Design, Synthesis, and Antibacterial Evaluation of Novel Isoindolin-1-ones Derivatives Containing Piperidine Fragments.

A series of novel isoindoline-1-one derivatives containing piperidine moiety were designed and synthesized using natural compounds as raw materials, and their biological activities were tested for three bacterial and three fungal pathogens. These derivatives exhibited good against phytopathogenic bacteria activities against Pseudomonas syringae pv actinidiae (Psa) and Xanthomonas axonopodis pv.citri (Xac). Some compounds exhibited excellent antibacterial activities against Xanthomonas oryzae pv oryzae (Xoo). The dose of Y8 against Xoo (the maximum half lethal effective concentration (EC50) = 21.3 µg/mL) was better than that of the thiediazole copper dose (EC50 = 53.3 µg/mL). Excitingly, further studies have shown that the molecular docking of Y8 with 2FBW indicates that it can fully locate the interior of the binding pocket through hydrogen bonding and hydrophobic interactions, thereby enhancing its anti-Xoo activity. Scanning electron microscopy (SEM) studies revealed that Y8 induced the Xoo cell membrane collapse. Moreover, the proteomic results also indicate that Y8 may be a multifunctional candidate as it affects the formation of bacterial Xoo biofilms, thereby exerting antibacterial effects.

Multiple lysine substitutions in the peptaibol trichogin GA IV enhance the antibiotic activity against plant pathogenic Pseudomonas syringae.

Plant diseases caused by Pseudomonas syringae are essentially controlled in the field with the use of copper-based products and antibiotics, raising environmental and safety concerns. Antimicrobial peptides (AMPs) derived from fungi may represent a sustainable alternative to those chemicals. Trichogin GA IV, a non-ribosomal, 11-residue long AMP naturally produced by the fungus Trichoderma longibrachiatum has the ability to insert into phospholipidic membranes and form water-filled pores, thereby perturbing membrane integrity and permeability. In previous studies, peptide analogs modified at the level of specific residues were designed to be water-soluble and active against plant pathogens. Here, we studied the role of glycine-to-lysine substitutions and of the presence of a C-terminal leucine amide on bioactivity against Pseudomonas syringae bacteria. P. syringae diseases affect a wide range of crops worldwide, including tomato and kiwifruit. Our results show that trichogin GA IV analogs containing two or three Gly-to-Lys substitutions are highly effective in vitro against P. syringae pv. tomato (Pst), displaying minimal inhibitory and minimal bactericidal concentrations in the low micromolar range. The same analogs are also able to inhibit in vitro the kiwifruit pathogen P. syringae pv. actinidiae (Psa) biovar 3. When sprayed on tomato plants 24 h before Pst inoculation, only tri-lysine containing analogs were able to significantly reduce bacterial titers and symptom development in infected plants. Our results point to a positive correlation between the number of lysine substitutions and the antibacterial activity. This correlation was supported by microscopy analyses performed with mono-, di- and tri-Lys containing analogs that showed a different degree of interaction with Pst cells and ultrastructural changes that culminated in cell lysis.

Carbon Nanomaterials for Plant Priming through Mechanostimulation: Emphasizing the Role of Shape.

The use of nanomaterials to improve plant immunity for sustainable agriculture is gaining increasing attention; yet, the mechanisms involved remain unclear. In contrast to metal-based counterparts, carbon-based nanomaterials do not release components. Determining how these carbon-based nanomaterials strengthen the resistance of plants to diseases is essential as well as whether shape influences this process. Our study compared single-walled carbon nanotubes (SWNTs) and graphene oxide (GO) infiltration against the phytopathogen Pseudomonas syringae pv tomato DC3000. Compared with plants treated with GO, plants primed with SWNTs showed a 29% improvement in the pathogen resistance. Upon nanopriming, the plant displayed wound signaling with transcriptional regulation similar to that observed under brushing-induced mechanostimulation. Compared with GO, SWNTs penetrated more greatly into the leaf and improved transport, resulting in a heightened wound response; this effect resulted from the tubular structure of SWNTs, which differed from the planar form of GO. The shape effect was further demonstrated by wrapping SWNTs with bovine serum albumin, which masked the sharp edges of SWNTs and resulted in a significant decrease in the overall plant wound response. Finally, we clarified how the local wound response led to systemic immunity through increased calcium ion signaling in distant plant areas, which increased the antimicrobial efficacy. In summary, our systematic investigation established connections among carbon nanomaterial priming, mechanostimulation, and wound response, revealing recognition patterns in plant immunity. These findings promise to advance nanotechnology in sustainable agriculture by strengthening plant defenses, enhancing resilience, and reducing reliance on traditional chemicals.

Evaluating the Stability of Lytic and Lysogenic Bacteriophages in Various Protectants.

Phage therapy has regained value as a potential alternative and a complementary anti-infective approach to antibiotics in the fight against bacterial pathogens. Due to their host specificity, non-pathogenic nature for humans, and low production cost, phages offer an effective opportunity for utilization in healthcare, agriculture, and food preservation. Well-defined storage conditions are essential for commercialization and dissemination of phage usage. For this purpose, in our study, after the isolation and characterization of two different phages, one lytic and the other lysogenic; storage and shelf-life studies of phages were evaluated in a presence of various protectants (glycerol, sodium azide, DMSO with chloroform) and without any protectant during 8-month period at four different temperatures. The short-time stability of the lytic P. syringae phage and lysogenic MRSA phage, which were determined by STEM analysis to belong to the Straboviridae and Siphoviridae families, respectively were also examined for the different temperatures and the pH levels ranging from 1.0 to 14.0. This study revealed the storage-model of phages that exhibit distinct lifecycles, for the first time and provided a theoretical basis for development and application of phages, has yielded valuable findings contributing to understanding of phage biology.

Identification of Indole-3-Acetic Acid-Regulated Genes in Pseudomonas syringae pv. tomato Strain DC3000.

The auxin indole-3-acetic acid (IAA) is a plant hormone that not only regulates plant growth and development but also plays important roles in plant-microbe interactions. We previously reported that IAA alters expression of several virulence-related genes in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000). To learn more about the impact of IAA on regulation of PtoDC3000 gene expression, we performed a global transcriptomic analysis of bacteria grown in culture, in the presence or absence of exogenous IAA. We observed that IAA repressed expression of genes involved in the type III secretion (T3S) system and motility and promoted expression of several known and putative transcriptional regulators. Several of these regulators are orthologs of factors known to regulate stress responses and accordingly expression of several stress response-related genes was also upregulated by IAA. Similar trends in expression for several genes were also observed by quantitative reverse transcription PCR. Using an Arabidopsis thaliana auxin receptor mutant that accumulates elevated auxin, we found that many of the P. syringae genes regulated by IAA in vitro were also regulated by auxin in planta. Collectively the data indicate that IAA modulates many aspects of PtoDC3000 biology, presumably to promote both virulence and survival under stressful conditions, including those encountered in or on plant leaves. IMPORTANCE Indole-3-acetic acid (IAA), a form of the plant hormone auxin, is used by many plant-associated bacteria as a cue to sense the plant environment. Previously, we showed that IAA can promote disease in interactions between the plant pathogen Pseudomonas syringae strain PtoDC000 and one of its hosts, Arabidopsis thaliana. However, the mechanisms by which IAA impacts the biology of PtoDC3000 and promotes disease are not well understood. Here, we demonstrate that IAA is a signal molecule that regulates gene expression in PtoDC3000. The presence of exogenous IAA affects expression of over 700 genes in the bacteria, including genes involved in type III secretion and genes involved in stress response. This work offers insight into the roles of auxin-promoting pathogenesis.

Natural imidazole alkaloids as antibacterial agents against Pseudomonas syringae pv. actinidiae isolated from kiwi endophytic fungus Fusarium tricinctum.

Kiwi (Actinidia chinensis) plants are severely destroyed by canker disease which is caused by the bacterium Pseudomonas syringae pv. actinidiae (Psa). This program tries to find anti-Psa agents among secondary metabolites of endophytic fungi from kiwi plant itself. The chemical investigation on one kiwi endophytic fungi, Fusarium tricinctum, resulted in the isolation of nine new imidazole alkaloids, fusaritricines A-I (1-9) together with seven known analogues (10-16). The structures of new compounds were established by extensive spectroscopic methods. Compounds 2, 3, 9, and 13 showed good antibacterial activity against Psa with MIC values between 25 and 50 µg/mL. It is suggested that imidazole alkaloids should be potential anti-Psa agents.

Enhanced Antimicrobial Activity and Low Phytotoxicity of Acoustically Synthesized Large Aspect Ratio Cu-BTC Metal-Organic Frameworks with Exposed Metal Sites.

Metal-organic frameworks (MOFs) have recently been shown to be effective antimicrobial agents, particularly if they comprise pathogenicidal metal ions. Nevertheless, the accessibility of these active metal sites to the pathogen, and hence the MOFs' antimicrobial activity itself, is often poor since the metal nodes are usually embedded deep within its three-dimensional (3D) structure. We show that a unique copper-based (copper(II)-benzene-1,3,5-tricarboxylate) MOF, whose quasi-two-dimensional (quasi-2D) swordlike structure facilitates exposure of the metal ions along its surface, exhibits enhanced antimicrobial properties against three representative plant pathogens: a bacterium (Pseudomonas syringae), a fungus (Fusarium solani), and a virus (Odontoglossum ringspot virus (ORSV)). Such superior antimicrobial activity results in low minimum inhibitory concentrations (MICs)─half that of a commercial pesticide and an eighth of its conventional 3D cubic MOF counterpart (HKUST-1)─and hence low phytotoxicity, which can be attributed to the accessibility of the surface copper sites to the pathogen, thereby facilitating their adhesion and physical contact with the MOF. Additionally, we observed that orchids treated with the quasi-2D MOF showed negligible phytotoxicity and 80% decreased viral load. This work constitutes the first study to demonstrate the antimicrobial properties of this novel MOF against bacterial, fungal, and viral plant pathogens, and the first chemical control of ORSV.

Sulfur Induces Resistance against Canker Caused by Pseudomonas syringae pv. actinidae via Phenolic Components Increase and Morphological Structure Modification in the Kiwifruit Stems.

Bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has led to considerable losses in all major kiwifruit-growing areas. There are no commercial products in the market to effectively control this disease. Therefore, the defense resistance of host plants is a prospective option. In our previous study, sulfur could improve the resistance of kiwifruit to Psa infection. However, the mechanisms of inducing resistance remain largely unclear. In this study, disease severity and protection efficiency were tested after applying sulfur, with different concentrations in the field. The results indicated that sulfur could reduce the disease index by 30.26 and 31.6 and recorded high protection efficiency of 76.67% and 77.00% after one and two years, respectively, when the concentration of induction treatments was 2.0 kg/m3. Ultrastructural changes in kiwifruit stems after induction were demonstrated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO), and the accumulation of lignin were determined by biochemical analyses. Our results showed that the morphological characteristics of trichomes and lenticels of kiwifruit stem were in the best defensive state respectively when the sulfur concentration was 3.0 kg/m3 and 1.5 kg/m3. Meanwhile, in the range of 0.5 to 2.0 kg/m3, the sulfur could promote the chloroplast and mitochondria of kiwifruit stems infected with Psa to gradually return to health status, increasing the thickness of the cell wall. In addition, sulfur increased the activities of PAL, POD and PPO, and promoted the accumulation of lignin in kiwifruit stems. Moreover, the sulfur protection efficiency was positively correlated with PPO activity (p < 0.05) and lignin content (p < 0.01), which revealed that the synergistic effect of protective enzyme activity and the phenolic metabolism pathway was the physiological effect of sulfur-induced kiwifruit resistance to Psa. This evidence highlights the importance of lignin content in kiwifruit stems as a defense mechanism in sulfur-induced resistance. These results suggest that sulfur enhances kiwifruit canker resistance via an increase in phenolic components and morphology structure modification in the kiwifruit stems. Therefore, this study could provide insights into sulfur to control kiwifruit canker caused by Psa.

Zopfiellasins A-D, Two Pairs of Epimeric Cytochalasins from Kiwi-Associated Fungus Zopfiella sp. and Their Antibacterial Assessment.

In our continuous search for antibacterial agents against Pseudomonas syringae pv. actinidiae (Psa) from kiwi-associated fungi, two pairs of epimeric cytochalasins, zopfiellasins A-D (1-4), were characterized from the fungus Zopfiella sp. The structures were established on the basis of spectroscopic data analysis, while the absolute configurations were determined by single-crystal X-ray diffraction. Compounds 1 and 3 exhibited antibacterial activity against Psa with MIC values of 25 and 50 µg/mL, respectively. This is the first report of anti-Psa activity of cytochalasin derivatives.

Direct Antibiotic Activity of Bacillibactin Broadens the Biocontrol Range of Bacillus amyloliquefaciens MBI600.

Bacillus amyloliquefaciens is considered the most successful biological control agent due to its ability to colonize the plant rhizosphere and phyllosphere where it outgrows plant pathogens by competition, antibiosis, and inducing plant defense. Its antimicrobial function is thought to depend on a diverse spectrum of secondary metabolites, including peptides, cyclic lipopeptides, and polyketides, which have been shown to target mostly fungal pathogens. In this study, we isolated and characterized the catecholate siderophore bacillibactin by B. amyloliquefaciens MBI600 under iron-limiting conditions and we further identified its potential antibiotic activity against plant pathogens. Our data show that bacillibactin production restrained in vitro and in planta growth of the nonsusceptible (to MBI600) pathogen Pseudomonas syringae pv. tomato. Notably, it was also related to increased antifungal activity of MBI600. In addition to bacillibactin biosynthesis, iron starvation led to upregulation of specific genes involved in microbial fitness and competition. IMPORTANCE Siderophores have mostly been studied concerning their contribution to the fitness and virulence of bacterial pathogens. In the present work, we isolated and characterized for the first time the siderophore bacillibactin from a commercial bacterial biocontrol agent. We proved that its presence in the culture broth has significant biocontrol activity against nonsusceptible bacterial and fungal phytopathogens. In addition, we suggest that its activity is due to a new mechanism of action, that of direct antibiosis, rather than by competition through iron scavenging. Furthermore, we showed that bacillibactin biosynthesis is coregulated with the transcription of antimicrobial metabolite synthases and fitness regulatory genes that maximize competition capability. Finally, this work highlights that the efficiency and range of existing bacterial biocontrol agents can be improved and broadened via the rational modification of the growth conditions of biocontrol organisms.

A Synergic Potential of Antimicrobial Peptides against Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) is the pathogenic agent responsible for the bacterial canker of kiwifruit (BCK) leading to major losses in kiwifruit productions. No effective treatments and measures have yet been found to control this disease. Despite antimicrobial peptides (AMPs) having been successfully used for the control of several pathogenic bacteria, few studies have focused on the use of AMPs against Psa. In this study, the potential of six AMPs (BP100, RW-BP100, CA-M, 3.1, D4E1, and Dhvar-5) to control Psa was investigated. The minimal inhibitory and bactericidal concentrations (MIC and MBC) were determined and membrane damaging capacity was evaluated by flow cytometry analysis. Among the tested AMPs, the higher inhibitory and bactericidal capacity was observed for BP100 and CA-M with MIC of 3.4 and 3.4-6.2 µM, respectively and MBC 3.4-10 µM for both. Flow cytometry assays suggested a faster membrane permeation for peptide 3.1, in comparison with the other AMPs studied. Peptide mixtures were also tested, disclosing the high efficiency of BP100:3.1 at low concentration to reduce Psa viability. These results highlight the potential interest of AMP mixtures against Psa, and 3.1 as an antimicrobial molecule that can improve other treatments in synergic action.

Pseudomonas syringae pv. phaseolicola Uses Distinct Modes of Stationary-Phase Persistence To Survive Bacteriocin and Streptomycin Treatments.

Antimicrobial treatment of bacteria often results in a small population of surviving tolerant cells, or persisters, that may contribute to recurrent infection. Antibiotic persisters are metabolically dormant, but the basis of their persistence in the presence of membrane-disrupting biological compounds is less well understood. We previously found that the model plant pathogen Pseudomonas syringae pv. phaseolicola 1448A (Pph) exhibits persistence to tailocin, a membrane-disrupting biocontrol compound with potential for sustainable disease control. Here, we compared physiological traits associated with persistence to tailocin and to the antibiotic streptomycin and established that both treatments leave similar frequencies of persisters. Microscopic profiling of treated populations revealed that while tailocin rapidly permeabilizes most cells, streptomycin treatment results in a heterogeneous population in the redox and membrane permeability state. Intact cells were sorted into three fractions according to metabolic activity, as indicated by a redox-sensing reporter dye. Streptomycin persisters were cultured from the fraction associated with the lowest metabolic activity, but tailocin persisters were cultured from a fraction associated with an active metabolic signal. Cells from culturable fractions were able to infect host plants, while the nonculturable fractions were not. Tailocin and streptomycin were effective in eliminating all persisters when applied sequentially, in addition to eliminating cells in other viable states. This study identifies distinct metabolic states associated with antibiotic persistence, tailocin persistence, and loss of virulence and demonstrates that tailocin is highly effective in eliminating dormant cells.IMPORTANCE Populations of genetically identical bacteria encompass heterogeneous physiological states. The small fraction of bacteria that are dormant can help the population survive exposure to antibiotics and other stresses, potentially contributing to recurring infection cycles in animal or plant hosts. Membrane-disrupting biological control treatments are effective in killing dormant bacteria, but these treatments also leave persister-like survivors. The current work demonstrates that in Pph, persisters surviving treatment with membrane-disrupting tailocin proteins have an elevated redox state compared to that of dormant streptomycin persisters. Combination treatment was effective in killing both persister types. Culturable persisters corresponded closely with infectious cells in each treated population, whereas the high-redox and unculturable fractions were not infectious. In linking redox states to heterogeneous phenotypes of tailocin persistence, streptomycin persistence, and infection capability, this work will inform the search for mechanisms and markers for each phenotype.

Leaf temperature responses to ABA and dead bacteria in wheat and Arabidopsis.

Stomatal densities, aperture openness and their responsiveness to environmental change determine plant water loss and regulate entry of pathogens. Stomatal responsiveness is usually assessed on restricted areas of leaves or isolated epidermal peels floated in solution. Analyzing these responses in the whole plant context could give valuable additional information, for example on the role of mesophyll in stomatal responses. We analyzed stomatal responses to the phytohormone abscisic acid (ABA) and pathogenic elicitors in intact plants by dynamic measurement of leaf temperature. We tested whether ABA-induced stomatal closure in wheat requires external nitrate and whether bacterial elicitor-induced stomatal closure can be detected by dynamic thermal imaging in intact Arabidopsis. We found that wheat was hypersensitive to all applied treatments, as even mock-treated leaves showed a strong increase in leaf temperature. Nevertheless, ABA activated stomatal closure in wheat independent of exogenous nitrate. Pathogenic elicitors triggered a fast and transient increase in leaf temperature in intact Arabidopsis, indicating short-term stomatal closure. The data suggest that the dynamics of pathogen-induced stomatal closure is different in whole plants compared to epidermal peels, where elicitor-induced stomatal closure persists longer. We propose that dynamic thermal imaging could be applied to address the effect of pathogenic elicitors on stomatal behavior in whole plants to complement detached sample assays and gain a better understanding of stomatal immunity.

Plant-produced bacteriocins inhibit plant pathogens and confer disease resistance in tomato.

Bacteriocins are a diverse group of bacterial antimicrobial peptides (AMPs) that represent potential replacements for current antibiotics due to their novel modes of action. At present, production costs are a key constraint to the use of bacteriocins and other AMPs. Here, we report the production of bacteriocins in planta - a potentially scalable and cost-effective approach for AMP production. Nine bacteriocin genes with three different modes of action and minimal or no post-translational modifications were synthesized, cloned and used to transform Arabidopsis thaliana. To confirm bacteriocin functionality and the potential to use these plants as biofactories, Arabidopsis T3 crude leaf extracts were subjected to inhibition assays against the bacterial pathogens Clavibacter michiganensis subsp. michiganensis (Cmm) and Pseudomonas syringae pv. tomato DC3000 (Pst). Six and seven of nine extracts significantly inhibited Cmm and Pst, respectively. Three bacteriocin genes (plantaricin, enteriocin, and leucocin) were then selected for over-expression in tomato (Solanum lycopersicum). In vitro plant pathogen inhibition assays of T0, T1 and T2 transgenic tomato leaf extracts confirmed antimicrobial activity against both pathogens for all three generations of plants, indicating their potential use as stable biopesticide biofactories. Plantaricin and leucocin-expressing T2 tomato plants were resistant to Cmm, and leucocin-expressing T2 plants were resistant to Pst. This study highlights that plants can be used as biofactories for AMP production and that the expression of bacteriocins in planta may offer new opportunities for disease control in agriculture.

Exocyst subunit Exo70B2 is linked to immune signaling and autophagy.

During the immune response, activation of the secretory pathway is key to mounting an effective response, while gauging its output is important to maintain cellular homeostasis. The Exo70 subunit of the exocyst functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst regulation remains challenging. We show that, in Arabidopsis thaliana, Exo70B2 behaves as a bona fide exocyst subunit. Conversely, treatment with the salicylic acid (SA) defence hormone analog benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We reveal that Exo70B2 interacts with AUTOPHAGY-RELATED PROTEIN 8 (ATG8) via two ATG8-interacting motives (AIMs) and its transport into the vacuole is dependent on autophagy. In line with its role in immunity, we discovered that Exo70B2 interacted with and was phosphorylated by the kinase MPK3. Mimicking phosphorylation had a dual impact on Exo70B2: first, by inhibiting localization at sites of active secretion, and second, it increased the interaction with ATG8. Phosphonull variants displayed higher effector-triggered immunity (ETI) and were hypersensitive to BTH, which induce secretion and autophagy. Our results suggest a molecular mechanism by which phosphorylation diverts Exo70B2 from the secretory into the autophagy pathway for its degradation, to dampen secretory activity.

Activity of Adesmia boronioides resinous exudate against phytopathogenic bacteria.

Resinous exudate obtained from the aerial parts of Adesmia boronioides Hook.f. were evaluated to determine anti-phytopathogenic effects. Briefly, resinous exudate was obtained by dipping fresh plant material in dichloromethane; chemical composition was determined by GC-MS; and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated against four phytopathogenic bacteria. Resinous exudate yield was 8.5% (resin/fresh plant), of which esquel-6-en-9-one (14.25%), esquel-7-en-9-one (5.86%), and veratric acid (2.59%) were the effective antibacterial compounds. Tested against Pectobacterium carotovorum subsp. carotovora, Erwinia amylovora, Bacillus subtilis, and Pseudomonas syringae, MICs and MBCs ranged from 16 to 128 µg/mL and 32-256 µg/mL, respectively. These results provide initial evidence that resinous bush A. boronioides is a new and alternative source of substances with agricultural interest.

Agromonas: a rapid disease assay for Pseudomonas syringae growth in agroinfiltrated leaves.

The lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth, and that transient depletion of a host immune gene and transient expression of a type-III effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure-function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers a reliable approach for further comprehensive analysis of plant immunity.

Antibacterial Functions and Proposed Modes of Action of Novel 1,2,3,4-Tetrahydro-ß-carboline Derivatives that Possess an Attractive 1,3-Diaminopropan-2-ol Pattern against Rice Bacterial Blight, Kiwifruit Bacterial Canker, and Citrus Bacterial Canker.

In recent years, naturally occurring tetrahydro-ß-carboline (THC) alkaloids and their derivatives have been of biological interest. However, few studies and developments have reported the use of such structures in managing plant bacterial diseases. Herein, an array of novel THC derivatives containing an attractive 1,3-diaminopropan-2-ol pattern were prepared to evaluate the antiphytopathogen activity in vitro and in vivo and explore innovative antibacterial frameworks. Notably, target compounds exhibited excellent activities against three rebellious phytopathogens, namely, Pseudomonas syringae pv. actinidiae (Psa), Xanthomonas axonopodis pv. citri, and Xanthomonas oryzae pv. oryzae, at related optimal EC50 values of 2.39 (II9), 2.06 (I23), and 1.69 (II9) µg/mL, respectively. These effects were superior to those of the parent structure 1,2,3,4-THC and positive controls. In vivo assays showed that II9 exhibited excellent control efficiencies of 51.89 and 65.45% at 200 µg/mL against rice bacterial blight and kiwifruit bacterial canker, respectively, and I23 substantially relieved the citrus canker on the leaves. Antibacterial mechanisms indicated that these THC compounds could induce the increment of reactive oxygen species and subsequently endow the tested bacteria with distinct apoptotic behavior. In addition, II9 could alleviate the hypersensitive response and pathogenicity of Psa. Overall, these simple THC derivatives can be further developed as versatile antibacterial agents.

Plant defensin antibacterial mode of action against Pseudomonas species.

BACKGROUND: Though many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5-lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. Also, a plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments. RESULTS: Plant defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. A defensin from Medicago truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. The ability for MtDef4 to damage bacterial outer membranes was also verified visually through fluorescent microscopy. Another defensin from M. truncatula, MtDef5, failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. The transposon insertion site on MtDef4 resistant P. syringae pv. syringae mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to plant defensin treatments. CONCLUSIONS: MtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. MtDef5, appears to have a different antibacterial MOA. Additionally, the MtDef4 antibacterial mode of action may also involve inhibition of translation.

Tomato Atypical Receptor Kinase1 Is Involved in the Regulation of Preinvasion Defense.

Tomato Atypical Receptor Kinase 1 (TARK1) is a pseudokinase required for postinvasion immunity. TARK1 was originally identified as a target of the Xanthomonas euvesicatoria effector protein Xanthomonas outer protein N (XopN), a suppressor of early defense signaling. How TARK1 participates in immune signal transduction is not well understood. To gain insight into TARK1's role in tomato (Solanum lycopersicum) immunity, we used a proteomics approach to isolate and identify TARK1-associated immune complexes formed during infection. We found that TARK1 interacts with proteins predicted to be associated with stomatal movement. TARK1 CRISPR mutants and overexpression (OE) lines did not display differences in light-induced stomatal opening or abscisic acid-induced stomatal closure; however, they did show altered stomatal movement responses to bacteria and biotic elicitors. Notably, we found that TARK1 CRISPR plants were resistant to Pseudomonas syringae pathovar tomato strain DC3000-induced stomatal reopening, and TARK1 OE plants were insensitive to P syringae pathovar tomato strain DC3118 (coronatine deficit)-induced stomatal closure. We also found that TARK1 OE in leaves resulted in increased susceptibility to bacterial invasion. Collectively, our results indicate that TARK1 functions in stomatal movement only in response to biotic elicitors and support a model in which TARK1 regulates stomatal opening postelicitation.