Kazal-type serine protease inhibitors (KPIs) interfere with microbial proteases and have been associated with antimicrobial activity, yet their specific role in plant protection remains poorly understood. In this work, we evaluated the antimicrobial potential of recombinant Arabidopsis thaliana KPI (rAtKPI-1T), recombinant Toxoplasma gondii KPI (rTgPI-1), and two truncated versions of rTgPI-1 (rTgPI-1NT and rTgPI-1CT). Both rAtKPI-1T and rTgPI-1 exhibited inhibitory effects against Pseudomonas syringae DC3000, P. syringae (AvRpm1), and P. viridiflava in a concentration-dependent manner, with significant inhibition at 3.5μM. The lower MIC50 values obtained for rTgPI-1 and its truncated forms compared to rAtKPI-1T suggest a higher antibacterial potency. Binding and immunofluorescence assays further revealed that rAtKPI-1T, rTgPI-1, and rTgPI-1NT associated with bacterial surfaces, while rTgPI-1CT displayed weaker or transient interactions. In addition to their antibacterial activity, rTgPI-1NT and rTgPI-1CT also inhibited the germination of Botrytis cinerea conidia. Both truncated proteins significantly reduced germination after 6 and 9h of incubation, with rTgPI-1CT exerting a markedly stronger antifungal effect than rTgPI-1NT. These findings suggest that specific Kazal-type domains, particularly those in the C-terminal region, could play a critical role in suppressing early infection processes of necrotrophic fungi. Overall, this study demonstrates that rKPIs display distinct and complementary antimicrobial profiles mediated by microbial binding and protease inhibition. The contrasting activities of rTgPI-1NT and rTgPI-1CT highlight the value of domain-level analysis to discern functional contributions within multidomain KPIs. These results expand current knowledge on plant-derived protease inhibitors and underscore their potential as biotechnological tools for sustainable crop protection.

Tomato pith necrosis is a damaging disease affecting greenhouse and field-grown tomato production globally, often associated with environmental stress and excessive nitrogen fertilization. This study investigates the bacterial etiology and the (inter)national context of disease outbreaks during the 2023-2024 growing seasons in Al Taif, Saudi Arabia (SA). Sixteen bacterial isolates were recovered from symptomatic plants, and phenotypic and molecular analyses identified Pseudomonas mediterranea (n = 12) and Pseudomonas viridiflava (n = 4) as the causal agents, although Pseudomonas corrugata and Pseudomonas fluorescens biotype I have also been reported from SA in the past. P. mediterranea was the most prevalent species, consistent with earlier findings in southern Europe. Genetic characterization using multilocus sequence typing and minimum spanning tree analysis revealed high genetic diversity in P. viridiflava, including a novel singleton lineage, indicating potential local adaptation, and all SA strains belonged to Pseudomonas syringae (sensu latu) phylogroup 7a. In contrast, P. mediterranea isolates exhibited lower genetic diversity, but six novel sequence types (ST8-ST13) were identified exclusively in SA strains, forming a distinct phylogenetic clade. Pathogenicity assays showed that P. viridiflava caused severe necrosis, while P. mediterranea caused internal browning without wilting, suggesting a difference in virulence. The co-occurrence of both species in the same greenhouses highlights the complexity of disease dynamics and potential interactions between these pathogens. Given the global relevance of P. syringae phylogroup 7a, mainly formed by P. viridiflava strains, and the risk of pathogen spread via plant trade, this study underscores the importance of stringent phytosanitary controls and accurate diagnostics tailored to regional pathogen variants.

In plants, multiple cell types contribute to immunity, but what division of labor exists among cell types when immunity is activated? We compared, at single-cell resolution, the response of Arabidopsis thaliana leaf cells during pattern-triggered and effector-triggered immunity (PTI/ETI), sampled at 3 and 5 h after infection with Pseudomonas syringae DC3000. Core defense modules were broadly shared across cell clusters, but their activation varied in timing and intensity, with key immune receptors also showing cell type-specific expression dynamics. Mesophyll cell populations could be distinguished based on their resilience patterns: after the initial response, some populations continue to express defense genes at high levels during both PTI and ETI, while others quickly reinitiate growth-related gene expression programs but only during PTI. Gene regulatory network inference revealed WRKY-regulated modules enriched in cells sensing effectors, while salicylic acid biosynthesis regulators were activated in complementary clusters. Analysis of cue1 mutants demonstrated that core immune responses are robust to altered leaf architecture. In addition, we uncovered cryptic defense pathways, including sucrose-responsive modules, in this mutant. By capturing early immune responses at high resolution, our study reveals cell type-specific coordination of plant immunity and provides a framework for decoding immune signaling networks.

Successful recognition of pathogen effectors by plant disease resistance proteins, or effector-triggered immunity (ETI), contains the invading pathogen through localized hypersensitive cell death. ETI also activates long-range signalling to establish broad-spectrum systemic acquired resistance (SAR). Here we describe a sensitive luciferase (LUC) reporter that captures the spatial-temporal dynamics of SAR signal generation, propagation and establishment in systemic responding leaves following ETI. JASMONATE-INDUCED SYSTEMIC SIGNAL 1 (JISS1) encodes an endoplasmic-reticulum-localized protein of unknown function. JISS1::LUC captured very early ETI-elicited SAR signalling, which surprisingly was not affected by classical SAR mutants but was dependent on calcium and was also wound responsive. Both jasmonate biosynthesis and perception mutants abolished JISS1::LUC signalling and SAR to Pseudomonas syringae. Furthermore, we discovered that ETI initiated jasmonate-dependent systemic surface electrical potentials. These surface potentials were dependent on both glutamate receptors and JISS1, despite neither JISS1 loss-of-function nor glutamate receptor mutants altering SAR to Pseudomonas syringae. We thus demonstrate that jasmonate signalling, usually associated with antagonism of defence against biotrophs, is crucial to the rapid initiation and establishment of SAR systemic defence responses (including the activation of systemic surface potentials) and that JISS1::LUC serves as a reporter to further dissect these pathways.

Pectobacterium carotovorum is a gram-negative phytopathogenic bacterium that causes soft rot disease on diverse plant species. It encodes the type III secretion system effector protein, DspE, and its chaperone, DspF. The DspE family proteins form water and solute channels in plant cells, flooding the apoplast to aid bacterial multiplication. In Pseudomonas syringae, the DspE ortholog, AvrE, upregulates abscisic acid (ABA) expression, leading to stomatal closure. In this study, a Pectobacterium carotovorum dspEF mutant did not cause leaf cell death in tobacco leaves. This observation is supported by the lower expression of PCWDE such as pelB, pelI, celV, prtW, and the quorum sensing system transcript expI in tobacco plants prior to visual symptoms (5 hours post-inoculation). Interestingly, neither dspE/F or hrpL mutation affected synthesis of QS signaling molecule AHL under microbiological settings. However, maceration symptoms occurred if leaves infiltrated with the dspEF mutant were kept under high humidity or detached post-infiltration. These leaves showed elevated transcription of ABA synthesis genes compared to infiltrated leaves maintained on the plant under ambient conditions. To validate this involvement, co-infiltration of ABA with the dspEF mutant restored its ability to cause maceration in attached leaves under ambient conditions. Overall, our data suggest that DspE/F facilitates host susceptibility by creating an aqueous apoplast, promoting ABA accumulation and stomata closure.

HighlightsWhat are the main findings?Cu-based sputtered films show strong antibacterial activity.Cu90Sn10, Cu90Zn10, and Cu80Ti20 targets give coatings with the best performance.Films exhibit high durability and corrosion resistance.Large-area multicomponent nanolayers were successfully produced.What are the implications of the main findings?Cu-alloy targets are promising for greenhouse glass surfaces.Industrial PVD enables uniform large-area antimicrobial films.Findings support future antibacterial greenhouse applications.Scalable nanolayer design enables reproducible industrial coating.Antibacterial thin-film coatings are of increasing interest for enhancing hygiene in controlled environments such as commercial greenhouses. Phytopathogens including Pseudomonas syringae, and human pathogens such as Escherichia coli, Micrococcus luteus, and Staphylococcus aureus, frequently contaminate greenhouse environments. The present study aimed to develop and evaluate multifunctional magnetron-sputtered glass coatings with strong antimicrobial performance, deposited by physical vapor deposition to achieve precise control of film composition and uniform coverage of large substrates (≥0.25 m2), ensuring industrial-scale applicability. Thin films were fabricated by magnetron sputtering using multi-alloy targets composed of Cu, Sn, Zn, Al, Ni, Fe, Ti, Mn, Nb, or Co. Fourteen distinct coating compositions were characterized using high-resolution transmission electron microscopy, scanning transmission electron microscopy, and energy-dispersive X-ray spectroscopy. Antibacterial performance was evaluated against the following strains: E. coli (PCM 2560), M. luteus (PCM 525), S. aureus (PCM 2602), and P. syringae pv. tomato (IOR2146). Coatings prepared from 90%Cu-10%Sn, 90%Cu-10%Zn, and 80%Cu-20%Ti targets exhibited one of the highest antibacterial efficiencies. These coatings also showed strong mechanical durability and corrosion resistance. Our results indicate that coatings obtained from Cu-based multi-alloy targets by magnetron sputtering are promising candidates for use as durable, antimicrobial inner glass surfaces in future greenhouse applications.

Time-series transcriptome and metabolome profiling Uncovers WRKY6 and WRKY23 as critical regulators in tobacco response to Pseudomonas syringae infection.

A qPCR assay for the detection and quantification of Pseudomonas syringae pv. syringae in susceptible tissues of sweet cherry.

Soybean maintained immunodiversity during domestication despite a significant loss in genetic diversity.

Difunctional pyrido[2,3-b]pyrazine derivatives: Biological activity and preliminary mechanism.

ABSTRACT To increase yield and productivity, plants are treated with chemicals; however, these interventions have inadvertently contributed to a decline in food security. To mitigate these challenges, plant growth-promoting rhizobacteria (PGPRs), which increase yield, have been widely used to modulate plant immunity. Some PGPRs have recently been reported to exhibit opportunistic pathogenicity in humans, raising safety concerns regarding their widespread application. Membrane vesicles (MVs) derived from PGPR may retain similar plant growth-promoting (PGP) traits and thus could aid in plant protection. This research tests the hypothesis that MVs derived from the PGPR Priestia megaterium VIT-2021 as an effective and sustainable alternative for combating bacterial infection. MVs were successfully isolated and characterized, and the results ensured that these MVs are efficient antimicrobial agents against Pseudomonas syringae. Proteomic analysis of the MVs revealed the presence of four antimicrobial peptides AMP underlying their antimicrobial effects. Furthermore, in vitro treatment with these MVs downregulated P. syringae virulence genes, including avrE, hrpS, and mgrA. In planta experiments conducted on Oryza sativa demonstrated that P. megaterium MVs effectively reduced P. syringae pathogenicity through immune modulation, as evidenced by the upregulation of WRKY13, IPA1, WKRY45, NPR1 and PR1, with fold increase of approximately 2.98, 3.55, 1.73, 2.44, and 5.51, respectively, at 4.2 × 10⁸ particle concentration. WRKY13 is involved in SA-mediated secondary cell wall synthesis, which resists pathogen ingress. Increased IPA1 promoted the upregulation of WRKY45, which could contribute to SA-mediated antimicrobial secondary metabolite synthesis. Increased expression of NPR1 and PR1 mediate cell wall rigidification and antimicrobial activity, ultimately contribute to robust immunity. Additionally, increased IPA1 is associated with multiple tiller outgrowths, panicle branching, and grain size development. Thus, P. megaterium MVs demonstrated dual functionality of immunomodulation and growth promotion in O. sativa. Therefore, the current research highlights a pioneering strategy for sustainable crop protection and yield enhancement.

Numerous communities of bacteria, fungi, viruses, and other microorganisms live inside plants and colonize their internal and external tissues, having a significant impact on the health and functionality of the plants. Studies on infection specific microbiome interactions remain restricted in spite of broad studies of plant microbiome interactions. An excellent model for examining these interactions is kiwifruit bacterial canker (KBC), which is caused by Pseudomonas syringae pv actinidiae (Psa). The present understanding of Psa pathogenicity and kiwifruit-associated microbiomes in various ecological niches is summarized in this review with a focus on their dual functions of either promoting or suppressing disease. We identify important microbial taxa and interaction mechanisms that serve as ecological leverage points for KBC control mediated by the microbiome by incorporating recent findings. This perspective advances the conceptual shift from pathogen elimination to microbiome balance restoration, offering new insights into sustainable KBC management.

A series of heteroaromatic Michael acceptors derived from camphor were synthesized via Claisen-Schmidt condensation and evaluated against bacterial phytopathogens. Antibacterial activity was determined in vitro against Pseudomonas syringae pv syringae (Pss), P. syringae pv actinidiae (Psa), and Agrobacterium tumefaciens. Compounds 3 (derived from (R)-camphor) and 4 (derived from (S)-camphor) showed the highest activity against Pss, with EC50 values of 23.29 and 16.68 μg/mL, respectively. In in vivo assays on cherry branches, compounds 3 and 4 significantly reduced the development of necrotic lesions caused by Pss (76.31% and 73.03% reduction, respectively). In silico toxicity assessments indicated favorable safety profiles. These functionalized camphor derivatives represent potential bactericides for disease management in fruit crops.

Goldenrod (Solidago gigantea Aiton) is a highly invasive species in Europe (e.g., Poland, Germany, and the Czech Republic) whose secondary metabolites can serve as potential sources of bioactive compounds. This study evaluated the phytochemical profile of S. gigantea extracts and evaluated their antibacterial, insecticidal, and phytotoxic activities. The extracts were found to be rich in flavonoids (TFC = 101 mg QE/g) and phenolics (TPC = 175 mg GAE/g), with chlorogenic acid and rutin as dominant constituents. Strong antibacterial activity was observed against Gram-positive bacteria, particularly Staphylococcus spp. (MIC90 = 2.3 mg/mL; MBC = 5 mg/mL), while Gram-negative bacteria were less sensitive, with moderate susceptibility in Rhizobium radiobacter and Pseudomonas syringae. The extract exhibited fungistatic activity against all tested filamentous fungi, with Fusarium species being the most sensitive (49-56% growth inhibition at 10 mg/mL). Insecticidal assays demonstrated significant mortality of Tribolium confusum adults at 2.5-7.0 mg/mL and feeding inhibition at concentrations as low as 0.5 mg/mL. Seedling growth tests showed dose-dependent effects-from mild suppression to moderate stimulation, varying by plant species. Foliar application revealed both stimulatory and inhibitory effects, with the strongest biomass reduction in cress at 10 mg/mL (-45%). These findings indicate that S. gigantea extracts possess potent antibacterial, antifungal, insecticidal, and allelopathic activities. Their concentration-dependent effects on pathogens and plants highlight potential applications in sustainable agriculture, including natural crop protection and integrated pest management.

Potato seedlings were challenged with two parasites, namely, the fall armyworm and Pseudomonas syringae pv. tomato, in combination and individually. Growth (plant height, stem diameter, total number of tubers and total tuber weight) and physiological function (photosynthesis rate, stomatal conductance, transpiration efficiency, the ratio of intercellular CO2 concentration to ambient CO2 concentration (Ci/Ca) and water use efficiency) were measured to assess the effects of the parasites on the plants. A correlation analysis of the measured growth and physiology parameters was done to understand the co-ordination of the parasite-attacked plant processes. Finally, plant metabolomic profiles were determined to assess the effects of the parasites on the metabolomes of the treated plants. Individually and in combination, the parasites had varied effects on the growth and physiology of the plants. The correlation analysis also revealed key associations between the growth and physiology aspects, and the parasites caused metabolomic reprogramming in the treated plants. Some of the results were expected but there were also unexpected outcomes. Surprisingly, the pest drastically reduced plant height when administered alone, but its ability to reduce height lessened when it was co-administered with the bacterium. The lessened ability of the pest to reduce plant height in the presence of the bacterium hints at parasite-to-parasite antagonism. This same pattern extended to stem diameter and total tuber weight. The pest individually reduced stem diameter and total tuber weight, but not when co-administered. This also hints at parasite-to-parasite antagonism. However, this matter warrants further investigation. In conclusion, the pest and the pathogenic bacterium induce morpho-physiological and metabolomic changes in potato seedlings, their effects on the measured parameters vary, and there is a possible parasite-to-parasite antagonism.

Receptor for activated C kinase 1 (RACK1), a scaffold protein, functions in different biological processes in plants through interacting with various receptor kinases/proteins and heterotrimeric G proteins. The functions of RACK1 have been investigated extensively in the model plant Arabidopsis. However, little is known about the roles of RACK1 homologs in soybean. Soybean is a paleotetraploidy plant and each gene has two copies in its genome. As a result, the forward genetic approaches are not suitable for studying the gene functions in soybean. To resolve the gene redundancy, we used Bean pod mottle virus-induced gene silencing approach to interrogate gene functions in soybean. Using this approach, we successfully silenced two homologous genes of GmRACK1 (GmRACK1A/1B) in soybean. The GmRACK1A/1B-silenced plants exhibited significantly compromised resistance to Soybean mosaic virus, Pseudomonas syringae pv. glycinea (Psg), and Xanthomonas campestris pv. glycinea (Xag). The compromised disease resistance was correlated with the reduced activation of GmMPK3/6 in response to Psg infection. Taken together, our results indicate that GmRACK1A/1B play positive roles in soybean immunity possibly through activating GmMPK3/6, demonstrate that GmRACK1 could serve as a potential target for molecular breeding, laying the foundation for enhancing broad-spectrum resistance in soybean through genetic engineering approaches.

Wheat (Triticum aestivum L.) is one of the most important cereals worldwide, but is affected by various bacterial diseases, including bacterial leaf blight caused by Pseudomonas syringae, that decrease productivity significantly. The indiscriminate use of synthetic pesticides for disease management has led to environmental contamination, development of resistance and health risks, highlighting the need for alternative and environmentally friendly solutions. In this study, phytopathogenic Pseudomonas were isolated from symptomatic wheat leaves collected in Córdoba province (Argentina). The isolates were phenotypically identified using biochemical tests and the LOPAT scheme, confirming their classification within Group I of P. syringae. Pathogenicity tests verified their ability to induce typical leaf blight symptoms, and nine isolates were positive for the production of syringomycin, a phytotoxin associated with host tissue necrosis. The antimicrobial activity of different phytochemical preparations obtained from Thymus vulgaris and Origanum vulgare (decoctions, alcoholic, hexanic, chloroformic and propanolic extracts, as well as essential oils (EOs)) against the isolated and reference strains of P. syringae was evaluated. Disk diffusion assays revealed that thyme and oregano EOs exhibited the strongest inhibitory effects compared to the other phytochemical preparations tested. Antimicrobial activity assays by broth microdilution technique confirmed the antibacterial potency of thyme EO (MIC = 1.43–22.99 mg/mL) and oregano EO (MIC = 2.89–23.13 mg/mL). These results demonstrated the efficacy of T. vulgaris and O. vulgare EOs as natural antimicrobial agents, suggesting their potential application as sustainable alternatives to synthetic pesticides for the control of bacterial leaf blight in wheat crops.

The growing resistance of phytopathogenic bacteria highlights the need for new bactericides. DNA gyrase, essential in bacteria but absent in plants and animals, is an underexploited target for agricultural bactericides. Here, we modeled the Ralstonia solanacearum GyrA-GyrB-DNA complex using AlphaFold3 and conducted structure-based virtual screening of 1.27 million compounds. Among 100 tested candidates, GI-A14 showed potent, broad-spectrum antibacterial activity against R. solanacearum, Pseudomonas syringae pv tomato, and Xanthomonas oryzae pv oryzae, with minimum inhibitory concentrations of 1-4 μg/mL. Enzymatic assays confirmed that GI-A14 stabilizes the gyrase-DNA cleavage complex, indicating a DNA-interfacial inhibition mechanism. Furthermore, GI-A14 significantly attenuated the pathogenicity and biofilm formation of R. solanacearum. In planta, GI-A14 provided strong protection against bacterial wilt, speck, and blight, outperforming the commercial agent zhongshengmycin. These results establish DNA gyrase as a viable molecular target for plant disease control and identify GI-A14 as a promising lead compound for next-generation green bactericides.

Bacterial phytopathogens, such as Pseudomonas syringae pv. tomato (Pst) DC3000, induce water-soaked lesions in the leaf apoplast under high humidity, facilitating infection. However, it remains largely unclear how plants regulate their resistance to restrict bacterial infection in response to humidity. Here, we demonstrate that abscisic acid (ABA)-catabolizing ABA 8'-hydroxylase, encoded by CYP707A3, plays a critical role in this resistance in Arabidopsis thaliana. Elevated humidity induces CYP707A3 expression, which is essential for reducing ABA levels and promoting stomatal opening, thereby limiting bacterial water-soaking and infection following leaf invasion. High humidity also increases cytosolic Ca2+ levels via the Ca2+ channels CNGC2 and CNGC4, with partial involvement from CNGC9, activating the calmodulin-binding transcription activator CAMTA3 to drive CYP707A3 induction. However, Pst DC3000 counteracts this defense response using type III secretion effectors, including AvrPtoB,facilitating water-soaking. These findings provide insights into the mechanisms underlying the competition between plants and bacteria for leaf water under elevated humidity.

Plant essential oils (EOs) are attracting interest as ecofriendly alternatives to antibiotics and copper-based control of kiwifruit bacterial canker (KBC), caused by Pseudomonas syringae pv. actinidiae (Psa). This study chemically profiled six EOs (anise, basil, cardamom, cumin, fennel, and laurel) and evaluated their antimicrobial activity both in vitro and in planta. The in vitro assay targeted four strains, two of Psa and two of the low-virulent P. syringae pv. actinidifoliorum (Pfm), whereas the in planta assay focused on the highly virulent Psa7286 strain, assessed under preventive and curative application regimes (i.e., 14 days pre- or post-inoculation, respectively). Cumin, with cuminaldehyde as its major component (48%), was the most effective EO in vitro, significantly inhibiting growth at 5–10% concentration, whereas anise, rich in anethole (89%), was consistently the least effective one. However, the in planta application of the EOs produced antimicrobial effects that differed markedly from in vitro results and showed strong dependence on the timing of application. Preventive treatment significantly reduced Psa endophytic populations in basil (70%), anise (54%), laurel (42%), and cumin (35%) compared to untreated plants. In contrast, when the EOs were applied post-inoculation (curative treatment), a significant decrease in Psa colonization was observed in laurel (81%), cardamon (70%), cumin (31%) and fennel (29%). Although plant EOs are gaining momentum in the control of Psa and other diseases, translation from in vitro to in planta efficacy is not direct and is strongly timing-dependent, which underscores the need to perform validation trials in planta and to fine-tune application schedules for the integrated management of KBC.