Xanthomonas citri subsp. citri type III effector PthA4 directs the dynamical expression of a putative citrus carbohydrate-binding protein gene for canker formation.

Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker, elicits canker symptoms in citrus plants because of the transcriptional activator-like (TAL) effector PthA4, which activates the expression of the citrus susceptibility gene CsLOB1. This study reports the regulation of the putative carbohydrate-binding protein gene Cs9g12620 by PthA4-mediated induction of CsLOB1 during Xcc infection. We found that the transcription of Cs9g12620 was induced by infection with Xcc in a PthA4-dependent manner. Even though it specifically bound to a putative TAL effector-binding element in the Cs9g12620 promoter, PthA4 exerted a suppressive effect on the promoter activity. In contrast, CsLOB1 bound to the Cs9g12620 promoter to activate its expression. The silencing of CsLOB1 significantly reduced the level of expression of Cs9g12620, which demonstrated that Cs9g12620 was directly regulated by CsLOB1. Intriguingly, PhtA4 interacted with CsLOB1 and exerted feedback control that suppressed the induction of expression of Cs9g12620 by CsLOB1. Transient overexpression and gene silencing revealed that Cs9g12620 was required for the optimal development of canker symptoms. These results support the hypothesis that the expression of Cs9g12620 is dynamically directed by PthA4 for canker formation through the PthA4-mediated induction of CsLOB1.

Comparative transcriptomics reveals a highly polymorphic Xanthomonas HrpG virulence regulon.

BACKGROUND: Bacteria of the genus Xanthomonas cause economically significant diseases in various crops. Their virulence is dependent on the translocation of type III effectors (T3Es) into plant cells by the type III secretion system (T3SS), a process regulated by the master response regulator HrpG. Although HrpG has been studied for over two decades, its regulon across diverse Xanthomonas species, particularly beyond type III secretion, remains understudied. RESULTS: In this study, we conducted transcriptome sequencing to explore the HrpG regulons of 17 Xanthomonas strains, encompassing six species and nine pathovars, each exhibiting distinct host and tissue specificities. We employed constitutive expression of plasmid-borne hrpG*, which encodes a constitutively active form of HrpG, to induce the regulon. Our findings reveal substantial inter- and intra-specific diversity in the HrpG* regulons across the strains. Besides 21 genes directly involved in the biosynthesis of the T3SS, the core HrpG* regulon is limited to only five additional genes encoding the transcriptional activator HrpX, the two T3E proteins XopR and XopL, a major facility superfamily (MFS) transporter, and the phosphatase PhoC. Interestingly, genes involved in chemotaxis and genes encoding enzymes with carbohydrate-active and proteolytic activities are variably regulated by HrpG*. CONCLUSIONS: The diversity in the HrpG* regulon suggests that HrpG-dependent virulence in Xanthomonas might be achieved through several distinct strain-specific strategies, potentially reflecting adaptation to diverse ecological niches. These findings enhance our understanding of the complex role of HrpG in regulating various virulence and adaptive pathways, extending beyond T3Es and the T3SS.

Engineering broad-spectrum resistance to rice bacterial blight by editing the OsETR susceptible haplotype using CRISPR/Cas9.

KEY MESSAGE: CRISPR/Cas9-mediated knockout of the susceptible haplotype of OsETR, encoding an embryogenesis transmembrane protein, confers broad-spectrum resistance to bacterial leaf blight in a susceptible rice cultivar without yield penalty.

Effect of mutation of phaC on carbon supply, extracellular polysaccharide production, and pathogenicity of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight in rice. Polyhydroxyalkanoates (PHAs) consitute a diverse group of biopolyesters synthesized by bacteria under nutrient-limited conditions. The phaC gene is important for PHA polymerization. We investigated the effects of phaC gene mutagensis in Xoo strain PXO99A. The phaC gene knock-out mutant exhibited reduced swarming ability relative to that of the wild-type. Under conditions where glucose was the sole sugar source, extracellular polysaccharide (EPS) production by ΔphaC declined by 44.8%. ΔphaC showed weak hypersensitive response (HR) induction in the leaves of non-host Nicotiana tabacum, concomitant with downregulation of hpa1 gene expression. When inoculated in rice leaves by the leaf-clipping method, ΔphaC displayed reduced virulence in terms of lesion length compared with the wild-type strain. The complemented strain showed no significant difference from the wild-type strain, suggesting that the deletion of phaC in Xoo induces significant alterations in various physiological and biological processes. These include bacterial swarming ability, EPS production, transcription of hrp genes, and glucose metabolism. These changes are intricately linked to the energy utilization and virulence of Xoo during plant infection. These findings revealed involvement of phaC in Xoo is in the maintaining carbon metabolism by functioning in the PHA metabolic pathway.

Hijacking of the methylglyoxal detoxification pathway: a new tactic of Xoo pathogenesis in rice.

Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial blight (BB), has developed a unique strategy to infect rice by hijacking the host's methylglyoxal (MG) detoxification pathway. This results in an over-accumulation of MG, which facilitates tissue colonization and evasion of host's immune responses. While MG role in abiotic stresses is well-documented, its involvement in biotic stresses has not been extensively explored. Recently, Fu et al. (2024) provided the first evidence of MG role in promoting Xoo pathogenesis in rice. This new virulence strategy contributes to the pathogen's remarkable adaptability and survival. In this mechanism of hijacking of MG detoxification pathway, Xoo induces OsWRKY62.1 to inhibit OsGLY II expression, leading to MG overaccumulation in infected rice cells. This excess MG hinders plant cell organelle function, creating a favorable environment for Xoo by compromising the rice defense system. In this article, we have presented our perspectives on how the BB pathogen adapts its virulence mechanisms to infect and cause disease in rice.

A cyclic di-GMP-binding adaptor protein interacts with a N5-glutamine methyltransferase to regulate the pathogenesis in Xanthomonas citri subsp. citri.

The second messenger cyclic diguanylate monophosphate (c-di-GMP) regulates a wide range of bacterial behaviours through diverse mechanisms and binding receptors. Single-domain PilZ proteins, the most widespread and abundant known c-di-GMP receptors in bacteria, act as trans-acting adaptor proteins that enable c-di-GMP to control signalling pathways with high specificity. This study identifies a single-domain PilZ protein, XAC3402 (renamed N5MapZ), from the phytopathogen Xanthomonas citri subsp. citri (Xcc), which modulates Xcc virulence by directly interacting with the methyltransferase HemK. Through yeast two-hybrid, co-immunoprecipitation and immunofluorescent staining, we demonstrated that N5MapZ and HemK interact directly under both in vitro and in vivo conditions, with the strength of the protein-protein interaction decreasing at high c-di-GMP concentrations. This finding distinguishes N5MapZ from other characterized single-domain PilZ proteins, as it was previously known that c-di-GMP enhances the interaction between those single-domain PilZs and their protein partners. This observation is further supported by the fact that the c-di-GMP binding-defective mutant N5MapZR10A can interact with HemK to inhibit the methylation of the class 1 translation termination release factor PrfA. Additionally, we found that HemK plays an important role in Xcc pathogenesis, as the deletion of hemK leads to extensive phenotypic changes, including reduced virulence in citrus plants, decreased motility, production of extracellular enzymes and stress tolerance. Gene expression analysis has revealed that c-di-GMP and the HemK-mediated pathway regulate the expression of multiple virulence effector proteins, uncovering a novel regulatory mechanism through which c-di-GMP regulates Xcc virulence by mediating PrfA methylation via the single-domain PilZ adaptor protein N5MapZ.

The underground world of plant disease: Rhizosphere dysbiosis reduces above-ground plant resistance to bacterial leaf spot and alters plant transcriptome.

Just as the human gut microbiome is colonized by a variety of microbes, so too is the rhizosphere of plants. An imbalance in this microbial community, known as dysbiosis, can have a negative impact on plant health. This study sought to explore the effect of rhizosphere dysbiosis on the health of tomato plants (Solanum lycopersicum L.), using them and the foliar bacterial spot pathogen Xanthomonas perforans as model organisms. The rhizospheres of 3-week-old tomato plants were treated with either streptomycin or water as a control, and then spray-inoculated with X. perforans after 24 h. Half of the plants that were treated with both streptomycin and X. perforans received soil microbiome transplants from uninfected plant donors 48 h after the streptomycin was applied. The plants treated with streptomycin showed a 26% increase in disease severity compared to those that did not receive the antibiotic. However, the plants that received the soil microbiome transplant exhibited an intermediate level of disease severity. The antibiotic-treated plants demonstrated a reduced abundance of rhizobacterial taxa such as Cyanobacteria from the genus Cylindrospermum. They also showed a down-regulation of genes related to plant primary and secondary metabolism, and an up-regulation of plant defence genes associated with induced systemic resistance. This study highlights the vital role that beneficial rhizosphere microbes play in disease resistance, even against foliar pathogens.

Novel trans-Resveratrol Derivatives: Design, Synthesis, Antibacterial Activity, and Mechanisms.

Rice bacterial leaf blight and rice bacterial leaf streak have induced tremendous damage to production of rice worldwide. To discover an effective novel antibacterial agent, a series of novel trans-resveratrol (RSV) derivatives containing 1,3,4-oxadiazole and amide moieties were designed and synthesized for the first time. Most of them showed excellent antibacterial activities against Xanthomonas oryzae pv oryzicola and Xanthomonas oryzae pv oryzae. Especially, compound J12 had the best inhibitory with the half-maximal effective concentration values of 4.2 and 5.0 mg/L, respectively, which were better than that of RSV (63.7 and 75.4 mg/L), bismerthiazol (79.5 and 89.6 mg/L), and thiodiazole copper (105.4 and 112.8 mg/L). Furthermore, compound J12 had an excellent control effect against rice bacterial leaf streak and rice bacterial leaf blight, with protective activities of 46.2 and 42.1% and curative activities of 44.5 and 41.7%, respectively. Preliminary mechanisms indicated that compound J12 could not only remarkably decrease biofilm formation, extracellular polysaccharide production, and the synthesis of extracellular enzymes but also destroy bacterial cell surface morphology, thereby reducing the pathogenicity of bacteria. In addition, compound J12 could increase the activity of defense-related enzymes and affect the expression of multiple pathogenic-related genes including plant-pathogen interaction, the MAPK signaling pathway, and phenylpropanoid biosynthesis, and this could improve the defense of rice against rice bacterial leaf streak infection. The present work indicates that the RSV derivatives can be used as promising candidates for the development of antibacterial agents.

VmsR, a LuxR-Type Regulator, Contributes to Virulence, Cell Motility, Extracellular Polysaccharide Production and Biofilm Formation in Xanthomonas oryzae pv. oryzicola.

LuxR-type regulators play pivotal roles in regulating numerous bacterial processes, including bacterial motility and virulence, thereby exerting a significant influence on bacterial behavior and pathogenicity. Xanthomonas oryzae pv. oryzicola, a rice pathogen, causes bacterial leaf streak. Our research has identified VmsR, which is a response regulator of the two-component system (TCS) that belongs to the LuxR family. These findings of the experiment reveal that VmsR plays a crucial role in regulating pathogenicity, motility, biofilm formation, and the production of extracellular polysaccharides (EPSs) in Xoc GX01. Notably, our study shows that the vmsR mutant exhibits a reduced swimming motility but an enhanced swarming motility. Furthermore, this mutant displays decreased virulence while significantly increasing EPS production and biofilm formation. We have uncovered that VmsR directly interacts with the promoter regions of fliC and fliS, promoting their expression. In contrast, VmsR specifically binds to the promoter of gumB, resulting in its downregulation. These findings indicate that the knockout of vmsR has profound effects on virulence, motility, biofilm formation, and EPS production in Xoc GX01, providing insights into the intricate regulatory network of Xoc.

Application of natural-products repurposing strategy to discover novel FtsZ inhibitors: Bactericidal evaluation and the structure-activity relationship of sanguinarine and its analogs.

The novel bactericidal target-filamentous temperature-sensitive protein Z (FtsZ)-has drawn the attention of pharmacologists to address the emerging issues with drug/pesticide resistance caused by pathogenic bacteria. To enrich the structural diversity of FtsZ inhibitors, the antibacterial activity and structure-activity relationship (SAR) of natural sanguinarine and its analogs were investigated by using natural-products repurposing strategy. Notably, sanguinarine and chelerythrine exerted potent anti-Xanthomonas oryzae pv. oryzae (Xoo) activity, with EC50 values of 0.96 and 0.93 mg L-1, respectively, among these molecules. Furthermore, these two compounds could inhibit the GTPase activity of XooFtsZ, with IC50 values of 241.49 µM and 283.14 µM, respectively. An array of bioassays including transmission electron microscopy (TEM), fluorescence titration, and Fourier transform infrared spectroscopy (FT-IR) co-verified that sanguinarine and chelerythrine were potential XooFtsZ inhibitors that could interfere with the assembly of FtsZ filaments by inhibiting the GTPase hydrolytic ability of XooFtsZ protein. Additionally, the pot experiment suggested that chelerythrine and sanguinarine demonstrated excellent curative activity with values of 59.52% and 54.76%, respectively. Excitedly, these two natural compounds also showed outstanding druggability, validated by acceptable drug-like properties and low toxicity on rice. Overall, the results suggested that chelerythrine was a new and potential XooFtsZ inhibitor to develop new bactericide and provided important guiding values for rational drug design of FtsZ inhibitors. Notably, our findings provide a novel strategy to discover novel, promising and green bacterial compounds for the management of plant bacterial diseases.

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.

Insights into bs5 resistance mechanisms in pepper against Xanthomonas euvesicatoria through transcriptome profiling.

BACKGROUND: Bacterial spot of pepper (BSP), caused by four different Xanthomonas species, primarily X. euvesicatoria (Xe), poses a significant challenge in pepper cultivation. Host resistance is considered the most important approach for BSP control, offering long-term protection and sustainability. While breeding for resistance to BSP for many years focused on dominant R genes, introgression of recessive resistance has been a more recent focus of breeding programs. The molecular interactions underlying recessive resistance remain poorly understood. RESULTS: In this study, transcriptomic analyses were performed to elucidate defense responses triggered by Xe race P6 infection by two distinct pepper lines: the Xe-resistant line ECW50R containing bs5, a recessive resistance gene that confers resistance to all pepper Xe races, and the Xe-susceptible line ECW. The results revealed a total of 3357 upregulated and 4091 downregulated genes at 0, 1, 2, and 4 days post-inoculation (dpi), with the highest number of differentially expressed genes (DEGs) observed at 2 dpi. Pathway analysis highlighted DEGs in key pathways such as plant-pathogen interaction, MAPK signaling pathway, plant hormone signal transduction, and photosynthesis - antenna proteins, along with cysteine and methionine metabolism. Notably, upregulation of genes associated with PAMP-Triggered Immunity (PTI) was observed, including components like FLS2, Ca-dependent pathways, Rboh, and reactive oxygen species (ROS) generation. In support of these results, infiltration of ECW50R leaves with bacterial suspension of Xe led to observable hydrogen peroxide accumulation without a rapid increase in electrolyte leakage, suggestive of the absence of Effector-Triggered Immunity (ETI). Furthermore, the study confirmed that bs5 does not disrupt the effector delivery system, as evidenced by incompatible interactions between avirulence genes and their corresponding dominant resistant genes in the bs5 background. CONCLUSION: Overall, these findings provide insights into the molecular mechanisms underlying bs5-mediated resistance in pepper against Xe and suggest a robust defense mechanism in ECW50R, primarily mediated through PTI. Given that bs5 provides early strong response for resistance, combining this resistance with other dominant resistance genes will enhance the durability of resistance to BSP.

Identification of candidate single-nucleotide polymorphisms (SNPs) and genes associated with sugarcane leaf scald disease.

Leaf scald, caused by Xanthomonas albilineans, is a severe disease affecting sugarcane worldwide. One of the most practical ways to control it is by developing resistant sugarcane cultivars. It is essential to identify genes associated with the response to leaf scald. A panel of 170 sugarcane genotypes was evaluated for resistance to leaf scald in field conditions for 2 years, followed by a 1-year greenhouse experiment. The phenotypic evaluation data showed a wide continuous distribution, with heritability values ranging from 0.58 to 0.84. Thirteen single nucleotide polymorphisms (SNPs) were identified, significantly associated with leaf scald resistance. Among these, eight were stable across multiple environments and association models. The candidate genes identified and validated based on RNA-seq and qRT-PCR included two genes that encode NB-ARC leucine-rich repeat (LRR)-containing domain disease-resistance protein. These findings provide a basis for developing marker-assisted selection strategies in sugarcane breeding programs.

Exploration of Type III effector Xanthomonas outer protein Q (XopQ) inhibitor from Picrasma quassioides as an antibacterial agent using chemoinformatics analysis.

The present study was focused on exploring the efficient inhibitors of closed state (form) of type III effector Xanthomonas outer protein Q (XopQ) (PDB: 4P5F) from the 44 phytochemicals of Picrasma quassioides using cutting-edge computational analysis. Among them, Kumudine B showed excellent binding energy (-11.0 kcal/mol), followed by Picrasamide A, Quassidine I and Quassidine J with the targeted closed state of XopQ protein compared to the reference standard drug (Streptomycin). The molecular dynamics (MD) simulations performed at 300 ns validated the stability of top lead ligands (Kumudine B, Picrasamide A, and Quassidine I)-bound XopQ protein complex with slightly lower fluctuation than Streptomycin. The MM-PBSA calculation confirmed the strong interactions of top lead ligands (Kumudine B and QuassidineI) with XopQ protein, as they offered the least binding energy. The results of absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis confirmed that Quassidine I, Kumudine B and Picrasamide A were found to qualify most of the drug-likeness rules with excellent bioavailability scores compared to Streptomycin. Results of the computational studies suggested that Kumudine B, Picrasamide A, and Quassidine I could be considered potential compounds to design novel antibacterial drugs against X. oryzae infection. Further in vitro and in vivo antibacterial activities of Kumudine B, Picrasamide A, and Quassidine I are required to confirm their therapeutic potentiality in controlling the X. oryzae infection.

Engineering a biomimicking strategy for discovering nonivamide-based quorum-sensing inhibitors for controlling bacterial infection.

The overuse of antibiotics over an extended period has led to increasing antibiotic resistance in pathogenic bacteria, culminating in what is now considered a global health crisis. To tackle the escalating disaster caused by multidrug-resistant pathogens, the development of new bactericides with new action mechanism is highly necessary. In this study, using a biomimicking strategy, a series of new nonivamide derivatives that feature an isopropanolamine moiety [the structurally similar to the diffusible signal factor (DSF) of Xanthomonas spp.] were prepared for serving as potential quorum-sensing inhibitors (QSIs). After screening and investigation of their rationalizing structure-activity relationships (SARs), compound A26 was discovered as the most optimal active molecule, with EC50 values of 9.91 and 7.04 µg mL-1 against Xanthomonas oryzae pv oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac). A docking study showed that compound A26 exhibited robust interactions with Glu A: 161 of RpfF, which was strongly evidenced by fluorescence titration assay (KA value for Xoo RpfF-A26 = 104.8709 M-1). Furthermore, various bioassays showed that compound A26 could inhibit various bacterial virulence factors, including biofilm formation, extracellular polysaccharides (EPS), extracellular enzyme activity, DSF production, and swimming motility. In addition, in vivo anti-Xoo results showed that compound A26 had excellent control efficiency (curative activity: 43.55 %; protective activity: 42.56 %), surpassing that of bismerthiazol and thiodiazole copper by approximately 8.0%-37.3 %. Overall, our findings highlight a new paradigm wherein nonivamide derivatives exhibit potential in combating pathogen resistance issues by inhibiting bacterial quorum sensing systems though attributing to their new molecular skeleton, novel mechanisms of action, and non-toxic features.

Identification of novel 4-substituted 7H-pyrrolo[2,3-d]pyrimidine derivatives as new FtsZ inhibitors: Bioactivity evaluation and computational simulation.

Bacterial infections and the consequent outburst of bactericide-resistance issues are fatal menace to both global health and agricultural produce. Hence, it is crucial to explore candidate bactericides with new mechanisms of action. The filamenting temperature-sensitive mutant Z (FtsZ) protein has been recognized as a new promising and effective target for new bactericide discovery. Hence, using a scaffold-hopping strategy, we designed new 7H-pyrrolo[2,3-d]pyrimidine derivatives, evaluated their antibacterial activities, and investigated their structure-activity relationships. Among them, compound B6 exhibited the optimal in vitro bioactivity (EC50 = 4.65 µg/mL) against Xanthomonas oryzae pv. oryzae (Xoo), which was superior to the references (bismerthiazol [BT], EC50 = 48.67 µg/mL; thiodiazole copper [TC], EC50 = 98.57 µg/mL]. Furthermore, the potency of compound B6 in targeting FtsZ was validated by GTPase activity assay, FtsZ self-assembly observation, fluorescence titration, Fourier-transform infrared spectroscopy (FT-IR) assay, molecular dynamics simulations, and morphological observation. The GTPase activity assay showed that the final IC50 value of compound B6 against XooFtsZ was 235.0 µM. Interestingly, the GTPase activity results indicated that the B6-XooFtsZ complex has an excellent binding constant (KA = 103.24 M-1). Overall, the antibacterial behavior suggests that B6 can interact with XooFtsZ and inhibit its GTPase activity, leading to bacterial cell elongation and even death. In addition, compound B6 showed acceptable anti-Xoo activity in vivo and low toxicity, and also demonstrated a favorable pharmacokinetic profile predicted by ADMET analysis. Our findings provide new chemotypes for the development of FtsZ inhibitors as well as insights into their underlying mechanisms of action.

The Identification and Gene Mapping of Spotted Leaf Mutant spl43 in Rice.

Our study investigates the genetic mechanisms underlying the spotted leaf phenotype in rice, focusing on the spl43 mutant. This mutant is characterized by persistent reddish-brown leaf spots from the seedling stage to maturity, leading to extensive leaf necrosis. Using map-based cloning, we localized the responsible locus to a 330 Kb region on chromosome 2. We identified LOC_Os02g56000, named OsRPT5A, as the causative gene. A point mutation in OsRPT5A, substituting valine for glutamic acid, was identified as the critical factor for the phenotype. Functional complementation and the generation of CRISPR/Cas9-mediated knockout lines in the IR64 background confirmed the central role of OsRPT5A in controlling this trait. The qPCR results from different parts of the rice plant revealed that OsRPT5A is constitutively expressed across various tissues, with its subcellular localization unaffected by the mutation. Notably, we observed an abnormal accumulation of reactive oxygen species (ROS) in spl43 mutants by examining the physiological indexes of leaves, suggesting a disruption in the ROS system. Complementation studies indicated OsRPT5A's involvement in ROS homeostasis and catalase activity regulation. Moreover, the spl43 mutant exhibited enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo), highlighting OsRPT5A's role in rice pathogen resistance mechanisms. Overall, our results suggest that OsRPT5A plays a critical role in regulating ROS homeostasis and enhancing pathogen resistance in rice.

Genome and transcriptome exploration reveals receptor-like kinases as potential resistance gene analogs against bacterial blight in pomegranate.

BACKGROUND: Pomegranate (Punica granatum L.) is a tropical fruit crop of pharma-nutritional importance. However, it faces farming challenges due to pests and diseases, particularly bacterial blight and wilt. Developing resistant cultivars is crucial for sustainable pomegranate cultivation, and understanding resistance's genetic basis is essential. METHODS AND RESULTS: We used an extensive resistance gene analogues (RGA) prediction tool to identify 958 RGAs, classified into Nucleotide Binding Site-leucine-rich repeat (NBS-LRR) proteins, receptor-like kinases (RLKs), receptor-like proteins (RLPs), Transmembrane coiled-coil (TM-CC), and nine non-canonical RGAs. RGAs were distributed across all eight chromosomes, with chromosome 02 containing the most RGAs (161), and chromosome 08 having the highest density (4.42 RGA/Mb). NBS-LRR genes were predominantly present on chromosomes 08 and 02, whereas RLKs and RLPs were primarily located on chromosomes 04 and 07. Gene ontology analysis revealed that 475 RGAs were associated with defence against various biotic stresses. Using RNAseq, we identified 120 differentially expressed RGAs, with RLKs (74) being prominent among the differentially expressed genes. CONCLUSION: The discovery of these RGAs is a significant step towards breeding pomegranates for pest and disease resistance. The differentially expressed RLKs hold promise for developing resistant cultivars against bacterial blight, thereby contributing to the sustainability of pomegranate cultivation.

Novel aurone-derived piperazine sulfonamides: Development and mechanisms of action as immunostimulants against plant bacterial diseases.

Bacterial diseases pose a significant threat to the sustainable production of crops. Given the unsatisfactory performance and poor eco-compatibility of conventional bactericides, here we present a series of newly structured bactericides that are inspiringly designed by aurone found in plants of the Asteraceae family. These aurone-derived compounds contain piperazine sulfonamide motifs and have shown promising in vitro performance against Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola and Xanthomonas axonopodis pv. citri, in particular, compound II23 achieved minimum half-maximal effective concentrations of 1.06, 0.89, and 1.78 µg/mL, respectively. In vivo experiments conducted in a greenhouse environment further revealed that II23 offers substantial protective and curative effects ranging between 68.93 and 70.29% for rice bacterial leaf streak and 53.17-64.43% for citrus bacterial canker, which stands in activity compared with lead compound aurone and commercial thiodiazole copper. Additional physiological and biochemical analyses, coupled with transcriptomics, have verified that II23 enhances defense enzyme activities and chlorophyll levels in rice. Significantly, it also stimulates the accumulation of abscisic acid (ABA) and upregulates the expression of key genes OsPYL/RCAR5, OsBIPP2C1, and OsABF1, thereby activating the ABA signaling pathway in rice plants under biological stress from bacterial infections.

Transcriptome analysis reveals the inhibitory mechanism of 3,4-Dimethoxyphenol from Streptomyces albidoflavus strain ML27 against Xanthomonas oryzae pv. oryzae.

Bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), poses a significant threat to rice cultivation across diverse regions. Growing concerns about pesticide resistance and environmental impact underscore the urgent necessity for eco-friendly biopesticides. Here, the complete genome sequence of Streptomyces albidoflavus strain ML27 revealed substantial antimicrobial activity and secondary metabolite production potential through genome mining. 3,4-dimethoxyphenol (purity 97%) was successfully isolated from the fermentation broth of S. albidoflavus strain ML27, exhibiting broad and pronounced inhibitory effects on the growth of seven different fungi and five tested bacteria. The efficacy of 3,4-dimethoxyphenol in controlling rice bacterial leaf blight was evaluated through pot tests, demonstrating substantial therapeutic (69.39%) and protective (84.53%) effects. Application of 3,4-dimethoxyphenol to Xoo resulted in cells displayed notable surface depressions, wrinkles, distortions, or even ruptures compared to their typical morphology. Transcriptome analysis revealed significant inhibition of membrane structures, protein synthesis and secretion, bacterial secretion system, two-component system, flagellar assembly, as well as various metabolic and biosynthetic pathways by 3,4-dimethoxyphenol. Notably, the down-regulation of the type III secretion system (T3SS) expression was a pivotal finding. Furthermore, validation via quantitative real-time polymerase chain reaction (qRT-PCR) analysis confirmed significant downregulation of 10 genes related to T3SS upon 3,4-dimethoxyphenol treatment. Based on these results, it is promising to develop 3,4-dimethoxyphenol as a novel biopesticide targeting the T3SS of Xoo for controlling bacterial leaf blight in rice.