The Type III Effector XopLXcc in Xanthomonas campestris pv. campestris Targets the Proton Pump Interactor 1 and Suppresses Innate Immunity in Arabidopsis.

Xanthomonas campestris pathovar campestris (Xcc) is a significant phytopathogen causing black rot disease in crucifers. Xcc injects a variety of type III effectors (T3Es) into the host cell to assist infection or propagation. A number of T3Es inhibit plant immunity, but the biochemical basis for a vast majority of them remains unknown. Previous research has revealed that the evolutionarily conserved XopL-family effector XopLXcc inhibits plant immunity, although the underlying mechanisms remain incompletely elucidated. In this study, we identified proton pump interactor (PPI1) as a specific virulence target of XopLXcc in Arabidopsis. Notably, the C-terminus of PPI1 and the Leucine-rich repeat (LRR) domains of XopLXcc are pivotal for facilitating this interaction. Our findings indicate that PPI1 plays a role in the immune response of Arabidopsis to Xcc. These results propose a model in which XopLXcc binds to PPI1, disrupting the early defense responses activated in Arabidopsis during Xcc infection and providing valuable insights into potential strategies for regulating plasma membrane (PM) H+-ATPase activity during infection. These novel insights enhance our understanding of the pathogenic mechanisms of T3Es and contribute to the development of effective strategies for controlling bacterial diseases.

Interactive regulation of immune-related resistance genes with salicylic acid and jasmonic acid signaling in systemic acquired resistance in the Xanthomonas-Brassica pathosystem.

Pathogen-responsive immune-related genes (resistance genes [R-genes]) and hormones are crucial mediators of systemic acquired resistance (SAR). However, their integrated functions in regulating SAR signaling components in local and distal leaves remain largely unknown. To characterize SAR in the Xanthomonas campestris pv. campestris (Xcc)-Brassica napus pathosystem, the responses of R-genes, (leaf and phloem) hormone levels, H2O2 levels, and Ca2+ signaling-related genes were assessed in local and distal leaves of plants exposed to four Xcc-treatments: Non-inoculation (control), only secondary Xcc-inoculation in distal leaves (C-Xcc), only primary Xcc-inoculation in local leaves (Xcc), and both primary and secondary Xcc-inoculation (X-Xcc). The primary Xcc-inoculation provoked disease symptoms as evidenced by enlarged destructive necrosis in the local leaves of Xcc and X-Xcc plants 7 days post-inoculation. Comparing visual symptoms in distal leaves 5 days post-secondary inoculation, yellowish necrotic lesions were clearly observed in non Xcc-primed plants (C-Xcc), whereas no visual symptom was developed in Xcc-primed plants (X-Xcc), demonstrating SAR. Pathogen resistance in X-Xcc plants was characterized by distinct upregulations in expression of the PAMP-triggered immunity (PTI)-related kinase-encoding gene, BIK1, the (CC-NB-LRR-type) R-gene, ZAR1, and its signaling-related gene, NDR1, with a concurrent enhancement of the kinase-encoding gene, MAPK6, and a depression of the (TIR-NB-LRR-type) R-gene, TAO1, and its signaling-related gene, SGT1, in distal leaves. Further, in X-Xcc plants, higher salicylic acid (SA) and jasmonic acid (JA) levels, both in phloem and distal leaves, were accompanied by enhanced expressions of the SA-signaling gene, NPR3, the JA-signaling genes, LOX2 and PDF1.2, and the Ca2+-signaling genes, CAS and CBP60g. However, in distal leaves of C-Xcc plants, an increase in SA level resulted in an antagonistic depression of JA, which enhanced only SA-dependent signaling, EDS1 and NPR1. These results demonstrate that primary Xcc-inoculation in local leaves induces resistance to subsequent pathogen attack by upregulating BIK1-ZAR1-mediated synergistic interactions with SA and JA signaling as a crucial component of SAR.

Discovery of Anomer-Inverting Transglycosylase: Cyclic Glucohexadecaose-Producing Enzyme from Xanthomonas, a Phytopathogen.

Various Xanthomonas species cause well-known plant diseases. Among various pathogenic factors, the role of α-1,6-cyclized ß-1,2-glucohexadecaose (CßG16α) produced by Xanthomonas campestris pv. campestris was previously shown to be vital for infecting model organisms, Arabidopsis thaliana and Nicotiana benthamiana. However, enzymes responsible for biosynthesizing CßG16α are essentially unknown, which limits the generation of agrichemicals that inhibit CßG16α synthesis. In this study, we discovered that OpgD from X. campestris pv. campestris converts linear ß-1,2-glucan to CßG16α. Structural and functional analyses revealed OpgD from X. campestris pv. campestris possesses an anomer-inverting transglycosylation mechanism, which is unprecedented among glycoside hydrolase family enzymes.

A large scale bacterial attraction assay: A new quantitative bacterial migration assay suitable for genetic screens.

Bacteria use various motility mechanisms to explore their environments. Chemotaxis is the ability of a motile bacterial cell to direct its movement in response to chemical gradients. A number of methods have been developed and widely used to study chemotactic responses to chemoeffectors including capillary, agar plug, microscopic slide, and microfluidic assays. While valuable, these assays are primarily designed to monitor rapid chemotactic responses to chemoeffectors on a small scale, which poses challenges in collecting large quantities of attracted bacteria. Consequently, these setups are not ideal for experiments like forward genetic screens. To overcome this limitation, we developed the Large Scale Bacterial Attraction assay (LSBA), which relies on the use of a Nalgene™ Reusable Filter Unit and other materials commonly found in laboratories. We validate the LSBA by investigating chemoeffector kinetics in the setup and by using chemoattractants to quantify the chemotactic response of wild-type, and motility impaired strains of the plant pathogenic bacterium Xanthomonas campestris pv. campestris and the environmental bacterium Shewanella oneidensis. We show that the LSBA establishes a long lasting chemoeffector gradient, that the setup can be used to quantify bacterial migration over time and that the LSBA offers the possibility to collect high numbers of attracted bacteria, making it suitable for genetic screens.

Design, synthesis and bactericidal activity of novel coumarin derivatives containing pyridinium salts.

Coumarin is a natural product known for its diverse biological activities. While its antifungal properties in agricultural chemistry have been extensively studied, there is limited research on its antibacterial potential. In this study, we developed several novel coumarin derivatives by combining coumarin with pyridinium salt through molecular hybridization and chemical synthesis. Our findings reveal that most of these derivatives exhibit promising antibacterial activity. Among them, derivative A25 has been identified as the most effective compound based on three-dimensional quantitative structure-activity relationships. It demonstrates significant in vitro and in vivo activity against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicola (Xoc), and Xanthomonas campestris pv. citri (Xac), outperforming the commercially available thiediazole copper. Initial investigations into its mechanism of action suggest that A25 disrupts the cell membranes of Xoc and Xoo, thereby inhibiting bacterial growth. Additionally, A25 enhances the activity of defense enzymes in rice and modulates the expression of proteins related to the pyruvate metabolism pathway. This dual action contributes to rice's resistance against bacterial infestation. We anticipate that this study will serve as a foundation for the development of coumarin-based bactericides.

Study of cabbage antioxidant system response on early infection stage of Xanthomonas campestris pv. campestris.

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) significantly affects the production of cabbage and other cruciferous vegetables. Plant antioxidant system plays an important role in pathogen invasion and is one of the main mechanisms underlying resistance to biological stress. Therefore, it is important to study the resistance mechanisms of the cabbage antioxidant system during the early stages of Xcc. In this study, 108 CFU/mL (OD600 = 0.1) Xcc race1 was inoculated on "zhonggan 11" cabbage using the spraying method. The effects of Xcc infection on the antioxidant system before and after Xcc inoculation (0, 1, 3, and 5 d) were studied by physiological indexes determination, transcriptome and metabolome analyses. We concluded that early Xcc infection can destroy the balance of the active oxygen metabolism system, increase the generation of free radicals, and decrease the scavenging ability, leading to membrane lipid peroxidation, resulting in the destruction of the biofilm system and metabolic disorders. In response to Xcc infection, cabbage clears a series of reactive oxygen species (ROS) produced during Xcc infection via various antioxidant pathways. The activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased after Xcc infection, and the ROS scavenging rate increased. The biosynthesis of non-obligate antioxidants, such as ascorbic acid (AsA) and glutathione (GSH), is also enhanced after Xcc infection. Moreover, the alkaloid and vitamin contents increased significantly after Xcc infection. We concluded that cabbage could resist Xcc invasion by maintaining the stability of the cell membrane system and improving the biosynthesis of antioxidant substances and enzymes after infection by Xcc. Our results provide theoretical basis and data support for subsequent research on the cruciferous vegetables resistance mechanism and breeding to Xcc.

Crystal structure of a newly identified M61 family aminopeptidase with broad substrate specificity that is solely responsible for recycling acidic amino acids.

Aminopeptidases with varied substrate specificities are involved in different crucial physiological processes of cellular homeostasis. They also have wide applications in food and pharma industries. Within the bacterial cell, broad specificity aminopeptidases primarily participate in the recycling of amino acids by degrading oligopeptides generated via primary proteolysis mediated by cellular ATP-dependent proteases. However, in bacteria, a truly broad specificity enzyme, which can cleave off acidic, basic, Gly and hydrophobic amino acid residues, is extremely rare. Here, we report structure-function of a putative glycyl aminopeptidase (M61xc) from Xanthomonas campestris pv campestris (Xcc) belonging to the M61 peptidase family. The enzyme exhibits broad specificity and cleaves Ala, Leu, Asp, Glu, Met, Ser, Phe, Tyr, Gly, Arg, and Lys at the N terminus, optimally of peptides with a length of 3-7 amino acids. Further, we report the high-resolution crystal structure of M61xc in the apo form (2.1 Å) and bestatin-bound form (1.95 Å), detailing its catalytic and substrate preference mechanisms. Comparative analysis of enzyme activity in crude cell extracts from both wild-type and m61xc-knockout mutant strains of Xcc has elucidated the unique intracellular role of M61xc. This study suggests that M61xc is the exclusive enzyme in these bacteria that is responsible for liberating Asp/Glu residues from the N-termini of peptides. Also, in view of its broad specificity and peptide degradation ability, it could be considered equivalent to M1 or other oligomeric peptidases from families like M17, M18, M42 or S9, who have an important auxiliary role in post-proteasomal protein degradation in prokaryotes.

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.

Characterisation of New Foxunavirus Phage Murka with the Potential of Xanthomonas campestris pv. campestris Control.

Phages of phytopathogenic bacteria are considered to be promising agents for the biological control of bacterial diseases in plants. This paper reports on the isolation and characterisation of a new Xanthomonas campestris pv. campestris phage, Murka. Phage morphology and basic kinetic characteristics of the infection were determined, and a phylogenomic analysis was performed. The phage was able to lyse a reasonably broad range (64%, 9 of the 14 of the Xanthomonas campestris pv. campestris strains used in the study) of circulating strains of the cabbage black rot pathogen. This lytic myovirus has a DNA genome of 44,044 bp and contains 83 predicted genes. Taxonomically, it belongs to the genus Foxunavirus. This bacteriophage is promising for use as a possible means of biological control of cabbage black rot.

The type VI secretion system of Stenotrophomonas rhizophila CFBP13503 limits the transmission of Xanthomonas campestris pv. campestris 8004 from radish seeds to seedlings.

Stenotrophomonas rhizophila CFBP13503 is a seedborne commensal bacterial strain, which is efficiently transmitted to seedlings and can outcompete the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc8004). The type VI secretion system (T6SS), an interference contact-dependent mechanism, is a critical component of interbacterial competition. The involvement of the T6SS of S. rhizophila CFBP13503 in the inhibition of Xcc8004 growth and seed-to-seedling transmission was assessed. The T6SS cluster of S. rhizophila CFBP13503 and nine putative effectors were identified. Deletion of two T6SS structural genes, hcp and tssB, abolished the competitive advantage of S. rhizophila against Xcc8004 in vitro. The population sizes of these two bacterial species were monitored in seedlings after inoculation of radish seeds with mixtures of Xcc8004 and either S. rhizophila wild-type (wt) strain or isogenic hcp mutant. A significant decrease in the population size of Xcc8004 was observed during confrontation with the S. rhizophila wt in comparison with T6SS-deletion mutants in germinated seeds and seedlings. We found that the T6SS distribution among 835 genomes of the Stenotrophomonas genus is scarce. In contrast, in all available S. rhizophila genomes, T6SS clusters are widespread and mainly belong to the T6SS group i4. In conclusion, the T6SS of S. rhizophila CFBP13503 is involved in the antibiosis against Xcc8004 and reduces seedling transmission of Xcc8004 in radish. The distribution of this T6SS cluster in the S. rhizophila complex could make it possible to exploit these strains as biocontrol agents against X. campestris pv. campestris.

Disinfection Efficacy of Electrohydraulic Discharge Plasma against Xanthomonas campestris pv campestris: A Sustainable Seed Treatment Approach.

The prevalence of plant diseases caused by pathogens such as Xanthomonas campestris pv campestris (Xcc) poses a significant challenge to sustainable agriculture, necessitating the development of effective and eco-friendly disinfection methods. In this study, we investigated the efficacy of electrohydraulic discharge plasma (EHDP) as a promising alternative for disinfection against Xcc, a pathogen responsible for black rot in cruciferous vegetables. Unlike conventional gas-phase plasma, EHDP introduces two pivotal components: gas-liquid interface plasma (GLIP) and its consequential byproduct, plasma-activated water (PAW). While GLIP enables dual-phase production of reactive oxygen and nitrogen species (RONS), PAW is a reservoir of liquid-phase long-lived RONS, thereby enhancing its bactericidal efficacy. In our evaluations, we tested EHDP-induced GLIP and EHDP-induced PAW against Xcc cells in both in vitro (Xcc suspension) and in vivo (Xcc-inoculated cabbage seeds) settings, achieving noteworthy results. Within 15 min, these methods eliminated ∼98% of the Xcc cells in suspension. For in vivo assessments, nontreated seeds exhibited an infection rate of 98%. In contrast, both EHDP treatments showed a significant reduction, with ∼60% fewer seeds infected while maintaining ∼90% germination rate. In addition, the liquid-phase RONS in EHDP-PAW may enhance seed vigor with a faster germination rate within the initial 5 days. Remarkably, around 90% of EHDP-PAW-treated seeds yielded healthy seedlings, indicating dual benefits in bacterial suppression and seed growth stimulation. In contrast, the percentage of healthy seedlings from nontreated, Xcc-inoculated seeds was approximately 70%. Our research demonstrates the feasibility of using eco-friendly EHDP in the seed disinfection process.

Characterization of Emerging Xanthomonas campestris Isolates on the Nonnative Weed Garlic Mustard (Alliaria petiolata).

Nonnative plant infestations provide unique opportunities to investigate pathogen emergence with evolutionarily recent plant introduction events. The widespread distribution of invasive plants and their proximity to genetically related crops highlights the risks of nonnative plants acting as ancillary hosts and fostering microbial recombination and pathogen selection. Garlic mustard (Alliaria petiolata) is a widespread, nonnative cruciferous weed that grows throughout North America and along the forested edges of diverse agricultural fields. The recent identification of a novel Xanthomonas campestris pv. incanae strain isolated from a diseased A. petiolata population led to the current investigation of the distribution and diversity of X. campestris isolates from naturally infected A. petiolata. A total of 14 diseased A. petiolata sites were sampled across three states, leading to the identification of diverse X. campestris pathotypes and genotypes. Pathogenicity assays and multilocus sequence analyses identified pathogenic X. c. pv. incanae and X. c. pv. barbareae strains collected from disparate A. petiolata populations. Moreover, independently collected X. c. pv. incanae strains demonstrated a broad cruciferous host range by infecting cabbage (Brassica oleracea var. capitata), garden stock (Matthiola incana), and the cover crop yellow mustard (Guillenia flavescens). This study highlights the genetic variability and host potential of natural X. campestris populations and the potential risks to Brassica crops via widespread, dense garlic mustard reservoirs.

Complete Genome Sequence of a Copper-Resistant Xanthomonas campestris pv. campestris Strain Isolated from Broccoli in Mauritius Suggests Adaptive Gene Gain Through Horizontal Gene Transfer.

Bacterial adaptation is facilitated by the presence of mobile genetic elements and horizontal gene transfer of genes, such as those coding for virulence factors or resistance to antimicrobial compounds. A hybrid assembly of Nanopore MinIon long-read and Illumina short-read data was produced from a copper-resistant Xanthomonas campestris pv. campestris strain isolated from symptomatic broccoli leaves in Mauritius. We obtained a 5.2-Mb high-quality chromosome and no plasmid. We found four genomic islands, three of which were characterized as integrative conjugative elements or integrative mobilizable elements. These genomic islands carried type III effectors and the copper resistance copLABMGF system involved in pathogenicity and environmental adaptation, respectively.

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.

Targeted knockout of early nodulin-like 3 (MusaENODL3) gene in banana reveals its function in resistance to Xanthomonas wilt disease.

Nodulins and nodulin-like proteins play an essential role in the symbiotic associations between legumes and Rhizobium bacteria. Their role extends beyond the leguminous species, as numerous nodulin-like proteins, including early nodulin-like proteins (ENODL), have been identified in various non-leguminous plants, implying their involvement in functions beyond nodulation, such as nutrient transport and growth modulation. Some ENODL proteins have been associated with plant defense against pathogens, as evident in banana infected with Xanthomonas campestris pv. musacearum (Xcm) causing banana Xanthomonas wilt (BXW) disease. Nonetheless, the specific role of ENODL in plant defense remains to be fully elucidated. The MusaENODL3 gene was found to be repressed in BXW-resistant banana progenitor 'Musa balbisiana' and 20-fold upregulated in BXW-susceptible cultivar 'Gonja Manjaya' upon early infection with Xcm. To further unravel the role of the ENODL gene in disease resistance, the CRISPR/Cas9 system was employed to disrupt the MusaENODL3 gene in 'Gonja Manjaya' precisely. Analysis of the enodl3 edited events confirmed the accurate manipulation of the MusaENODL3 gene. Disease resistance and gene expression analysis demonstrated that editing the MusaENODL3 gene resulted in resistance to BXW disease, with 50% of the edited plants remaining asymptomatic. The identification and manipulation of the MusaENODL3 gene highlight its potential as a critical player in plant-pathogen interactions, offering new opportunities for enhancing disease resistance in crops like banana, an important staple food crop and source of income for resource-poor farmers in the tropics. This study provides the first evidence of the direct role of the ENODL3 gene in developing disease-resistant plants.

Characterization of multiple lysophosphatidic acid acyltransferases in the plant pathogen Xanthomonas campestris.

Phosphatidic acid (PA) is the precursor of most phospholipids like phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In bacteria, its biosynthesis begins with the acylation of glycerol-3-phosphate to lysophosphatidic acid (LPA), which is further acylated to PA by the PlsC enzyme. Some bacteria, like the plant pathogen Xanthomonas campestris, use a similar pathway to acylate lysophosphatidylcholine to phosphatidylcholine (PC). Previous studies assigned two acyltransferases to PC formation. Here, we set out to study their activity and found a second much more prominent function of these enzymes in LPA to PA conversion. This PlsC-like activity was supported by the functional complementation of a temperature-sensitive plsC-deficient Escherichia coli strain. Biocomputational analysis revealed two further PlsC homologs in X. campestris. The cellular levels of the four PlsC-like proteins varied with respect to growth phase and growth temperature. To address the question whether these enzymes have redundant or specific functions, we purified two recombinant, detergent-solubilized enzymes in their active form, which enabled the first direct biochemical comparison of PlsC isoenzymes from the same organism. Overlapping but not identical acyl acceptor and acyl donor preferences suggest redundant and specialized functions of the X. campestris PlsC enzymes. The altered fatty acid composition in plsC mutant strains further supports the functional differentiation of these enzymes.

Complete genome sequence of Xanthomonas phage M29, a new member of Foxunavirus isolated in the Czech Republic.

The newly discovered Xanthomonas phage M29 (Xp M29) is the first lytic phage infecting Xanthomonas campestris pv. campestris (Xcc) that was isolated from cabbage leaves in the Czech Republic. The phage consists of icosahedral head approximately 60 nm in diameter and a probably contractile tail of 170 nm. The complete genome size was 42 891 bp, with a G + C content of 59.6%, and 69 ORFs were predicted on both strands. Pairwise nucleotide comparison showed the highest similarity with the recently described Xanthomonas phage FoX3 (91.2%). Bacteriophage Xp M29 has a narrow host range infecting 5 out of 21 isolates of Xcc. Xp M29 is a novel species in a newly formed genus Foxunavirus assigned directly to the class Caudoviricetes.