Imprimatins A and B: Novel plant activators targeting salicylic acid metabolism in Arabidopsis thaliana

Plant activators are agrochemicals that protect plants from a broad range of pathogens by activating the plant immune system. Unlike pesticides, they do not target pathogens; therefore, plant activators provide durable effects that are not overcome by pathogenic microbes. Although certain plant activators have been applied to paddy fields for more than 30 years, the molecular basis of the underlying immune induction are unclear. From the screening of 10,000 diverse chemicals by a high-throughput screening procedure to identify compounds that specifically enhance pathogen-induced cell death in Arabidopsis cultured cells, we identified 7 compounds, which we designated as immune priming chemicals (Imprimatins). These compounds increased disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants. Pretreatments increased the accumulation of endogenous salicylic acid (SA) but reduced its metabolite, SA-O-β-D-glucoside (SAG). Imprimatins inhibited the enzymatic activities of 2 SA glucosyltransferases (SAGTs) in vitro at concentrations effective for immune priming. Single and double knockout Arabidopsis plants for both SAGTs consistently exhibited enhanced disease resistance and SA accumulation. Our results demonstrate that the control of the free SA pool through SA-inactivating enzymes can be a useful methodology to confer disease resistance in plants. SAGTs can pave the way for target-based discovery of novel crop protectants.     

Novel Plant Immune-Priming Compounds Identified via High-Throughput Chemical Screening Target Salicylic Acid Glucosyltransferases in Arabidopsis

Plant activators are compounds, such as analogs of the defense hormone salicylic acid (SA), that protect plants from pathogens by activating the plant immune system. Although some plant activators have been widely used in agriculture, the molecular mechanisms of immune induction are largely unknown. Using a newly established high-throughput screening procedure that screens for compounds that specifically potentiate pathogen-activated cell death in Arabidopsis thaliana cultured suspension cells, we identified five compounds that prime the immune response. These compounds enhanced disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants. Pretreatments increased the accumulation of endogenous SA, but reduced its metabolite, SA-O-β-d-glucoside. Inducing compounds inhibited two SA glucosyltransferases (SAGTs) in vitro. Double knockout plants that lack both SAGTs consistently exhibited enhanced disease resistance. Our results demonstrate that manipulation of the active free SA pool via SA-inactivating enzymes can be a useful strategy for fortifying plant disease resistance and may identify useful crop protectants.     

One shot-two pathogens blocked: Exposure of Arabidopsis to hexadecane,a long chain volatile organic compound,confers induced resistance against both Pectobacterium carotovorum and Pseudomonas syringae

Bacteria and plant derived volatile organic compounds have been reported as the chemical triggers that elicit induced resistance in plants. Previously, volatile organic compounds (VOCs), including acetoin and 2,3-butanediol, were found to be emitted from plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis GB03, which had been shown to elicit ISR and plant growth promotion. More recently, we reported data that stronger induced resistance could be elicited against Pseudomonas syringae pv maculicola ES4326 in plants exposed to C13 VOC from another PGPR Paenibacillus polymyxa E681 compared with that of strain GB03. Here, we assessed whether another long hydrocarbon C16 hexadecane (HD) conferred protection to Arabidopsis from infection of a biotrophic pathogen, P. syringae pv maculicola and a necrotrophic pathogen, Pectobacterium carotovorum subsp carotovorum. Collectively, long-chain VOCs can be linked to a plant resistance activator for protecting plants against both biotrophic and necrotrophic pathogens at the same time.     

Isolation and characterization of the plant immune-priming compounds Imprimatin B3 and -B4, potentiators of disease resistance in Arabidopsis thaliana

Plant activators are chemical crop protectants that fortify the immune system in plants. Unlike pesticides that target pathogens, plant activators provide durable effects against a broad spectrum of diseases, which have not been overcome by pathogenic microbes. Plant activators are not only useful agrochemicals, but can also help to elucidate the details of the plant immune system. Using an established high-throughput screening procedure, we previously identified 5 compounds, designated as Imprimatins, which prime plant immune response. These compounds increased disease resistance against pathogenic Pseudomonas bacteria in Arabidopsis plants by inhibiting 2 salicylic acid (SA) glucosyltransferases (SAGTs), resulting in accumulation of the phytohormone SA. Here, we report the isolation of 2 additional Imprimatins, B3 and B4, which are structurally similar to Imprimatin B1 and B2. Because these compounds did not have strong inhibitory effects on SAGTs in vitro, they may exert their function after metabolic conversion in vivo.     

Arabidopsis DAL1 and DAL2, two RING finger proteins homologous to Drosophila DIAP1, are involved in regulation of programmed cell death

Programmed cell death (PCD) is a precise, genetically controlled cellular process with important roles in plant growth, development, and response to biotic and abiotic stress. However, the genetic mechanisms that control PCD in plants are unclear. Two Arabidopsis genes, DAL1 and DAL2 (for Drosophila DIAP1 like 1 and 2), encoding RING finger proteins with homology to DIAP1 were identified, and a series of experiments were performed to elucidate their roles in the regulation of PCD and disease resistance. Expression of DAL1 and DAL2 genes was induced in Arabidopsis plants after inoculation with virulent and avirulent strains of Pseudomonas syrinage pv. tomato (Pst) DC3000 or after infiltration with fumonisin B1 (FB1). Plants with mutations in the DAL1 and DAL2 genes displayed more severe disease after inoculation with an avirulent strain of Pst DC3000, but they showed similar disease severity as the wild-type plant after inoculation with a virulent strain of Pst DC3000. Significant accumulations of reactive oxygen species (ROS) and increased cell death were observed in the dal1 and dal2 mutant plants after inoculation with the avirulent strain of Pst DC3000. The dal mutant plants underwent extensive PCD upon infiltration of FB1 and displayed higher levels of ROS accumulation, callose deposition, and autofluorescence than the wild-type plants. Our data suggest that DAL1 and DAL2 may act as negative regulators of PCD in Arabidopsis.     

Endophytic bacteria enhancing growth and disease resistance of potato (Solanum tuberosum L.)

Priming plants by non-pathogenic bacteria allows the host to save energy and to reduce time needed for development of defense reaction during a pathogen attack. However, information on the role of endophytes in plant defense is limited. Here, the ability of endophytic bacteria to promote growth and resistance of potato plants towards infection by the necrotroph Pectobacterium atrosepticum was studied. A Pseudomonas sp. strain was selected due to antagonism towards bacterial pathogens and a Methylobacterium sp. strain because of efficient plant colonization. The aim of this study was to find if there is any correlation between plant growth promotion and induction of resistance by endophytes of potato, as well as to study the putative mechanisms of endophytes interacting with the plant during resistance induction. Both tested strains promoted growth of potato shoots but only the Pseudomonas sp. increased potato resistance towards the soft rot disease. Induction of disease resistance by the Methylobacterium sp. was inversely proportional to the size of bacterial population used for inoculation. The plant antioxidant system was moderately activated during the induction of resistance by the biocontrol strains. qPCR data on expression of marker genes of induced systemic resistance and acquired systemic resistance in endophyte-infected Arabidopsis plants showed activation of both salicylic acid and jasmonate/ethylene-dependent pathways after challenge inoculation with the pathogen. We suggest that some endophytes have the potential to activate both basal and inducible plant defense systems, whereas the growth promotion by biocontrol strains may not correlate with induction of disease resistance.     

Use of Arabidopsis thaliana and Pseudomonas syringae in the Study of Plant Disease Resistance and Tolerance

The interaction between Arabidopsis thaliana and the bacterium Pseudomonas syringae is being developed as a model experimental system for plant pathology research. Race-specific ("gene-for-gene") resistance has been demonstrated for this interaction, and pathogen genes that determine avirulence have been isolated and characterized. Because certain lines of both Arabidopsis and soybean are resistant to bacteria carrying the avirulence genes avrRpt2 and avrB, extremely similar pathogen recognition mechanisms are apparently present in these two plant species. Isogenic bacterial strains that differ by the presence of single avirulence genes are being used to analyze plant resistance. Plant resistance genes have been identified in crosses between resistant and susceptible lines. The extensive map-based cloning tools available in Arabidopsis are being used to isolate these resistance genes. In a related project, ethylene-insensitive Arabidopsis mutants are being used to examine the role of ethylene in disease development. Ethylene apparently mediates symptom formation in susceptible plants and is not required for resistance, suggesting possible strategies for enhancement of disease tolerance in crops.     

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes,in <Emphasis Type="Italic">Arabidopsis</Emphasis> infected with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Selective Detoxification of Phenols by Pichia pastoris and Arabidopsis thaliana Heterologously Expressing the PtUGT72B1 from Populus trichocarpa

Phenols are present in the environment and commonly in contact with humans and animals because of their wide applications in many industries. In a previous study, we reported that uridine diphosphate-glucose-dependent glucosyltransferase PtUGT72B1 from Populus trichocarpa has high activity in detoxifying trichlorophenol by conjugating glucose. In this study, more experiments were performed to determine the substrate specificity of PtUGT72B1 towards phenolic compounds. Among seven phenols tested, three were glucosylated by PtUGT72B1 including phenol, hydroquinone, and catechol. Transgenic Arabidopsis plants expressing the enzyme PtUGT72B1 showed higher resistance to hydroquinone and catechol but more sensitivity to phenol than wild type plants. Transgenic Pichia pastoris expressing PtUGT72B1 showed enhanced resistance to all three phenols. Compared with wild type Arabidopsis plants, transgenic Arabidopsis plants showed higher removal efficiencies and exported more glucosides of phenol, phenyl β-D-glucopyranoside, to the medium after cultured with the three phenols. Protein extracts from transgenic Arabidopsis plants showed enhanced conjugating activity towards phenol, hydroquinone and catechol. PtUGT72B1 showed much higher expression level in Pichia pastoris than in Arabidopsis plants. Kinetic analysis of the PtUGT72B1 was also performed.     

Thaxtomin A-deficient endophytic Streptomyces sp. enhances plant disease resistance to pathogenic Streptomyces scabies

Each plant species in nature harbors endophytes, a community of microbes living within host plants without causing any disease symptom. However, the exploitation of endophyte-based phytoprotectants is hampered by the paucity of mechanistic understandings of endophyte-plant interaction. We here reported two endophytic Streptomyces isolates IFB-A02 and IFB-A03 recovered from a stress-tolerant dicotyledonous plant Artemisia annua L. After the determination of their non-pathogenicity at the genomic level and from the toxin (thaxtomin A, TXT) level, the endophytism of both isolates was supported by their successful colonization in planta. Of the two endophytes, IFB-A03 was further studied for the mechanism of endophyte-conferred phytoprotection owing to its plant growth promotion in model eudicot Arabidopsis thaliana. Using the endophyte-Arabidopsis co-cultivation system into which pathogenic Streptomyces scabies was introduced, we demonstrated that IFB-A03 pre-inoculation could activate the salicylic acid (SA)-mediated plant defense responses upon pathogen challenge. Moreover, IFB-A03 was shown to partially rescue the defense deficiency in eds5 (enhanced disease susceptibility 5) Arabidopsis mutants, putatively acting at the upstream of SA accumulation in the defense signaling pathway associated with the systemic acquired resistance (SAR). These data suggest that endophytic Streptomyces sp. IFB-A03 could be a promising candidate for biocontrol agents against S. scabies—a causative pathogen of common scab diseases prevailing in agronomic systems.     

Stress resistance and C1 metabolism involved in plant colonization of a methanotroph Methylosinus sp. B4S

Methanotrophs are widespread and have been isolated from various environments including the phyllosphere. In this study, we characterized the plant colonization by Methylosinus sp. B4S, an α-proteobacterial methanotroph isolated from plant leaf. The gfp-tagged Methylosinus sp. B4S cells were observed to colonize Arabidopsis leaf surfaces by forming aggregates. We cloned and sequenced the general stress response genes, phyR, nepR and ecfG, from Methylosinus sp. B4S. In vitro analysis showed that the phyR expression level was increased after heat shock challenge, and phyR was shown to be involved in resistance to heat shock and UV light. In the phyllospheric condition, the gene expression level of phyR as well as mmoX and mxaF was found to be relatively high, compared with methane-grown liquid cultures. The phyR-deletion strain as well as the wild-type strain inoculated on Arabidopsis leaves proliferated at the initial phase and then gradually decreased during plant colonization. These results have shed light firstly on the importance of general stress resistance and C1 metabolism in methanotroph living in the phyllosphere.     

Integration of plant growth-promoting rhizobacteria and chemical elicitors for induction of systemic resistance in mulberry against multiple diseases

Abstract

In mulberry (Morus alba L.), various individual strains of plant growth-promoting rhizobacteria (PGPR) and synthetic analogs of naturally occurring plant activators have demonstrated their potential to elicit induced systemic resistance (ISR) against either brown leaf spot (Cercospora moricola) or leaf rust (Cerotelium fici) diseases. However, these biological and chemical elicitors have not been evaluated so far against multiple infections of both these diseases which commonly occur during the post-rainy season. The present study was therefore aimed to assess the capability of PGPR strains and chemical plant activators, as individual and in integration, in elicitation of ISR against multiple infections. Three PGPR strains, Azotobacter chroococcum strain Azc-3, Bacillus megaterium strain Bm-1 and Pseudomonas fluorescens strain Psf-4, and plant activators, acetyl-salicylic acid (ASA), sodium salicylate (NaS) and 4-amino-n-butyric acid (ABA) were selected for the study. Under in vitro tests, all the plant activators up to 2000 ppm concentration exhibited their compatibility with the PGPR strains tested. Upon assaying of elicitors with plant-pathosystem, disease suppression was significantly (p = 0.05) high with integrated application of PGPR strains and plant activators when compared to their individual applications. All the elicitors at individual application varied in their response to multiple infections with the plant age. However, integration of Azc-3 + ASA provided greater suppression to multiple infections of brown leaf spot and leaf rust diseases during the entire growth period of mulberry plants. Thus, this combination of biological and chemical elicitors holds great promise to provide an effective ecofriendly alternative to the toxic chemical fungicides presently recommended for the control of brown leaf spot and leaf rust diseases in mulberry.     

Fungal elicitor protein PebC1 from Botrytis cinerea improves disease resistance in Arabidopsis thaliana

We previously identified a novel protein elicitor, PebC1, from Botrytis cinerea and described its enhancement of plant growth, drought tolerance and disease resistance in tomato. Here, we have investigated the defense-associated molecular responses in Arabidopsis thaliana after treatment with recombinant PebC1. PebC1 was expressed in Escherichia coli. Recombinant protein treatments improved plant resistance to Botrytis infection and maintained plant defenses for more than 21 days. The purified protein at 10 μg ml^?1 activated extracellular medium alkalization (pH) and reactive oxygen species and nitric oxide generation and also induced defense gene expression. Arabidopsis mutants that are insensitive to salicylic acid had increased resistance to Botrytis infection after PebC1 treatment but PebC1 did not affect the resistance of mutants with jasmonic acid and ethylene transduction pathways. The results suggest that PebC1 can function as an activator of plant disease resistance and can promote disease resistance to Botrytis in A. thaliana through the ethylene signal transduction pathway.     

Quercetin-induced H2O2 mediates the pathogen resistance against Pseudomonas syringae pv. Tomato DC3000 in Arabidopsis thaliana

Quercetin is a potent antioxidant and has been extensively used as a therapy intervention to prevent age-associated diseases. However, emerging studies showed it can also act as a prooxidant and induce H₂O₂ under certain conditions. In the current study, our results showed that quercetin contributed to the pathogen resistance in Arabidopsis thaliana (Arabidopsis) in response to the infection of virulent strain Pseudomonas syringae pv. Tomato DC3000 (Pst). Various defense responses, such as H₂O₂ burst, callose deposition, cell death, PR1 (pathogenesis-related 1) and PAL1 (Phe ammonia-lyase 1) gene expression, have been investigated in quercetin-pretreated Pst-inoculated Arabidopsis Col-0 and there was a strong defensive response in quercetin-pretreated Arabidopsis against virulent Pst. However, with the presence of catalase, the protective effects of quercetin on pathogen resistance to virulent Pst disappeared in Arabidopsis, suggesting that H₂O₂ may play a key role in plant defense responses. In addition, we confirmed that quercetin did not show any beneficial effect on pathogen-free leaves in Arabidopsis, indicating that pathogen challenge is also required to induce the defense responses in quercetin-pretreated Arabidopsis. Furthermore, strong defense responses have been observed in quercetin-pretreated Arabidopsis mutant jar1, ein2, and abi1-2 under Pst challenge, whereas no protective effect has been observed in quercetin-pretreated Arabidopsis mutant NahG and npr1. These findings indicate that quercetin induces the resistance to Pst in Arabidopsis via H₂O₂ burst and involvement of SA and NPR1.     

Promotion of plant growth, biological control and induced systemic resistance in maize by Pseudomonas aurantiaca JD37

Some Pseudomonas aurantiaca strains have been found to facilitate plant growth. A P. aurantiaca JD37 strain isolated from a suburb of Shanghai, China, was found to effectively colonize the rhizosphere soil and internal roots of maize (Zea mays L.) and promote maize growth. Agar diffusion assays and biocontrol effect experiments showed that strain JD37 had significant antagonistic activity against Bipolaris maydis, as well as a high biocontrol effect on southern maize leaf blight caused by B. maydis. PCR detection, associated with reverse-phase high-performance liquid chromatography assays, showed that strain JD37 might produce a number of important antibacterial substances, such as phenazine-1-carboxylic acid, pyrrolnitrin and 2,4-diacetylphloroglucinol. The crude bacterial extracts and the cell-free supernatant of strain JD37 were found to induce resistance in maize against B. maydis and reduce plant disease. Our results indicate the potential of some bacteria for producing bacterial compounds that serve as inducers of disease resistance, which is an attractive alternative to the application of chemical fertilizers, pesticides and supplement in agricultural practices.     

Endophytic blastospores of Beauveria bassiana provide high resistance against plant disease caused by Botrytis cinerea

The entomopathogenic fungus Beauveria bassiana is widely used for insect pest control and can produce three distinct infective unit types under different nutritional and environmental conditions: aerial conidia, blastospores, and submerged conidia. Here, we identified endophytic colonization ability and existing forms of the three types of B. bassiana infective units after inoculating Arabidopsis plants via soil drenching, and tested their effects on their presence mold disease caused by Botrytis cinerea. We found that all B. bassiana infective unit types colonized Arabidopsis leaves, with germinating and producing hyphae by hydrophilic blastopores and submerged conidia; further, we showed that blastospores were more effective in defending against B. cinerea, compared with aerial conidia. These findings suggest that in addition to aerial conidia, the colonization of other two types of entomopathogenic fungal infective units could also have important impacts on plant resistance. This study contributes to better understanding on the function of B. bassiana as fungal endophytes, which could lead to a new paradigm for how to successfully use these organisms in biological control against plant diseases.     

OsCPK20 positively regulates Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against Magnaporthe grisea

Calcium-dependent protein kinases are important decoders of calcium signals in plants, which are involved in plant immunity. We report isolation and functional characterization of a pathogen-responsive OsCPK20 gene in rice. The expression of OsCPK20 in rice was significantly induced following treatment with a Magnaporthe grisea elicitor. Overexpression of constitutively active OsCPK20 in Arabidopsis enhanced the resistance to infection with Pseudomonas syringae pv. tomato, associated with elevated expression of both SA- and JA-related defense genes. Similarly, transgenic rice plants containing constitutively active OsCPK20 exhibited enhanced resistance to blast fungus M. grisea. The enhanced resistance in the transgenic Arabidopsis and rice was associated with activated expression of both SA- and JA-related defense genes. We also found that OsCPK20 was significantly induced by drought stress, indicating that OsCPK20 might be involved in plant response to drought stress. Taken together, our results indicate that rice OsCPK20 positively regulates Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against M. grisea, and that it may enhance disease resistance by activating both SA- and JA-dependent defense responses.     

Expression of a crown gall biological control phenotype in an avirulent strain of Agrobacterium vitis by addition of the trifolitoxin production and resistance genes

Background

Agrobacterium vitis is a causal agent of crown-gall disease. Trifolitoxin (TFX) is a peptide antibiotic active only against members of a specific group of α-proteobacteria that includes Agrobacterium and its close relatives. The ability of TFX production by an avirulent strain of Agrobacterium to reduce crown gall disease is examined here.

Results

TFX was shown to be inhibitory in vitro against several A. vitis strains. TFX production, expressed from the stable plasmid pT2TFXK, conferred biological control activity to an avirulent strain of A. vitis. F2/5, against three virulent, TFX-sensitive strains of A. vitis tested on Nicotiana glauca. F2/5(pT2TFXK) is significantly reduces number and size of galls when co-inoculated with tumorigenic strain CG78 at a 10:1 ratio, but is ineffective at 1:1 or 1:10 ratios. F2/5(pT2TFXK) is effective when co-inoculated with tumorigenic strain CG435 at 10:1 and 1:1 ratios, but not at a 1:10 ratio. When F2/5(pT2TFXK) is co-inoculated with CG49 at a 10:1 ratio, the incidence of gall formation does not decline but gall size decreases by more than 70%. A 24 h pre-inoculation with F2/5(pT2TFXK) does not improve biological control at the 1:10 ratio.

Conclusions

TFX production by an avirulent strain of Agrobacterium does confer in that strain the ability to control crown gall disease on Nicotiana glauca. This is the first demonstration that the production of a ribosomally synthesized, post-translationally modified peptide antibiotic can confer reduction in plant disease incidence from a bacterial pathogen.     

Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway

Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1.