[Genetic dissection of rice resistance to bacterial blight].

Bacterial blight, a major disease in rice, poses a serious impact on rice production. In this study, a doubled haploid (DH) population derived from a cross between the introduced japonica cultivar 'Maybelle' and the indica landrace 'Baiyeqiu' was used to investigate the pathogenicity of four pathogen races causing bacterial blight. The results showed that the pathogenicity of all the pathogen races exhibited continuous, transgressive distribution in the DH population. Moreover, strong correlations existed between every two pathogen races, with the correlation coefficients ranging from 0.3 to 0.6. A total of 12 quantitative trait loci (QTLs) distributed on chromosomes 1, 2, 3, 5, 6, 7, 9, and 12 were detected for rice bacterial blight, explaining 4.95% to 16.05% of the phenotype. Among these QTLs, a major QTL located in the interval RM6024-RM163 on chromosome 5 was detected in three pathogen races. In addition, the pyramiding of the positive alleles can apparently improve the rice resistance to bacterial blight. This study is of great significance for broadening the genetic resources with resistance to bacterial blight in China.

The timing of bacterial mesophyll infection shapes the leaf chemical landscape.

Chemistry in eukaryotic intercellular spaces is shaped by both hosts and symbiotic microorganisms such as bacteria. Pathogenic microorganisms like barley-associated Xanthomonas translucens (Xt) swiftly overtake the inner leaf tissue becoming the dominant microbial community member during disease development. The dynamic metabolic changes due to Xt pathogenesis in the mesophyll spaces remain unknown. Genomic group I of Xt consists of two barley-infecting lineages: pathovar translucens (Xtt) and pathovar undulosa (Xtu). Xtu and Xtt, although genomically distinct, cause similar water-soaked lesions. To define the metabolic signals associated with inner leaf colonization, we used untargeted metabolomics to characterize Xtu and Xtt metabolism signatures associated with mesophyll growth. We found that mesophyll apoplast fluid from infected tissue yielded a distinct metabolic profile and shift from catabolic to anabolic processes over time compared to water-infiltrated control. The pathways with the most differentially expressed metabolites by time were glycolysis, tricarboxylic acid cycle, sucrose metabolism, pentose interconversion, amino acids, galactose, and purine metabolism. Hierarchical clustering and principal component analysis showed that metabolic changes were more affected by the time point rather than the individual colonization of the inner leaves by Xtt compared to Xtu. Overall, in this study, we identified metabolic pathways that explain carbon and nitrogen usage during host-bacterial interactions over time for mesophyll tissue colonization. This foundational research provides initial insights into shared metabolic strategies of inner leaf colonization niche occupation by related but phylogenetically distinct phyllosphere bacteria. IMPORTANCE: The phyllosphere is a habitat for microorganisms including pathogenic bacteria. Metabolic shifts in the inner leaf spaces for most plant-microbe interactions are unknown, especially for Xanthomonas species in understudied plants like barley (Hordeum vulgare). Xanthomonas translucens pv. translucens (Xtt) and Xanthomonas translucens pv. undulosa (Xtu) are phylogenomically distinct, but both colonize barley leaves for pathogenesis. In this study, we used untargeted metabolomics to shed light on Xtu and Xtt metabolic signatures. Our findings revealed a dynamic metabolic landscape that changes over time, rather than exhibiting a pattern associated with individual pathovars. These results provide initial insights into the metabolic mechanisms of X. translucens inner leaf pathogenesis.

Application and Mechanism of Cryptolepine and Neocryptolepine Derivatives as T3SS Inhibitors for Control of Bacterial Leaf Blight on Rice.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv oryzae (Xoo) is extremely harmful to rice production. The traditional control approach is to use bactericides that target key bacterial growth factors, but the selection pressure on the pathogen makes resistant strains the dominant bacterial strains, leading to a decline in bactericidal efficacy. Type III secretion system (T3SS) is a conserved and critical virulence factor in most Gram-negative bacteria, and its expression or absence does not affect bacterial growth, rendering it an ideal target for creating drugs against Gram-negative pathogens. In this work, we synthesized a range of derivatives from cryptolepine and neocryptolepine. We found that compound Z-8 could inhibit the expression of Xoo T3SS-related genes without affecting the growth of bacteria. an in vivo bioassay showed that compound Z-8 could effectively reduce the hypersensitive response (HR) induced by Xoo in tobacco and reduce the pathogenicity of Xoo in rice. Furthermore, it exhibited synergy in control of bacterial leaf blight when combined with the quorum quenching bacterial F20.

Xanthomonas Phage PBR31: Classifying the Unclassifiable.

The ability of bacteriophages to destroy bacteria has made them the subject of extensive research. Interest in bacteriophages has recently increased due to the spread of drug-resistant bacteria, although genomic research has not kept pace with the growth of genomic data. Genomic analysis and, especially, the taxonomic description of bacteriophages are often difficult due to the peculiarities of the evolution of bacteriophages, which often includes the horizontal transfer of genes and genomic modules. The latter is particularly pronounced for temperate bacteriophages, which are capable of integration into the bacterial chromosome. Xanthomonas phage PBR31 is a temperate bacteriophage, which has been neither described nor classified previously, that infects the plant pathogen Xanthomonas campestris pv. campestris. Genomic analysis, including phylogenetic studies, indicated the separation of phage PBR31 from known classified bacteriophages, as well as its distant relationship with other temperate bacteriophages, including the Lederbervirus group. Bioinformatic analysis of proteins revealed distinctive features of PBR31, including the presence of a protein similar to the small subunit of D-family DNA polymerase and advanced lysis machinery. Taxonomic analysis showed the possibility of assigning phage PBR31 to a new taxon, although the complete taxonomic description of Xanthomonas phage PBR31 and other related bacteriophages is complicated by the complex evolutionary history of the formation of its genome. The general biological features of the PBR31 phage were analysed for the first time. Due to its presumably temperate lifestyle, there is doubt as to whether the PBR31 phage is appropriate for phage control purposes. Bioinformatics analysis, however, revealed the presence of cell wall-degrading enzymes that can be utilised for the treatment of bacterial infections.

Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri.

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease.

Ectopic Expression of the Executor-Type R Gene Paralog Xa27B in Rice Leads to Spontaneous Lesions and Enhanced Disease Resistance.

Plant disease resistance (R) gene-mediated effector-triggered immunity (ETI) is usually associated with hypersensitive response (HR) and provides robust and race-specific disease resistance against pathogenic infection. The activation of ETI and HR in plants is strictly regulated, and improper activation will lead to cell death. Xa27 is an executor-type R gene in rice induced by the TAL effector AvrXa27 and confers disease resistance to Xanthomonas oryzae pv. oryzae (Xoo). Here we reported the characterization of a transgenic line with lesion mimic phenotype, designated as Spotted leaf and resistance 1 (Slr1), which was derived from rice transformation with a genomic subclone located 5,125 bp downstream of the Xa27 gene. Slr1 develops spontaneous lesions on its leaves caused by cell death and confers disease resistance to both Xoo and Xanthomonas oryzae pv. oryzicola. Further investigation revealed that the Slr1 phenotype resulted from the ectopic expression of an Xa27 paralog gene, designated as Xa27B, in the inserted DNA fragment at the Slr1 locus driven by a truncated CaMV35Sx2 promoter in reverse orientation. Disease evaluation of IRBB27, IR24, and Xa27B mutants with Xoo strains expressing dTALE-Xa27B confirmed that Xa27B is a functional executor-type R gene. The functional XA27B-GFP protein was localized to the endoplasmic reticulum and apoplast. The identification of Xa27B as a new functional executor-type R gene provides additional genetic resources for studying the mechanism of executor-type R protein-mediated ETI and developing enhanced and broad-spectrum disease resistance to Xoo through promoter engineering. [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.

Leaf microbiome dysbiosis triggered by T2SS-dependent enzyme secretion from opportunistic Xanthomonas pathogens.

In healthy plants, the innate immune system contributes to maintenance of microbiota homoeostasis, while disease can be associated with microbiome perturbation or dysbiosis, and enrichment of opportunistic plant pathogens like Xanthomonas. It is currently unclear whether the microbiota change occurs independently of the opportunistic pathogens or is caused by the latter. Here we tested if protein export through the type-2 secretion system (T2SS) by Xanthomonas causes microbiome dysbiosis in Arabidopsis thaliana in immunocompromised plants. We found that Xanthomonas strains secrete a cocktail of plant cell wall-degrading enzymes that promote Xanthomonas growth during infection. Disease severity and leaf tissue degradation were increased in A. thaliana mutants lacking the NADPH oxidase RBOHD. Experiments with gnotobiotic plants, synthetic bacterial communities and wild-type or T2SS-mutant Xanthomonas revealed that virulence and leaf microbiome composition are controlled by the T2SS. Overall, a compromised immune system in plants can enrich opportunistic pathogens, which damage leaf tissues and ultimately cause microbiome dysbiosis by facilitating growth of specific commensal bacteria.

Analysis of CRISPR-Cas loci distribution in Xanthomonas citri and its possible control by the quorum sensing system.

Xanthomonas is an important genus of plant-associated bacteria that causes significant yield losses of economically important crops worldwide. Different approaches have assessed genetic diversity and evolutionary interrelationships among the Xanthomonas species. However, information from clustered regularly interspaced short palindromic repeats (CRISPRs) has yet to be explored. In this work, we analyzed the architecture of CRISPR-Cas loci and presented a sequence similarity-based clustering of conserved Cas proteins in different species of Xanthomonas. Although absent in many investigated genomes, Xanthomonas harbors subtype I-C and I-F CRISPR-Cas systems. The most represented species, Xanthomonas citri, presents a great diversity of genome sequences with an uneven distribution of the CRISPR-Cas systems among the subspecies/pathovars. Only X. citri subsp. citri and X. citri pv. punicae have these systems, exclusively of subtype I-C system. Moreover, the most likely targets of the X. citri CRISPR spacers are viruses (phages). At the same time, few are plasmids, indicating that CRISPR/Cas system is possibly a mechanism to control the invasion of foreign DNA. We also showed in X. citri susbp. citri that the cas genes are regulated by the diffusible signal factor, the quorum sensing (QS) signal molecule, according to cell density increases, and under environmental stress like starvation. These results suggest that the regulation of CRISPR-Cas by QS occurs to activate the gene expression only during phage infection or due to environmental stresses, avoiding a possible reduction in fitness. Although more studies are needed, CRISPR-Cas systems may have been selected in the Xanthomonas genus throughout evolution, according to the cost-benefit of protecting against biological threats and fitness maintenance in challenging conditions.

The CsAP2-09-CsWRKY25-CsRBOH2 cascade confers resistance against citrus bacterial canker by regulating ROS homeostasis.

Citrus bacterial canker (CBC) is a serious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that adversely impacts the global citrus industry. In a previous study, we demonstrated that overexpression of an Xcc-inducible apetala 2/ethylene response factor encoded by Citrus sinensis, CsAP2-09, enhances CBC resistance. The mechanism responsible for this effect, however, is not known. In the present study, we showed that CsAP2-09 targeted the promoter of the Xcc-inducible WRKY transcription factor coding gene CsWRKY25 directly, activating its transcription. CsWRKY25 was found to localize to the nucleus and to activate transcriptional activity. Plants overexpressing CsWRKY25 were more resistant to CBC and showed higher expression of the respiratory burst oxidase homolog (RBOH) CsRBOH2, in addition to exhibiting increased RBOH activity. Transient overexpression assays in citrus confirmed that CsWRKY25 and CsRBOH2 participated in the generation of reactive oxygen species (ROS) bursts, which were able to restore the ROS degradation caused by CsAP2-09 knockdown. Moreover, CsWRKY25 was found to bind directly to W-box elements within the CsRBOH2 promoter. Notably, CsRBOH2 knockdown had been reported previously to reduce the CBC resistance, while demonstrated in this study, CsRBOH2 transient overexpression can enhance the CBC resistance. Overall, our results outline a pathway through which CsAP2-09-CsWRKY25 transcriptionally reprograms CsRBOH2-mediated ROS homeostasis in a manner conducive to CBC resistance. These data offer new insight into the mechanisms and regulatory pathways through which CsAP2-09 regulates CBC resistance, highlighting its potential utility as a target for the breeding of CBC-resistant citrus varieties.

Involvement of oxidative stress in localization of bacterial spot infection in pepper plants.

Xanthomonas euvesicatoria is a major cause of bacterial spot disease in various crops. The present study was focused on the pathosystem pepper (Capsicum annuum L.) - X. euvesicatoria 269p (wild strain). The infectious process was studied using several different modes of in vivo inoculation under controlled conditions. The spread of the pathogen in different parts of the plants was monitored by a new qPCR procedure developed for the detection of X. euvesicatoria, as well as by re-isolation of viable bacterial cells. Photosynthesis, the number of viable pathogens, oxidative stress markers, activities of the main antioxidant enzymes, and levels of nonenzymatic antioxidants in the novel single-leaf model system were studied. The most important observation is that the invasion of the pathogen causes local infection and the dissemination of bacteria to the healthy parts of the host is blocked. The plants limit bacterial colonization around the entry points. Oxidative burst and alterations in antioxidant defenses are detected in infectious leaf lesions. Localized ROS overproduction resembles a hypersensitive response, but several differences can be observed. We assumed that pepper plants are more likely to manifest an intermediate phenotype, similar to lesions simulating disease or leaf flecking. By localizing the infection, possibly involving oxidative stress, the plant survives. However, the same applies to bacteria. The pathogen multiplies at the infection spots and is transmitted to other plants. Our conclusion is that the intermediate phenotype in the studied pathosystem is an example of long and successful co-evolution for both species.

Xanthomonas chitinilytica sp. nov., a novel chitinolytic bacterium isolated from a microbial fermentation bed material.

A Gram-stain-negative bacterium, H13-6T, was isolated from a microbial fermentation bed material collected from a pig farm located in Yan'an, Shaanxi, China. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain H13-6T was affiliated with the genus Xanthomonas and showed highest similarity to strain Xanthomonas maliensis M97T (98.38%), Xanthomonas prunicola CFBP 8353T (98.26%) and Xanthomonas oryzae ATCC 35933T (98.11%). The pairwise ortho Average Nucleotide Identity values and the digital DNA-DNA hybridization values between strain H13-6T and the other Xanthomonas species were all below their respective cut-offs. Two genes encoding for chitinase were found and the strain showed a strong chitin-degrading activity. The major fatty acids were Iso-C15:0 (55.9%), Antesio-C15:0 (7.4%) and Iso-C11:0 (5.5%) and the major polar lipids were diphosphatidylglycerol, phosphatidyglycerol and phosphatidylethanolamine. Based on the phenotypic properties and phylogenetic distinctiveness, Xanthomonas chitinilytica was proposed as a novel species of the genus Xanthomonas, with strain H13-6T (= CGMCC 1.61317T = NBRC 115641T) as type strain.

Live tracking of a plant pathogen outbreak reveals rapid and successive, multidecade plasmid reduction.

Quickly understanding the genomic changes that lead to pathogen emergence is necessary to launch mitigation efforts and reduce harm. In this study, we tracked in real time a 2022 bacterial plant disease outbreak in U.S. geraniums (Pelargonium × hortorum) caused by Xhp2022, a novel lineage of Xanthomonas hortorum. Genomes from 31 Xhp2022 isolates from seven states showed limited chromosomal variation and all contained a single plasmid (p93). Time tree and single nucleotide polymorphism whole-genome analysis estimated that Xhp2022 emerged within the last decade. The phylogenomic analysis determined that p93 resulted from the cointegration of three plasmids (p31, p45, and p66) found sporadically across isolates from previous outbreaks. Although p93 had a 49 kb nucleotide reduction, it retained putative fitness genes, which became predominant in the 2022 outbreak. Overall, we demonstrated, through rapid whole-genome sequencing and analysis, a recent, traceable event of genome reduction for niche adaptation typically observed over millennia in obligate and fastidious pathogens.IMPORTANCEThe geranium industry, valued at $4 million annually, faces an ongoing Xanthomonas hortorum pv. pelargonii (Xhp) pathogen outbreak. To track and describe the outbreak, we compared the genome structure across historical and globally distributed isolates. Our research revealed Xhp population has not had chromosome rearrangements since 1974 and has three distinct plasmids. In 2012, we found all three plasmids in individual Xhp isolates. However, in 2022, the three plasmids co-integrated into one plasmid named p93. p93 retained putative fitness genes but lost extraneous genomic material. Our findings show that the 2022 strain group of the bacterial plant pathogen Xanthomonas hortorum underwent a plasmid reduction. We also observed several Xanthomonas species from different years, hosts, and continents have similar plasmids to p93, possibly due to shared agricultural settings. We noticed parallels between genome efficiency and reduction that we see across millennia with obligate parasites with increased niche specificity.

Rice foliar-adapted Pantoea species: Promising microbial biostimulants enhancing rice resilience against foliar pathogens, Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae.

Foliar fungal blast and bacterial leaf blight have significant impacts on rice production, and their management through host resistance and agrochemicals has proven inadequate. To achieve their sustainable management, innovative approaches like leveraging the foliar microbiome, which collaborates with plants and competes against pathogens, are essential. In our study, we isolated three Pantoea strains (P. agglomerans Os-Ep-PPA-1b, P. vagans Os-Ep-PPA-3b, and P. deleyi Os-Ep-VPA-9a) from the rice phylloplane. These isolates exhibited antimicrobial action through their metabolome and volatilome, while also promoting rice growth. Our analysis, using Gas Chromatography-Mass Spectrometry (GC-MS), revealed the presence of various antimicrobial compounds such as esters and fatty acids produced by these Pantoea isolates. Inoculating rice seedlings with P. agglomerans and P. vagans led to increased root and shoot growth. Additionally, bacterized seedlings displayed enhanced immunocompetence, as evidenced by upregulated expressions of defense genes (OsEDS1, OsFLS2, OsPDF2.2, OsACO4, OsICS OsPR1a, OsNPR1.3, OsPAD4, OsCERK1.1), along with heightened activities of defense enzymes like Polyphenol Oxidase and Peroxidase. These plants also exhibited elevated levels of total phenols. In field trials, the Pantoea isolates contributed to improved plant growth, exemplified by increased flag-leaf length, panicle number, and grains per panicle, while simultaneously reducing the incidence of chaffy grains. Hypersensitivity assays performed on a model plant, tobacco, confirmed the non-pathogenic nature of these Pantoea isolates. In summary, our study underscores the potential of Pantoea bacteria in combatting rice foliar diseases. Coupled with their remarkable growth-promoting and biostimulant capabilities, these findings position Pantoea as promising agents for enhancing rice cultivation.

Transcriptome profiling of type VI secretion system core gene tssM mutant of Xanthomonas perforans highlights regulators controlling diverse functions ranging from virulence to metabolism.

IMPORTANCE: T6SS has received attention due to its significance in mediating interorganismal competition through contact-dependent release of effector molecules into prokaryotic and eukaryotic cells. Reverse-genetic studies have indicated the role of T6SS in virulence in a variety of plant pathogenic bacteria, including the one studied here, Xanthomonas. However, it is not clear whether such effect on virulence is merely due to a shift in the microbiome-mediated protection or if T6SS is involved in a complex virulence regulatory network. In this study, we conducted in vitro transcriptome profiling in minimal medium to decipher the signaling pathways regulated by tssM-i3* in X. perforans AL65. We show that TssM-i3* regulates the expression of a suite of genes associated with virulence and metabolism either directly or indirectly by altering the transcription of several regulators. These findings further expand our knowledge on the intricate molecular circuits regulated by T6SS in phytopathogenic bacteria.

Interrogating two extensively self-targeting Type I CRISPR-Cas systems in Xanthomonas albilineans reveals distinct anti-CRISPR proteins that block DNA degradation.

CRISPR-Cas systems store fragments of invader DNA as spacers to recognize and clear those same invaders in the future. Spacers can also be acquired from the host's genomic DNA, leading to lethal self-targeting. While self-targeting can be circumvented through different mechanisms, natural examples remain poorly explored. Here, we investigate extensive self-targeting by two CRISPR-Cas systems encoding 24 self-targeting spacers in the plant pathogen Xanthomonas albilineans. We show that the native I-C and I-F1 systems are actively expressed and that CRISPR RNAs are properly processed. When expressed in Escherichia coli, each Cascade complex binds its PAM-flanked DNA target to block transcription, while the addition of Cas3 paired with genome targeting induces cell killing. While exploring how X. albilineans survives self-targeting, we predicted putative anti-CRISPR proteins (Acrs) encoded within the bacterium's genome. Screening of identified candidates with cell-free transcription-translation systems and in E. coli revealed two Acrs, which we named AcrIC11 and AcrIF12Xal, that inhibit the activity of Cas3 but not Cascade of the respective system. While AcrF12Xal is homologous to AcrIF12, AcrIC11 shares sequence and structural homology with the anti-restriction protein KlcA. These findings help explain tolerance of self-targeting through two CRISPR-Cas systems and expand the known suite of DNA degradation-inhibiting Acrs.

Population Genomics Reveals an Emerging Lineage of Xanthomonas perforans on Pepper.

Xanthomonas perforans-the dominant causal agent of bacterial leaf spot of tomato-is an emerging pathogen of pepper, indicative of a potential host expansion across the southeastern United States. However, studies of the genetic diversity and evolution of X. perforans from pepper remain limited. In this study, the whole-genome sequences of 35 X. perforans strains isolated from pepper from four fields and two transplant facilities across southwest Florida between 2019 and 2021 were used to compare genomic divergence, evolution, and variation in type III secreted effectors. Phylogenetic analysis based on core genes revealed that all 35 X. perforans strains formed one genetic cluster with pepper and tomato strains from Alabama and Turkey and were closely related to strains isolated from tomato in Indiana, Mexico, and Louisiana. The in planta population growth of tomato strains isolated from Indiana, Mexico, Louisiana, and Turkey in pepper leaf mesophyll was on par with pepper X. perforans and X. euvesicatoria strains. Molecular clock analysis of the 35 Florida strains dated their emergence to approximately 2017. While strains varied in copper tolerance, all sequenced strains harbored the avrHah1 transcription activation-like effector located on a conjugative plasmid, not previously reported in Florida. Our findings suggest that there is a geographically distributed lineage of X. perforans strains on tomato that has the genetic background to cause disease on pepper. Moreover, this study clarifies potential adaptive variants of X. perforans on pepper that could help forecast the emergence of such strains and enable immediate or preemptive intervention.

Tal6b/AvrXa27A, a hidden TALE targeting the susceptibility gene OsSWEET11a and the resistance gene Xa27 in rice.

Xanthomonas oryzae pv. oryzae (Xoo) secretes transcription activator-like effectors (TALEs) to activate rice susceptibility (S) genes, causing bacterial blight (BB), as well as resistance (R) genes, leading to defense against BB. This activation follows a gene-for-gene paradigm that results in an arms race between the TALE of the pathogen and effector-binding elements (EBEs) in the promoters of host genes. In this study, we characterized a novel TALE, designated Tal6b/AvrXa27A, that activates the rice S gene OsSWEET11a and the rice R gene Xa27. Tal6b/AvrXa27A is a member of the AvrXa27/TalAO class and contains 16 repeat variable diresidues (RVDs); one RVD is altered and one is deleted in Tal6b/AvrXa27A compared with AvrXa27, a known avirulence (avr) effector of Xa27. Tal6b/AvrXa27A can transcriptionally activate the expression of Xa27 and OsSWEET11a via EBEs in their corresponding promoters, leading to effector-triggered immunity and susceptibility, respectively. The 16 RVDs in Tal6b/AvrXa27A have no obvious similarity to the 24 RVDs in the effector PthXo1, but EBETal6b and EBEPthXo1 are overlapped in the OsSWEET11a promoter. Tal6b/AvrXa27A is prevalent among Asian Xoo isolates, but PthXo1 has only been reported in the Philippine strain PXO99A. Genome editing of EBETal6b in the OsSWEET11a promoter further confirmed the requirement for OsSWEET11a expression in Tal6b/AvrXa27A-dependent susceptibility to Xoo. Moreover, Tal6b/AvrXa27A resulted in higher transcription of Xa27 than of OsSWEET11a, which led to a strong, rapid resistance response that blocked disease development. These findings suggest that Tal6b/AvrXa27A has a dual function: triggering resistance by activating Xa27 gene expression as an avirulence factor and inducing transcription of the S gene OsSWEET11a, resulting in virulence. Intriguingly, Tal6b/AvrXa27A, but not AvrXa27, can bind to the promoter of OsSWEET11a. The underlying recognition mechanism for this binding remains unclear but appears to deviate from the currently accepted TALE code.

Pan-Genomic Analysis Identifies the Chinese Strain as a New Subspecies of Xanthomonas fragariae.

Xanthomonas fragariae is classified as a quarantine pathogen by the European and Mediterranean Plant Protection Organization. It commonly induces typical angular leaf spot (ALS) symptoms in strawberry leaves. X. fragariae strains from China (YL19, SHAQP01, and YLX21) exhibit ALS symptoms in leaves and more severe symptoms of dry cavity rot in strawberry crowns. Conversely, strains from other countries do not cause severe dry cavity rot symptoms in strawberries. After employing multilocus sequence analysis (MLSA), average nucleotide identity (ANI), and amino acid identity (AAI), we determined that Chinese strains of X. fragariae are genetically distinct from other strains and can be considered a new subspecies. Subsequent analysis of 63 X. fragariae genomes published at NCBI using IPGA and EDGAR3.0 revealed the pan-genomic profile, with 1,680 shared genes present in all 63 strains, including 71 virulence-related genes. Additionally, we identified 123 genes exclusive to all the Chinese strains, encompassing 12 virulence-related genes. The qRT-PCR analysis demonstrated that the expression of XopD, XopG1, CE8, GT2, and GH121 out of 12 virulence-related genes of Chinese strains (YL19) exhibited a constant increase in the early stages (6, 24, 54, and 96 hours postinoculation [hpi]) of strawberry leaf infected by YL19. So, the presence of XopD, XopG1, CE8, GT2, and GH121 in Chinese strains may play important roles in the early infection process of Chinese strains. These findings offer novel insights into comprehending the population structure and variation in the pathogenic capacity of X. fragariae.

Flagellar interference with plasmid uptake in biofilms: a joint experimental and modeling study.

Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of Xanthomonas retroflexus (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated fliM mutants of the X. retroflexus wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of Pseudomonas putida suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.IMPORTANCEBiofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species, Xanthomonas retroflexus and Pseudomonas putida. Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms.

A Community-Curated DokuWiki Resource on Diagnostics, Diversity, Pathogenicity, and Genetic Control of Xanthomonads.

Xanthomonads, including Xanthomonas and Xylella species, constitute a large and significant group of economically and ecologically important plant pathogens. Up-to-date knowledge of these pathogens and their hosts is essential for the development of suitable control measures. Traditional review articles or book chapters have inherent limitations, including static content and rapid obsolescence. To address these challenges, we have developed a Web-based knowledge platform dedicated to xanthomonads, inspired by the concept of living systematic reviews. This platform offers a dynamic resource that encompasses bacterial virulence factors, plant resistance genes, and tools for diagnostics and genetic diversity studies. Our goal is to facilitate access for newcomers to the field, provide continuing education opportunities for students, assist plant protection services with diagnostics, provide valuable information to breeders on sources of resistance and breeding targets, and offer comprehensive expert knowledge to other stakeholders interested in plant-pathogenic xanthomonads. This resource is available for queries and updates at https://euroxanth.ipn.pt. [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.