoter regions of gene ghy in vitro. Both HpaR1 and Clp also bind to the promoter regions of gene ghy in vivo by chromatin immunoprecipitation (ChIP) assays. DNase I footprinting and 5'-RACE assays showed that both HpaR1 and Clp bind to the -35 region upstream of the ghy promoter. The HpaR1 binding site was located upstream of the Clp binding site. RT-qPCR and in vitro transcription assays showed that HpaR1 negatively while Clp positively regulates the transcription of gene ghy. Furthermore, HpaR1 inhibits the activation of Clp on the transcription of gene ghy in vitro. Our findings indicate that HpaR1 and Clp exhibit opposite effect on the transcription of gene ghy. It is speculated that HpaR1 may regulate the expression of gene ghy by inhibiting the activity of RNA polymerase.