Activate Plant Defense Research Articles

A two-in-one molybdenum disulfide-chitosan nanoparticles system for activating plant defense mechanisms and reactive oxygen species to treat Citrus Huanglongbing

Citrus Huanglongbing (HLB) poses an enormous challenge to Citrus cultivation worldwide, necessitating groundbreaking interventions beyond conventional pharmaceutical methods. In this study, we propose molybdenum disulfide-chitosan nanoparticles (MoS2-CS NPs) through electrostatic adsorption, preserving the plant-beneficial properties of molybdenum disulfide (MoS2), while enhancing its antibacterial effectiveness through chitosan modification. MoS2-CS NPs exhibited significant antibacterial efficacy against RM1021, and the closest relatives to Candidatus Liberibacter asiaticus (CLas), Erwinia carotovora, and Xanthomonas citri achieved survival rates of 7.40 % ± 1.74 %, 8.94 % ± 1.40 %, and 6.41 % ± 0.56 %, respectively. In vivo results showed, CLas survival rate of 10.42 % ± 3.51 %. Furthermore, treatment with MoS2-CS NPs resulted in an increase in chlorophyll and carotenoid content. Concomitantly, a significant reduction in malondialdehyde (MDA), soluble sugar, hydrogen peroxide (H2O2), and starch contents was also observed. Mechanistically, MoS2-CS NPs enhanced the activity of antioxidant-related enzymes by upregulating the expression of antioxidant genes, thereby galvanizing the antioxidant system to alleviate oxidative stress. Collectively, this dual functionality—combining direct antibacterial action with the activation of plant defense mechanisms—marks a promising strategy for managing Citrus Huanglongbing and suggests potential agricultural applications for MoS2-based antibacterial treatments.

Plant extracts and commercial products for controlling black spot and inducing resistance in rose bushes

Rose is one of the most appreciated flowers in the world; however, fungal diseases can affect its production, decreasing its commercial value. Thus, the objective of this work was to assess the potential of extracts of medicinal plants and commercial products based on plant extracts, plant oils, and potassium phosphite to control black spot (Diplocarpon rosae) in roses by evaluating the activation of plant defense mechanisms. The treatments consisted of using crude aqueous extract (CAE) of Rosmarinus officinalis leaves; CAE of Equisetum arvense stems; CAE of Moringa oleifera seeds; commercial product (CP) based on fermented plant extracts; CP based on potassium phosphite; CP based on plant oils; CP based on citrus mass; and fungicide. Severity of black spot disease, and yield, biometry, chlorophyll contents, and enzyme activity of peroxidase, catalase, and polyphenoloxidase in rose plants were evaluated. The treatments presented no difference from the control (water) for disease incidence and severity and yield. However, the longest stem was found in rose plants in the treatment with potassium phosphite-based commercial product, whereas the thickest stems were found in the three treatments with commercial products. The highest enzyme specific activities of peroxidase, catalase, and polyphenoloxidase were found in plants treated with potassium phosphite, CAE of M. oleifera, and CAE of R. officinalis. The results found showed that the tested natural products did not control black spot in rose bushes, despite their positive effect on plant physiology.

Mitogen-Activated Protein Kinase Kinase OsMEK2 Positively Regulates Ca2+ Influx and Ferroptotic Cell Death during Rice Immune Responses.

Mitogen-activated protein (MAP) kinase (MAPK) signaling pathway is important in plant immune responses, involved in iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death mediated by Ca2+. High Ca2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). Apoplastic Ca2+ chelation by EGTA during avirulent Magnaporthe oryzae infection altered Ca2+, ROS, and Fe2+ accumulation, increasing rice susceptibility to infection. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca2+ influx, and H2O2 accumulation, triggering rice ferroptotic cell death during virulent Magnaporthe oryzae infection. Here, we report a novel role of the MAPK signaling pathway in regulating cytoplasmic Ca2+ increase during ferroptotic cell death in rice immunity, using the ΔOsmek2 knockout mutant rice. The knockout of rice OsMEK2 impaired the ROS accumulation, lipid peroxidation, and iron accumulation during avirulent M. oryzae infection. This study has shown that OsMEK2 could positively regulate iron- and ROS-dependent ferroptotic cell death in rice by modulating the expression of OsNADP-ME, OsRBOHB, OsPLC, and OsCNGC. This modulation indicates a possible mechanism for how OsMEK2 participates in Ca2+ regulation in rice ferroptotic cell death, suggesting its broader role in plant immune responses in response to M. oryzae infection.

A conserved oomycete effector RxLR23 triggers plant defense responses by targeting ERD15La to release NbNAC68

Oomycete pathogens deliver many effectors to enhance virulence or suppress plant immunity. Plant immune networks are interconnected, in which a few effectors can trigger a strong defense response when recognized by immunity-related proteins. How effectors activate plant defense response remains poorly understood. Here we report Phytophthora capsici effector RxLR23KM can induce plant cell death and plant immunity. RxLR23KM specifically binds to ERD15La, a regulator of abscisic acid and salicylic acid pathway, and the binding intensity depends on the amino acid residues (K93 and M320). NbNAC68, a downstream protein of ERD15La, can stimulate plant immunity that is compromised after binding with ERD15La. Silencing of NbNAC68 substantially prevents the activation of plant defense response. RxLR23KM binds to ERD15La, releasing NbNAC68 to activate plant immunity. These findings highlight a strategy of plant defense response that ERD15La as a central regulator coordinates RxLR23KM to regulate NbNAC68-triggered plant immunity.

Green Routes: Exploring Protein-Based Virus-like Nanoparticle Transport and Immune Activation in Nicotiana benthamiana for Biotechnological Applications.

Viral, bacterial, fungal, and nematode infections cause significant agricultural losses, with limited treatment options, necessitating novel approaches to enhance plant defense systems and protection against pathogens. Virus-like nanoparticles (VLPs), extensively used in animal and human therapies (e.g., vaccines and immune enhancers), hold potential for novel agricultural solutions and advancing plant nanotechnology. This study employed various methodologies, including VLP production, confocal microscopy, and real-time qPCR. Our findings demonstrated the presence of 30 nm Qβ-VLPs, fluorescently labeled, within the intercellular space of Nicotiana benthamiana leaves one hour post-infiltration. Furthermore, infiltration with Qβ-VLPs led to an upregulation of key defense genes (NbPR1a, NbPR5, NbNPR, NbERF1, NbMYC2, and NbLRR2) in treated plants. Using RT-qPCR, a significant increase in the relative expression levels of defense genes was observed, with sustained high levels of NbERF1 and NbLRR2 even after 24 h. These findings suggest that Qβ-VLPs effectively upregulate genes crucial for pathogen defense in N. benthamiana, initiating PAMP-triggered immunity and launching signaling cascades that enhance defense mechanisms. This innovative application of VLPs to activate plant defense programs advances plant nanobiotechnology, offering new agricultural solutions.

The role of reactive oxygen species in plant-virus interactions.

Reactive oxygen species (ROS) play a complex role in interactions between plant viruses and their host plants. They can both help the plant defend against viral infection and support viral infection and spread. This review explores the various roles of ROS in plant-virus interactions, focusing on their involvement in symptom development and the activation of plant defense mechanisms. The article discusses how ROS can directly inhibit viral infection, as well as how they can regulate antiviral mechanisms through various pathways involving miRNAs, virus-derived small interfering RNAs, viral proteins, and host proteins. Additionally, it examines how ROS can enhance plant resistance by interacting with hormonal pathways and external substances. The review also considers how ROS might promote viral infection and transmission, emphasizing their intricate role in plant-virus dynamics. These insights offer valuable guidance for future research, such as exploring the manipulation of ROS-related gene expression through genetic engineering, developing biopesticides, and adjusting environmental conditions to improve plant resistance to viruses. This framework can advance research in plant disease resistance, agricultural practices, and disease control.

Field evaluations of plant defense activators and sulfur as alternatives to copper bactericides for the management of cabbage black rot in Florida

Field evaluations of plant defense activators and sulfur as alternatives to copper bactericides for the management of cabbage black rot in Florida

Biocontrol of Southern Blight Caused by Sclerotium rolfsii in Pepper Plants Using Bacillus subtilis GJ6-14

Southern blight, caused by the soil-borne fungus Sclerotium rolfsii, is a serious disease that affects many economically important crops. In this study, we selected Bacillus subtilis GJ6-14, from a total of 260 strains, to control Southern blight in pepper plants. In both seedling and plant tests, GJ6-14 significantly suppressed disease incidence and severity compared to control, furthermore, GJ6-14 demonstrated efficient colonization in the rhizosphere by maintaining the population from log 5.41 to log 3.92 in the pathogen-inoculated plants, indicating its potential as a biocontrol agent. Molecular analysis revealed up-regulation of defense-related genes, such as a 7.6-fold increase in LOX1 and 15.5-fold increase in PR1, at 72 hr after inoculation of S. rolfsii in GJ6-14-treated plants, suggesting activation of plant defense mechanisms. Overall, our findings highlight the promising role of B. subtilis GJ6-14 as a potential biocontrol agent in sustainable management of Southern blight in pepper plants.

The growth-promoting and disease-suppressing mechanisms of Trichoderma inoculation on peanut seedlings.

Trichoderma spp. is known for its ability to enhance plant growth and suppress disease, but the mechanisms for its interaction with host plants and pathogens remain unclear. This study investigated the transcriptomics and metabolomics of peanut plants (Arachis hypogaea L.) inoculated with Trichoderma harzianum QT20045, in the absence and presence of the stem rot pathogen Sclerotium rolfsii JN3011. Under the condition without pathogen stress, the peanut seedlings inoculated with QT20045 showed improved root length and plant weight, increased indole acetic acid (IAA) production, and reduced ethylene level, with more active 1-aminocyclopropane-1-carboxylate acid (ACC) synthase (ACS) and ACC oxidase (ACO), compared with the non-inoculated control. Under the pathogen stress, the biocontrol efficacy of QT20045 against S. rolfsii was 78.51%, with a similar effect on plant growth, and IAA and ethylene metabolisms to the condition with no biotic stress. Transcriptomic analysis of peanut root revealed that Trichoderma inoculation upregulated the expression of certain genes in the IAA family but downregulated the genes in the ACO family (AhACO1 and AhACO) and ACS family (AhACS3 and AhACS1) consistently in the absence and presence of pathogens. During pathogen stress, QT20045 inoculation leads to the downregulation of the genes in the pectinesterase family to keep the host plant's cell wall stable, along with upregulation of the AhSUMM2 gene to activate plant defense responses. In vitro antagonistic test confirmed that QT20045 suppressed S. rolfsii growth through mechanisms of mycelial entanglement, papillary protrusions, and decomposition. Our findings highlight that Trichoderma inoculation is a promising tool for sustainable agriculture, offering multiple benefits from pathogen control to enhanced plant growth and soil health.

Sustainable Remediation of Soil and Water Utilizing Arbuscular Mycorrhizal Fungi: A Review.

Phytoremediation is recognized as an environmentally friendly technique. However, the low biomass production, high time consumption, and exposure to combined toxic stress from contaminated media weaken the potential of phytoremediation. As a class of plant-beneficial microorganisms, arbuscular mycorrhizal fungi (AMF) can promote plant nutrient uptake, improve plant habitats, and regulate abiotic stresses, and the utilization of AMF to enhance phytoremediation is considered to be an effective way to enhance the remediation efficiency. In this paper, we searched 520 papers published during the period 2000-2023 on the topic of AMF-assisted phytoremediation from the Web of Science core collection database. We analyzed the author co-authorship, country, and keyword co-occurrence clustering by VOSviewer. We summarized the advances in research and proposed prospective studies on AMF-assisted phytoremediation. The bibliometric analyses showed that heavy metal, soil, stress tolerance, and growth promotion were the research hotspots. AMF-plant symbiosis has been used in water and soil in different scenarios for the remediation of heavy metal pollution and organic pollution, among others. The potential mechanisms of pollutant removal in which AMF are directly involved through hyphal exudate binding and stabilization, accumulation in their structures, and nutrient exchange with the host plant are highlighted. In addition, the tolerance strategies of AMF through influencing the subcellular distribution of contaminants as well as chemical form shifts, activation of plant defenses, and induction of differential gene expression in plants are presented. We proposed that future research should screen anaerobic-tolerant AMF strains, examine bacterial interactions with AMF, and utilize AMF for combined pollutant removal to accelerate practical applications.

Non-Pathogenic Pythium Induced the Resistance in Hydroponic Lettuce Against Root Rot Disease

Hy droponics is a common soilless type of culture. It is clean and gives high-yield crop production. Lettuce is the most widely grown leafy vegetable in hydroponics. The Pythium species causes root rot, a serious disease in lettuce. In this study, we investigated an effective elicitor to promote resistance in lettuce after pathogen infection. To evaluate the plant defense gene response profile, the expression stability of five candidate genes was studied using RT-qPCR. The LsPR1 and LsLTC2 gene expression were higher in lettuce leaves treated with non-pathogenic Pythium PyASR23 than in roots compared to untreated plants. To assess the disease, the rate of relative gene expression of LsLTC2, LsPR-1b like, LsERF, LsAOS, and LsDEF was associated with Pythium root rot pathogenesis. As a result, it was revealed that alterations in these gene expressions were a defense response to Pythium infection, which increased after pre-treatment with various elicitors for 24 hours. When the chemically stimulated lettuce was infected with pathogenic PySR31, the expression of these genes increased dramatically. These results suggested that non-pathogenic PyASR23 might be employed as an elicitor in lettuce roots and contribute to disease control by activating plant defense gene expression against the Pythium pathogen.

Biochar in Agriculture: Enhancing Crop Productivity and Disease Resistance

Biochar, derived through pyrolysis, presents a promising solution to the challenges faced in sustainable agriculture. This review delves into the diverse advantages of employing biochar to enhance crop yields while promoting environmental responsibility. Its cost-effectiveness and eco-friendly nature not only enrich soil fertility but also contribute to carbon capture, aiding in the fight against climate change. Additionally, while its effectiveness in disease control may vary, biochar shows potential in bolstering crops against environmental pressures. By altering soil characteristics, it encourages the growth of beneficial microbes and improves nutrient availability, ultimately supporting plant vitality. Moreover, integrating biochar into agricultural systems may prompt biochemical and physiological changes that activate plant defences against pathogens. This study thoroughly assesses biochar's impacts on soil health, crop output, and disease prevention, emphasizing its crucial role in advancing sustainable farming practices. Embracing biochar as a strategic resource offers great potential for cultivating resilient and environmentally friendly farming methods, marking a significant step towards sustainable crop disease management.

Cytoplasmic Ca2+ influx mediates iron- and reactive oxygen species-dependent ferroptotic cell death in rice immunity.

Iron- and reactive oxygen species (ROS)-dependent ferroptosis occurs in plant cells. Ca2+ acts as a conserved key mediator to control plant immune responses. Here, we report a novel role of cytoplasmic Ca2+ influx regulating ferroptotic cell death in rice immunity using pharmacological approaches. High Ca2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). During Magnaporthe oryzae infection, 14 different Ca2+ influx regulators altered Ca2+, ROS and Fe2+ accumulation, glutathione reductase (GR) expression, glutathione (GSH) depletion and lipid peroxidation, leading to ferroptotic cell death in rice. High Ca2+ levels inhibited the reduction of glutathione isulphide (GSSG) to GSH in vitro. Ca2+ chelation by ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetra-acetic acid (EGTA) suppressed apoplastic Ca2+ influx in rice leaf sheaths during infection. Blocking apoplastic Ca2+ influx into the cytoplasm by Ca2+ chelation effectively suppressed Ca2+-mediated iron-dependent ROS accumulation and ferroptotic cell death. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca2+ influx, as well as ROS and iron accumulation to trigger ferroptotic cell death in rice. The cytoplasmic Ca2+ influx through calcium-permeable cation channels, including the putative resistosomes, could mediate iron- and ROS-dependent ferroptotic cell death under reduced GR expression levels in rice immune responses.

Co-overexpression of SWEET sucrose transporters modulates sucrose synthesis and defence responses to enhance immunity against bacterial blight in rice.

Enhancing carbohydrate export from source to sink tissues is considered to be a realistic approach for improving photosynthetic efficiency and crop yield. The rice sucrose transporters OsSUT1, OsSWEET11aand OsSWEET14 contribute to sucrose phloem loading and seed filling. Crucially, Xanthomonas oryzae pv. oryzae (Xoo) infection in rice enhances the expression of OsSWEET11a and OsSWEET14 genes, and causes leaf blight. Here we show that co-overexpression of OsSUT1, OsSWEET11aand OsSWEET14 in rice reduced sucrose synthesis and transport leading to lower growth and yield but reduced susceptibility to Xoo relative to controls. The immunity-related hypersensitive response (HR) was enhanced in the transformed lines as indicated by the increased expression of defence genes, higher salicylic acidcontentand presence of HR lesions on the leaves. The results suggest that the increased expression of OsSWEET11a and OsSWEET14 in rice is perceived as a pathogen (Xoo) attack that triggers HR and results in constitutive activation of plant defences that are related to the signalling pathways of pathogen starvation. These findings provide a mechanistic basis for the trade-off between plant growth and immunity because decreased susceptibility against Xoo compromised plant growth and yield.

Alternative products for controlling Sclerotinia sclerotiorum in vivo AND in vitro in canola crops

Products that activate plant defense mechanisms, such as white mold, may increase the disease control spectrum in canola crops as an alternative to chemical control for disease management, aiming at lower environmental impacts and selection pressure on pathogens. This study evaluated Sclerotinia sclerotiorum control and resistance induction in canola crops with alternative products. In vitro and detached leaf experiments were conducted in a laboratory, and in vivo experiments occurred in a greenhouse. High Roots®; V6®; Maxi Flor®; Wert Plus®; potassium, manganese, and copper phosphites; manganese; procymidone fungicide; and Ascophyllum nodosum, Bacillus subtilis, and Bacillus thuringiensis extracts assessed S. sclerotiorum mycelial growth in vitro in detached leaves, white mold severity in a greenhouse, and enzymatic analysis in leaf tissues. The last two experiments also received the acibenzolar-S-methyl and Trichoderma asperellum treatments. S. sclerotiorum mycelial growth showed 100% inhibition under B. subtilis and Wert Plus® at 1000 ppm and procymidone at 100 and 1000 ppm in vitro. The procymidone and B. subtilis treatments in detached leaves inhibited 100% of fungus growth. The greenhouse experiments with lower severity were procymidone, copper phosphite, B. thuringiensis, B. subtilis, manganese phosphite, potassium phosphite, High Roots®, and V6®. Higher peroxidase enzyme activity occurred in plants treated with acibenzolar-S-methyl, copper phosphite, and fungicide 48 hours after pathogen inoculation. The polyphenol oxidase enzyme did not show activity. Alternative products were responsive and may aid the chemical control of white mold in canola crops.

Mitigating the Trade-Off between Growth and Stress Resistance in Plants by Fungal Volatile Compounds.

Plants grow in association with diverse microorganisms. During communication between plants and microbes, beneficial and phytopathogenic bacteria and fungi emit various volatile compounds (VCs). These microbial VCs (mVCs) are typically small, odorous compounds with low boiling point, high vapor pressure, and a lipophilic moiety (Schulz and Dickschat, 2007). Based on recent studies, mVCs appear to regulate plant nutrient acquisition, photosynthesis, phytohormone actions, and metabolic processes, leading to an improvement in plant performance (Fincheira et al., 2021). Since mVCs can activate plant defenses, and/or promote plant growth and development at low concentrations, their application in agriculture is attractive to efficiently grow crops and improve plant health in the absence of excess usage of chemical fertilizers and pesticides. To this end, mVCs have been used for integrated pest management (IPM) to enhance both crop defense and production in field conditions (Brilli et al., 2019). Understanding the molecular action of mVCs in plants will pave the way toward improved crop protection and ecosystem management.

Harnessing chickpea bacterial endophytes for improved plant health and fitness.

Endophytic bacteria live asymptomatically inside the tissues of host plants without inflicting any damage. Endophytes can confer several beneficial traits to plants, which can contribute to their growth, development, and overall health. They have been found to stimulate plant growth by enhancing nutrient uptake and availability. They can produce plant growth-promoting substances such as auxins, cytokinins, and gibberellins, which regulate various aspects of plant growth and development. Endophytes can also improve root system architecture, leading to increased nutrient and water absorption. Some endophytes possess the ability to solubilize nutrients, such as phosphorus and potassium, making them more available for plant uptake, and fixing atmospheric nitrogen. Chickpea (Cicer arietinum) is a major legume crop that has mutualistic interactions with endophytes. These endophytes can benefit the chickpea plant in various ways, including higher growth, improved nutrient uptake, increased tolerance to abiotic and biotic stressors, and disease suppression. They can produce enzymes and metabolites that scavenge harmful reactive oxygen species, thus reducing oxidative stress. Moreover, several studies reported that endophytes produce antimicrobial compounds, lytic enzymes, and volatile organic compounds that inhibit the growth of fungal pathogens and trigger systemic defense responses in plants, leading to increased resistance against a broad range of pathogens. They can activate plant defense pathways, including the production of defense-related enzymes, phytoalexins, and pathogenesis-related proteins, thereby providing long-lasting protection. It is important to note that the diversity and function of chickpea-associated endophytes can vary depending on factors such as variety, geographical location, and environmental conditions. The mechanisms behind the plant-beneficial interactions are still being intensively explored. In this review, new biotechnologies in agricultural production and ecosystem stability were presented. Thus, harnessing chickpea endophytes could be exploited in developing drought-resistant cultivars that can maintain productivity in arid and semi-arid environments, crucial for meeting the global demand for chickpeas.

Plant microRNAs regulate the defense response against pathogens.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, typically 20-25 nucleotides in length, that play a crucial role in regulating gene expression post-transcriptionally. They are involved in various biological processes such as plant growth, development, stress response, and hormone signaling pathways. Plants interact with microbes through multiple mechanisms, including mutually beneficial symbiotic relationships and complex defense strategies against pathogen invasions. These defense strategies encompass physical barriers, biochemical defenses, signal recognition and transduction, as well as systemic acquired resistance. MiRNAs play a central role in regulating the plant's innate immune response, activating or suppressing the transcription of specific genes that are directly involved in the plant's defense mechanisms against pathogens. Notably, miRNAs respond to pathogen attacks by modulating the balance of plant hormones such as salicylic acid, jasmonic acid, and ethylene, which are key in activating plant defense mechanisms. Moreover, miRNAs can cross boundaries into fungal and bacterial cells, performing cross-kingdom RNA silencing that enhances the plant's disease resistance. Despite the complex and diverse roles of miRNAs in plant defense, further research into their function in plant-pathogen interactions is essential. This review summarizes the critical role of miRNAs in plant defense against pathogens, which is crucial for elucidating how miRNAs control plant defense mechanisms.

INDUCTION OF RESISTANCE IN MUNGBEAN AGAINST CERCOSPORA LEAF SPOT THROUGH PLANT DEFENSE ACTIVATORS

Mungbean (Vigna radiata L. Wilczek), is a popular short-duration legume crop which is mostly cultivated in South and Southeast Asian countries. It is a rich source of proteins, carbohydrates, fats, minerals, and fibers. As an important source of dietary protein, it is usually consumed as whole seed or flour, or as sprouts. Cercospora Leaf Spot of Mungbean (CLSM) is a destructive disease caused by Cercospora canescens, which affects the whole crop, and causing 95% of yield losses. To mitigate these yield losses, five plant defense activators dipotassium hydrogen phosphate (K2HPO4), salicylic acid, carboxylic acid, citric acid, and benzoic acid were used in current study with 3 different concentrations (0.25, 0.5, and 0.75%) in a field trial arranged in randomized complete block design. Our field experiments revealed salicylic acid treated plants with minimum disease incidence (21.798%) followed by citric acid (25.131%), dipotassium hydrogen phosphate (27.466%), benzoic acid (29.064%), and carboxylic acid (35.043%) compared to untreated check. This revelation suggested the salicylic acid as a potent antifungal agent by activating the Mungbean defense systems for the management of CLSM.

Novel in silico screening system for plant defense activators using deep learning-based prediction of reactive oxygen species accumulation

BackgroundPlant defense activators offer advantages over pesticides by avoiding the emergence of drug-resistant pathogens. However, only a limited number of compounds have been reported. Reactive oxygen species (ROS) act as not only antimicrobial agents but also signaling molecules that trigger immune responses. They also affect various cellular processes, highlighting the potential ROS modulators as plant defense activators. Establishing a high-throughput screening system for ROS modulators holds great promise for identifying lead chemical compounds with novel modes of action (MoAs).ResultsWe established a novel in silico screening system for plant defense activators using deep learning-based predictions of ROS accumulation combined with the chemical properties of the compounds as explanatory variables. Our screening strategy comprised four phases: (1) development of a ROS inference system based on a deep neural network that combines ROS production data in plant cells and multidimensional chemical features of chemical compounds; (2) in silico extensive-scale screening of seven million commercially available compounds using the ROS inference model; (3) secondary screening by visualization of the chemical space of compounds using the generative topographic mapping; and (4) confirmation and validation of the identified compounds as potential ROS modulators within plant cells. We further characterized the effects of selected chemical compounds on plant cells using molecular biology methods, including pathogenic signal-triggered enzymatic ROS induction and programmed cell death as immune responses. Our results indicate that deep learning-based screening systems can rapidly and effectively identify potential immune signal-inducible ROS modulators with distinct chemical characteristics compared with the actual ROS measurement system in plant cells.ConclusionsWe developed a model system capable of inferring a diverse range of ROS activity control agents that activate immune responses through the assimilation of chemical features of candidate pesticide compounds. By employing this system in the prescreening phase of actual ROS measurement in plant cells, we anticipate enhanced efficiency and reduced pesticide discovery costs. The in-silico screening methods for identifying plant ROS modulators hold the potential to facilitate the development of diverse plant defense activators with novel MoAs.