Seed pretreatment with brassinosteroids stimulates sunflower immunity against parasitic weed (Orobanche cumana) infection.

Broomrape (Orobanche cumana) negatively affects sunflower, causing severe yield losses, and thus, there is a need to control O. cumana infestation. Brassinosteroids (BRs) play key roles in plant growth and provide resilience to weed infection. This study aims to evaluate the mechanisms by which BRs ameliorate O. cumana infection in sunflower (Helianthus annuus). Seeds were pretreated with BRs (1, 10, and 100 nM) and O. cumana inoculation for 4 weeks under soil conditions. O. cumana infection significantly reduced plant growth traits, photosynthesis, endogenous BRs and regulated the plant defence (POX, GST), BRs signalling (BAK1, BSK1 to BSK4) and synthesis (BRI1, BR6OX2) genes. O. cumana also elevated the levels of malondialdehyde (MDA), hydroxyl radical (OH-), hydrogen peroxide (H2O2) and superoxide (O2 •-) in leaves/roots by 77/112, 63/103, 56/97 and 54/89%, as well as caused ultrastructural cellular damages in both leaves and roots. In response, plants activated a few enzymes, superoxide dismutase (SOD), peroxidase (POD) and reduced glutathione but were unable to stimulate the activity of ascorbate peroxidase (APX) and catalase (CAT) enzymes. The addition of BRs (especially at 10 nM) notably recovered the ultrastructural cellular damages, lowered the production of oxidative stress, activated the key enzymatic antioxidants and induced the phenolic and lignin contents. The downregulation in the particular genes by BRs is attributed to the increased resilience of sunflower via a susceptible reaction. In a nutshell, BRs notably enhanced the sunflower resistance to O. cumana infection by escalating the plant immunity responses, inducing systemic acquired resistance, reducing oxidative or cellular damages, and modulating the expression of BR synthesis or signalling genes.

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.

LORE receptor homomerization is required for 3-hydroxydecanoic acid-induced immune signaling and determines the natural variation of immunosensitivity within the Arabidopsis genus.

The S-domain-type receptor-like kinase (SD-RLK) LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) from Arabidopsis thaliana is a pattern recognition receptor that senses medium-chain 3-hydroxy fatty acids, such as 3-hydroxydecanoic acid (3-OH-C10:0), to activate pattern-triggered immunity. Here, we show that LORE homomerization is required to activate 3-OH-C10:0-induced immune signaling. Fluorescence lifetime imaging in Nicotiana benthamiana demonstrates that AtLORE homomerizes via the extracellular and transmembrane domains. Co-expression of AtLORE truncations lacking the intracellular domain exerts a dominant negative effect on AtLORE signaling in both N. benthamiana and A. thaliana, highlighting that homomerization is essential for signaling. Screening for 3-OH-C10:0-induced reactive oxygen species production revealed natural variation within the Arabidopsis genus. Arabidopsis lyrata and Arabidopsis halleri do not respond to 3-OH-C10:0, although both possess a putative LORE ortholog. Both LORE orthologs have defective extracellular domains that bind 3-OH-C10:0 to a similar level as AtLORE, but lack the ability to homomerize. Thus, ligand binding is independent of LORE homomerization. Analysis of AtLORE and AlyrLORE chimera suggests that the loss of AlyrLORE homomerization is caused by several amino acid polymorphisms across the extracellular domain. Our findings shed light on the activation mechanism of LORE and the loss of 3-OH-C10:0 perception within the Arabidopsis genus.

Salicylic Acid Is Required for Broad-Spectrum Disease Resistance in Rice.

Rice plants contain high basal levels of salicylic acid (SA), but some of their functions remain elusive. To elucidate the importance of SA homeostasis in rice immunity, we characterized four rice SA hydroxylase genes (OsSAHs) and verified their roles in SA metabolism and disease resistance. Recombinant OsSAH proteins catalyzed SA in vitro, while OsSAH3 protein showed only SA 5-hydroxylase (SA5H) activity, which was remarkably higher than that of other OsSAHs that presented both SA3H and SA5H activities. Amino acid substitutions revealed that three amino acids in the binding pocket affected SAH enzyme activity and/or specificity. Knockout OsSAH2 and OsSAH3 (sahKO) genes conferred enhanced resistance to both hemibiotrophic and necrotrophic pathogens, whereas overexpression of each OsSAH gene increased susceptibility to the pathogens. sahKO mutants showed increased SA and jasmonate levels compared to those of the wild type and OsSAH-overexpressing plants. Analysis of the OsSAH3 promoter indicated that its induction was mainly restricted around Magnaporthe oryzae infection sites. Taken together, our findings indicate that SA plays a vital role in immune signaling. Moreover, fine-tuning SA homeostasis through suppression of SA metabolism is an effective approach in studying broad-spectrum disease resistance in rice.

Activation of the plant mevalonate pathway by extracellular ATP.

The mevalonate pathway plays a critical role in multiple cellular processes in both animals and plants. In plants, the products of this pathway impact growth and development, as well as the response to environmental stress. A forward genetic screen of Arabidopsis thaliana using Ca2+-imaging identified mevalonate kinase (MVK) as a critical component of plant purinergic signaling. MVK interacts directly with the plant extracellular ATP (eATP) receptor P2K1 and is phosphorylated by P2K1 in response to eATP. Mutation of P2K1-mediated phosphorylation sites in MVK eliminates the ATP-induced cytoplasmic calcium response, MVK enzymatic activity, and suppresses pathogen defense. The data demonstrate that the plasma membrane associated P2K1 directly impacts plant cellular metabolism by phosphorylation of MVK, a key enzyme in the mevalonate pathway. The results underline the importance of purinergic signaling in plants and the ability of eATP to influence the activity of a key metabolite pathway with global effects on plant metabolism.

Bacterial type III effector-induced plant C8 volatiles elicit antibacterial immunity in heterospecific neighbouring plants via airborne signalling.

Upon sensing attack by pathogens and insect herbivores, plants release complex mixtures of volatile compounds. Here, we show that the infection of lima bean (Phaseolus lunatus L.) plants with the non-host bacterial pathogen Pseudomonas syringae pv. tomato led to the production of microbe-induced plant volatiles (MIPVs). Surprisingly, the bacterial type III secretion system, which injects effector proteins directly into the plant cytosol to subvert host functions, was found to prime both intra- and inter-specific defense responses in neighbouring wild tobacco (Nicotiana benthamiana) plants. Screening of each of 16 effectors using the Pseudomonas fluorescens effector-to-host analyser revealed that an effector, HopP1, was responsible for immune activation in receiver tobacco plants. Further study demonstrated that 1-octen-3-ol, 3-octanone and 3-octanol are novel MIPVs emitted by the lima bean plant in a HopP1-dependent manner. Exposure to synthetic 1-octen-3-ol activated immunity in tobacco plants against a virulent pathogen Pseudomonas syringae pv. tabaci. Our results show for the first time that a bacterial type III effector can trigger the emission of C8 plant volatiles that mediate defense priming via plant-plant interactions. These results provide novel insights into the role of airborne chemicals in bacterial pathogen-induced inter-specific plant-plant interactions.

Short Peptides Make a Big Difference: The Role of Botany-Derived AMPs in Disease Control and Protection of Human Health.

Botany-derived antimicrobial peptides (BAMPs), a class of small, cysteine-rich peptides produced in plants, are an important component of the plant immune system. Both in vivo and in vitro experiments have demonstrated their powerful antimicrobial activity. Besides in plants, BAMPs have cross-kingdom applications in human health, with toxic and/or inhibitory effects against a variety of tumor cells and viruses. With their diverse molecular structures, broad-spectrum antimicrobial activity, multiple mechanisms of action, and low cytotoxicity, BAMPs provide ideal backbones for drug design, and are potential candidates for plant protection and disease treatment. Lots of original research has elucidated the properties and antimicrobial mechanisms of BAMPs, and characterized their surface receptors and in vivo targets in pathogens. In this paper, we review and introduce five kinds of representative BAMPs belonging to the pathogenesis-related protein family, dissect their antifungal, antiviral, and anticancer mechanisms, and forecast their prospects in agriculture and global human health. Through the deeper understanding of BAMPs, we provide novel insights for their applications in broad-spectrum and durable plant disease prevention and control, and an outlook on the use of BAMPs in anticancer and antiviral drug design.

The small molecule Zaractin activates ZAR1-mediated immunity in Arabidopsis.

Pathogenic effector proteins use a variety of enzymatic activities to manipulate host cellular proteins and favor the infection process. However, these perturbations can be sensed by nucleotide-binding leucine-rich-repeat (NLR) proteins to activate effector-triggered immunity (ETI). Here we have identified a small molecule (Zaractin) that mimics the immune eliciting activity of the Pseudomonas syringae type III secreted effector (T3SE) HopF1r and show that both HopF1r and Zaractin activate the same NLR-mediated immune pathway in Arabidopsis Our results demonstrate that the ETI-inducing action of pathogenic effectors can be harnessed to identify synthetic activators of the eukaryotic immune system.

iTRAQ-Based Proteomics Analysis of Response to Solanum tuberosum Leaves Treated with the Plant Phytotoxin Thaxtomin A.

Thaxtomin A (TA) is a phytotoxin secreted by Streptomyces scabies that causes common scab in potatoes. However, the mechanism of potato proteomic changes in response to TA is barely known. In this study, the proteomic changes in potato leaves treated with TA were determined using the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique. A total of 693 proteins were considered as differentially expressed proteins (DEPs) following a comparison of leaves treated with TA and sterile water (as a control). Among the identified DEPs, 460 and 233 were upregulated and downregulated, respectively. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, many DEPs were found to be involved in defense and stress responses. Most DEPs were grouped in carbohydrate metabolism, amino acid metabolism, energy metabolism, and secondary metabolism including oxidation-reduction process, response to stress, plant-pathogen interaction, and plant hormone signal transduction. In this study, we analyzed the changes in proteins to elucidate the mechanism of potato response to TA, and we provided a molecular basis to further study the interaction between plant and TA. These results also offer the option for potato breeding through analysis of the resistant common scab.

CaWRKY30 Positively Regulates Pepper Immunity by Targeting CaWRKY40 against Ralstonia solanacearum Inoculation through Modulating Defense-Related Genes.

The WRKY transcription factors (TFs) network is composed of WRKY TFs' subset, which performs a critical role in immunity regulation of plants. However, functions of WRKY TFs' network remain unclear, particularly in non-model plants such as pepper (Capsicum annuum L.). This study functionally characterized CaWRKY30-a member of group III Pepper WRKY protein-for immunity of pepper against Ralstonia solanacearum infection. The CaWRKY30 was detected in nucleus, and its transcriptional expression levels were significantly upregulated by R. solanacearum inoculation (RSI), and foliar application ethylene (ET), abscisic acid (ABA), and salicylic acid (SA). Virus induced gene silencing (VIGS) of CaWRKY30 amplified pepper's vulnerability to RSI. Additionally, the silencing of CaWRKY30 by VIGS compromised HR-like cell death triggered by RSI and downregulated defense-associated marker genes, like CaPR1, CaNPR1, CaDEF1, CaABR1, CaHIR1, and CaWRKY40. Conversely, transient over-expression of CaWRKY30 in pepper leaves instigated HR-like cell death and upregulated defense-related maker genes. Furthermore, transient over-expression of CaWRKY30 upregulated transcriptional levels of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40. On the other hand, transient over-expression of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40 upregulated transcriptional expression levels of CaWRKY30. The results recommend that newly characterized CaWRKY30 positively regulates pepper's immunity against Ralstonia attack, which is governed by synergistically mediated signaling by phytohormones like ET, ABA, and SA, and transcriptionally assimilating into WRKY TFs networks, consisting of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40. Collectively, our data will facilitate to explicate the underlying mechanism of crosstalk between pepper's immunity and response to RSI.

Faba bean root exudates alter pea root colonization by the oomycete Aphanomyces euteiches at early stages of infection.

Aphanomyces euteiches is an oomycete pathogen that causes the pea root rot. We investigated the potential role of early belowground defense in pea (susceptible plant) and faba bean (tolerant plant) at three days after inoculation. Pea and faba bean were inoculated with A. euteiches zoospores. Root colonization was examined. Root exudates from pea and faba bean were harvested and their impact on A. euteiches development were assessed by using in vitro assays. A. euteiches root colonization and the influence of the oomycete inoculation on specialized metabolites patterns and arabinogalactan protein (AGP) concentration of root exudates were also determined. In faba bean root, A. euteiches colonization was very low as compared with that of pea. Whereas infected pea root exudates have a positive chemotaxis index (CI) on zoospores, faba bean exudate CI was negative suggesting a repellent effect. While furanoacetylenic compounds were only detected in faba bean exudates, AGP concentration was specifically increased in pea.This work showed that early in the course of infection, host susceptibility to A. euteiches is involved via a plant-species specific root exudation opening new perspectives in pea root rot disease management.

Cytokinin Modulates Cellular Trafficking and the Cytoskeleton, Enhancing Defense Responses.

The plant hormone cytokinin (CK) plays central roles in plant development and throughout plant life. The perception of CKs initiating their signaling cascade is mediated by histidine kinase receptors (AHKs). Traditionally thought to be perceived mostly at the endoplasmic reticulum (ER) due to receptor localization, CK was recently reported to be perceived at the plasma membrane (PM), with CK and its AHK receptors being trafficked between the PM and the ER. Some of the downstream mechanisms CK employs to regulate developmental processes are unknown. A seminal report in this field demonstrated that CK regulates auxin-mediated lateral root organogenesis by regulating the endocytic recycling of the auxin carrier PIN1, but since then, few works have addressed this issue. Modulation of the cellular cytoskeleton and trafficking could potentially be a mechanism executing responses downstream of CK signaling. We recently reported that CK affects the trafficking of the pattern recognition receptor LeEIX2, influencing the resultant defense output. We have also recently found that CK affects cellular trafficking and the actin cytoskeleton in fungi. In this work, we take an in-depth look at the effects of CK on cellular trafficking and on the actin cytoskeleton in plant cells. We find that CK influences the actin cytoskeleton and endomembrane compartments, both in the context of defense signaling-where CK acts to amplify the signal-as well as in steady state. We show that CK affects the distribution of FLS2, increasing its presence in the plasma membrane. Furthermore, CK enhances the cellular response to flg22, and flg22 sensing activates the CK response. Our results are in agreement with what we previously reported for fungi, suggesting a fundamental role for CK in regulating cellular integrity and trafficking as a mechanism for controlling and executing CK-mediated processes.

Effectors of Puccinia striiformis f. sp. tritici Suppressing the Pathogenic-Associated Molecular Pattern-Triggered Immune Response Were Screened by Transient Expression of Wheat Protoplasts.

Puccinia striiformis f. sp. tritici (Pst) is an important pathogen of wheat (Triticum aestivum L.) stripe rust, and the effector protein secreted by haustoria is a very important component involved in the pathogenic process. Although the candidate effector proteins secreted by Pst haustoria have been predicted to be abundant, few have been functionally validated. Our study confirmed that chitin and flg22 could be used as elicitors of the pathogenic-associated molecular pattern-triggered immune (PTI) reaction in wheat leaves and that TaPr-1-14 could be used as a marker gene to detect the PTI reaction. In addition, the experimental results were consistent in wheat protoplasts. A rapid and efficient method for screening and identifying the effector proteins of Pst was established by using the wheat protoplast transient expression system. Thirty-nine Pst haustorial effector genes were successfully cloned and screened for expression in the protoplast. We identified three haustorial effector proteins, PSEC2, PSEC17, and PSEC45, that may inhibit the response of wheat to PTI. These proteins are localized in the somatic cytoplasm and nucleus of wheat protoplasts and are highly expressed during the infection and parasitism of wheat.

An efficient direct screening system for microorganisms that activate plant immune responses based on plant-microbe interactions using cultured plant cells.

Microorganisms that activate plant immune responses have attracted considerable attention as potential biocontrol agents in agriculture because they could reduce agrochemical use. However, conventional methods to screen for such microorganisms using whole plants and pathogens are generally laborious and time consuming. Here, we describe a general strategy using cultured plant cells to identify microorganisms that activate plant defense responses based on plant-microbe interactions. Microbial cells were incubated with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses secreted by an oomycete. Cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells served as a marker to evaluate the potential of microorganisms to activate plant defense responses. Twenty-nine bacterial strains isolated from the interior of Brassica rapa var. perviridis plants were screened, and 8 strains that enhanced cryptogein-induced ROS production in BY-2 cells were selected. Following application of these strains to the root tip of Arabidopsis seedlings, two strains, Delftia sp. BR1R-2 and Arthrobacter sp. BR2S-6, were found to induce whole-plant resistance to bacterial pathogens (Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovora subsp. carotovora NBRC 14082). Pathogen-induced expression of plant defense-related genes (PR-1, PR-5, and PDF1.2) was enhanced by the pretreatment with strain BR1R-2. This cell-cell interaction-based platform is readily applicable to large-scale screening for microorganisms that enhance plant defense responses under various environmental conditions.

Chitosan and EDTA conjugated graphene oxide antinematodes in Eggplant: Toward improving plant immune response.

A new strategy regarding the fabrication of chitosan (CS) or ethylene diamine tetraacetic acid (EDTA) on graphene oxide (GO) was performed. The nematocidal potential against Meloidogyne incognita causing root-knot infection in eggplant was tested. The plant immune response was investigated through measuring the photosynthetic pigments, phenols and proline contents, oxidative stress, and antioxidant enzymes activity. Results indicating that, the treatment by pure GO recorded the most mortality percentages of M. incognita 2nd juveniles followed by GO-CS then GO-EDTA. In vivo greenhouse experiments reveals that, the most potent treatment in reducing nematodes was GO-CS which recorded 85.42%, 75.3%, 55.5%, 87.81%, and 81.32% in numbers of 2nd juveniles, galls, females, egg masses and the developmental stage, respectively. The highest chlorophyll a (104%), chlorophyll b (46%), total phenols (137.5%), and free proline (145.2%) were recorded in GO-CS. The highest malondialdehyde (MDA) value was achieved by GO-EDTA (7.22%), and hydrogen peroxide (H2O2) content by 47.51% after the treatment with pure GO. Treatment with GO-CS increased the activities of catalase (CAT) by 98.3%, peroxidase (POD) by 97.52%, polyphenol oxidase (PPO) by 113.8%, and superoxide dismutase (SOD) by 42.43%. The synthesized nanocomposites increases not only the nematocidal activity but also the plant systematic immune response.

Possible role of small secreted peptides (SSPs) in immune signaling in bryophytes.

Plants utilize a plethora of peptide signals to regulate their immune response. Peptide ligands and their cognate receptors involved in immune signaling share common motifs among many species of vascular plants. However, the origin and evolution of immune peptides is still poorly understood. Here, we searched for genes encoding small secreted peptides in the genomes of three bryophyte lineages-mosses, liverworts and hornworts-that occupy a critical position in the study of land plant evolution. We found that bryophytes shared common predicted small secreted peptides (SSPs) with vascular plants. The number of SSPs is higher in the genomes of mosses than in both the liverwort Marchantia polymorpha and the hornwort Anthoceros sp. The synthetic peptide elicitors-AtPEP and StPEP-specific for vascular plants, triggered ROS production in the protonema of the moss Physcomitrella patens, suggesting the possibility of recognizing peptide ligands from angiosperms by moss receptors. Mass spectrometry analysis of the moss Physcomitrella patens, both the wild type and the Δcerk mutant secretomes, revealed peptides that specifically responded to chitosan treatment, suggesting their role in immune signaling.

Chitin nanofibers trigger membrane bound defense signaling and induce elicitor activity in plants.

The present study demonstrated that chitin-based nanofibers (CNFs) trigger the chitinase genes (PGIP1 and CaChi2), while elevating salicylic acid that can protect plants against pathogens. Cross-talk between this genetic induction and salicylic-acid-mediated immune response was also observed, which may arm a plant against multiple pathovars. Crab and mushroom based CNFs were synthesized by electrospinning and ball milling techniques. Plants (mung bean, Vigna radiata) (pepper, Capsicum annuum) were pre-inoculated with CNFs and treated with the pathogens Scrolotium rolfsii for pepper and Macrophomina phaseolina for mung bean and shrimp-based CNFs were used as a control. Treated plants had elevated levels of chitinase genes in response to CNFs at inoculation concentrations <10 mg/mL both in soil and media, to protect them against the pathogenic fungal disease. After 24 h of exposure to the pathogens, qRT-PCR showed genes class II chitinase gene (CaChi2) and polygalacturonase inhibitor protein 1 (PGIP1) to be up-regulated in both root and shoot at 0.1 and 1 mg/mL of inoculation, respectively. The ball milled mushroom CNFs were sufficient to trigger the membrane based enzymes with less diameter (≥15 nm) to be most efficient versus others. In vitro analysis showed IC50 of ball milled mushroom CNFs to be most efficient in limiting the growth of fungal biomass. Further trigger-like effects were prominent in reducing pathogenic fungal spread in both species.

Spermine and Spermidine Priming against Botrytis cinerea Modulates ROS Dynamics and Metabolism in Arabidopsis.

Polyamines (PAs) are ubiquitous small aliphatic polycations important for growth, development, and environmental stress responses in plants. Here, we demonstrate that exogenous application of spermine (Spm) and spermidine (Spd) induced cell death at high concentrations, but primed resistance against the necrotrophic fungus Botrytis cinerea in Arabidopsis. At low concentrations, Spm was more effective than Spd. Treatments with higher exogenous Spd and Spm concentrations resulted in a biphasic endogenous PA accumulation. Exogenous Spm induced the accumulation of H2O2 after treatment but also after infection with B. cinerea. Both Spm and Spd induced the activities of catalase, ascorbate peroxidase, and guaiacol peroxidase after treatment but also after infection with B. cinerea. The soluble sugars glucose, fructose, and sucrose accumulated after treatment with high concentrations of PAs, whereas only Spm induced sugar accumulation after infection. Total and active nitrate reductase (NR) activities were inhibited by Spm treatment, whereas Spd inhibited active NR at low concentrations but promoted active NR at high concentrations. Finally, γaminobutyric acid accumulated after treatment and infection in plants treated with high concentrations of Spm. Phenylalanine and asparagine also accumulated after infection in plants treated with a high concentration of Spm. Our data illustrate that Spm and Spd are effective in priming resistance against B. cinerea, opening the door for the development of sustainable alternatives for chemical pesticides.