Polyamine uptake transporter 2 is essential for systemic acquired resistance establishment in Arabidopsis.
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ABSTRACT: Plants have evolved a tightly regulated and inducible immune system to resist pathogen attack. After the initial contact with the pathogen, plants are significantly more resistant to future challenges, even from unrelated pathogens, an event known as systemic acquired resistance (SAR). In plants polyamines are involved in the regulation and establishment of the defense response by modulating their metabolism, specifically their biosynthesis, conjugation and catabolism. However, it is not known whether polyamine transport is involved in the plant defense response. Herein, we demonstrate that the PUT2/LAT4 transporter is required to establish local immunity and systemic defense responses in the A. thaliana-Pseudomonas syringae pv. tomato DC3000 (Pst) pathosystem. Our data revealed a dual phenotype in the put2-1 mutant after Pst inoculation, on one side, SAR was abolished in the systemic leaves while on the other, basal resistance to Pst was enhanced. Remarkably, this phenotype persisted even upon inoculation with unrelated pathogens such as Botrytis cinerea, highlighting a broad-spectrum SAR deficiency. To gain deeper insights into the involvement of PA transport in SAR, transcriptomic and metabolomic data of the put2-1 mutant were obtained.
Project description:The goal of the microarray experiment was to identify defense genes that were differentially expressed in the Arabidopsis mutant At-med16-1 and wild type in response to infection of the avirulent bacterial pathogen Pst DC3000/avrRpt2. Results indicated that, compared with the wild type, several positive regulators of SAR (systemic acquired resistance) were downregulated and a group of SAR negative regulators were upregulated in At-med16-1. Three biological replicates with leaves from 8 plants per sample were collected at 0, 4, 8, and 24 hours after inoculation with the avirulent bacterial pathogen Pst DC3000/avrRpt2. After extraction, RNA concentration was determined on a NanoDrop Spectrophotometer (Thermofisher Scientific, Waltham, MA) and sample quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Equal amount of RNA from the 3 biological replicates were pooled as one sample for microarray analysis.
Project description:The goal of the microarray experiment was to identify defense genes that were differentially expressed in the Arabidopsis mutant At-med16-1 and wild type in response to infection of the avirulent bacterial pathogen Pst DC3000/avrRpt2. Results indicated that, compared with the wild type, several positive regulators of SAR (systemic acquired resistance) were downregulated and a group of SAR negative regulators were upregulated in At-med16-1.
Project description:Recent evidence suggests that the ubiquitin-proteasome system (UPS) is involved in several aspects of plant immunity and a range of plant pathogens subvert the UPS to enhance their virulence. Here we show that proteasome activity is strongly induced during basal defense in Arabidopsis. Mutant lines of the proteasome subunits RPT2a and RPN12a support increased bacterial growth of virulent Pseudomonas syringae pv. tomato DC3000 (Pst) and Pseudomonas syringae pv. maculicola ES4326. Both proteasome subunits are required for Pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) responses. Analysis of bacterial growth after a secondary infection of systemic leaves revealed that the establishment of systemic-acquired resistance (SAR) is impaired in proteasome mutants, suggesting that the proteasome also plays an important role in defense priming and SAR. In addition, we show that Pst inhibits proteasome activity in a type-III secretion dependent manner. A screen for type-III effector proteins from Pst for their ability to interfere with proteasome activity revealed HopM1, HopAO1, HopA1 and HopG1 as putative proteasome inhibitors. Biochemical characterization of HopM1 by mass-spectrometry indicates that HopM1 interacts with several E3 ubiquitin ligases and proteasome subunits. This supports the hypothesis that HopM1 associates with the proteasome leading to its inhibition. Thus, the proteasome is an essential component of PTI and SAR, which is targeted by multiple bacterial effectors.
Project description:Rice Xa21 resistance gene, which encodes a protein with predicted leucine-rich repeat (LRR), transmembrane, juxtamembrane, and intracellular kinase domains, conferred immunity to diverse strains of Xanthomonas oryzae pv. oryzae (Xoo). We generated Xa21 plant on TP309 background (Oryza Sativa Japonica). Systemic Acquired Resistance (SAR) in plants confers durable broad-spectrum resistance to pathogens and requires a phytohormone, salicylic acid (SA). Arabidopsis NPR1/NIM1 is a key regulator of the SAR response. Recently, we found that rice NPR1 homolog 1 (NH1) mediated enhanced resistance responses for Xoo (Chern et al., 2005b). We further investigated relating pathways in rice by identifying proteins that interact with NH1. One of them, constitutive over-expression of NH1 mediated negative regulator of resistance (NRR) gene caused enhanced susceptibility to Xoo , indicating that this gene product negatively affects to basal resistance response (Chern et al., 2005a). To dissect defense responses for rice bacterial blight pathogen, we planed microarray using two resistant mutant named with Xa21-TP309, NH1ox and one super-susceptible mutant (NRRox) before pathogen inoculation and one day post pathogen inoculation. Keywords: Biotic stress response
Project description:Rice Xa21 resistance gene, which encodes a protein with predicted leucine-rich repeat (LRR), transmembrane, juxtamembrane, and intracellular kinase domains, conferred immunity to diverse strains of Xanthomonas oryzae pv. oryzae (Xoo). We generated Xa21 plant on TP309 background (Oryza Sativa Japonica). Systemic Acquired Resistance (SAR) in plants confers durable broad-spectrum resistance to pathogens and requires a phytohormone, salicylic acid (SA). Arabidopsis NPR1/NIM1 is a key regulator of the SAR response. Recently, we found that rice NPR1 homolog 1 (NH1) mediated enhanced resistance responses for Xoo (Chern et al., 2005b). We further investigated relating pathways in rice by identifying proteins that interact with NH1. One of them, constitutive over-expression of NH1 mediated negative regulator of resistance (NRR) gene caused enhanced susceptibility to Xoo , indicating that this gene product negatively affects to basal resistance response (Chern et al., 2005a). To dissect defense responses for rice bacterial blight pathogen, we planed microarray using two resistant mutant named with Xa21-TP309, NH1ox and one super-susceptible mutant (NRRox) before pathogen inoculation and one day post pathogen inoculation. Keywords: Biotic stress response Two or Three-condition experiment, NH1ox vs wild type control (LG) at two durations of Xoo inoculation (0d and 1d); NRRox vs wild type control (LG) at two durations of Xoo inoculation (0d and 1d); and Xa21vs wild type control (TP309) at three durations of Xoo inoculation (0d,1d and 2d);. Biological replicates: 2 or 4, independently grown and harvested.
Project description:After localized invasion by bacterial pathogens, systemic acquired resistance (SAR) is induced in uninfected plant tissues, resulting in enhanced defense against a broad range of pathogens. Although SAR requires mobilization of signaling molecules via the plant vasculature, the specific mechanisms remain elusive. The lipid transfer protein-defective in induced resistance 1-1 (DIR1-1) was identified in Arabidopsis thaliana by screening for mutants that were defective in SAR. Since then, the structure and lipid-binding properties of DIR1 have been determined, showing that its barrel structure can bind two, long-chain fatty-acid molecules. Several SAR mobile signals, including dehydroabietinal (DA), azelaic acid (AzA), and glycerol-3-phosphate (G3P) are dependent on DIR1 for transport to systemic leaves. Closure of stomata, controlled by guard cells, is a local response to bacteria. Here we demonstrate that stomatal response to pathogens is altered in systemic leaves by SAR, and this guard cell SAR defense requires DIR1. Using a multi-omics approach, we have determined potential SAR signaling mechanisms specific for guard cells in systemic leaves by profiling metabolite, lipid, and protein differences between guard cells in wild type and dir1-1 mutant during SAR. We identified two 18C fatty actyls and two 16C wax esters as putative SAR-related molecules dependent on DIR1. Proteins and metabolites related to amino acid biosynthesis and response to stimulus were altered in guard cells of dir1-1 compared to wild type. Identification of guard cell-specific SAR-related molecules will lead to new avenues of genetic modifications/molecular breeding for disease resistant plants.
Project description:Systemic Acquired Resistance (SAR) as a plant immune response improves immunity of distal tissue after local exposure to a pathogen. Stomata pores on leaf surfaces are formed by guard cells that recognize bacterial pathogens via pattern recognition receptors. We found that Arabidopsis thaliana plants that have been previously exposed to the pathogenic bacteria Pseudomonas syringae pv. tomato DC3000 (Pst) exhibit an altered stomatal response compared to control plants when systemic leaves are later exposed to the bacteria. Reduced stomatal apertures of SAR primed plants lead to decreases in the number of bacteria that enter the apoplastic space of the leaves. To examine how SAR affects molecular processes in guard cells of distal leaves, we collected Arabidopsis guard cell samples and extracted lipids, metabolites and proteins using a 3-in-1 method. Multi-omic results have revealed molecular components of SAR response specific to the functions of guard cells and roles of ROS and fatty acid signaling in guard cell SAR response. Additionally, our results show an increase in palmitic acid and its derivative 9-PAHSA in primed guard cells. Palmitic acid may play a role as an agonist to Flagellin Sensitive 2 (FLS2), which initiates stomatal closure upon perception of bacterial flagella. The improved understanding of how SAR immune signals affect stomatal movement and immunity can aid biotechnology and marker-based breeding of crops for enhanced disease resistance.
Project description:We investigated the relationships of the two immune-regulatory plant metabolites salicylic acid (SA) and pipecolic acid (Pip) in the establishment of plant systemic acquired resistance (SAR) in Arabidopsis thaliana induced by the bacterial pathogen Pseudomonas syringae. To characterize the transcriptional SAR response, we used wild-type Col-0 plants, SA-deficient sid2 plants and Pip-deficient ald1 plants and performed RNA-sequencing analyses (Bernsdorff et al., Plant Cell, 2016). SAR establishment in the wild-type is characterized by a strong transcriptional response systemically induced in the foliage that prepares plants for future pathogen attack by pre-activating multiple stages of defense signaling. Whereas systemic Pip elevations are indispensable for SAR and necessary for virtually the whole transcriptional SAR response, a moderate but significant SA-independent component of SAR activation and SAR gene expression is revealed. Arabidopsis thaliana plants were grown individually in pots containing a mixture of soil, vermiculite and sand (8:1:1) in a controlled cultivation chamber with a 10-h day (9 AM to 7 PM; photon flux density 100 mol m-2 s-1) / 14-h night cycle and a relative humidity of 70 %. Day and night temperatures were set to 21C and 18C, respectively. Experiments were performed with 5- to 6-week-old, naive plants exhibiting a uniform appearance. To activate SAR, plants were infiltrated between 10 AM and 12 AM into three lower (1) leaves with suspensions of the bacterial pathogen Pseudomonas syringae pv. maculicola (OD600 = 0.005). Infiltration with 10 mM MgCl2 served as the mock-control treatment. Upper (2) leaves were harvested 48 h after the primary treatment for the determination of systemic gene expression by RNA-seq analyses. Three biologically independent, replicate SAR induction experiments were performed with Col-0 and sid2 plants (experimental set 1), and three other biologically independent experiments with Col-0 and ald1 plants (experimental set 2). In each SAR experiment, at least 6 upper (2) leaves from 6 different plants pre-treated in 1 leaves with Psm (MgCl2) were pooled for one biological Psm- (mock-control) replicate. In this way, 3 biologically independent, replicate samples per treatment and plant genotype were obtained within each SAR set.
Project description:The goal of the microarray experiment was to determine the induction kinetics of transcriptome changes and to identify differentially expressed genes in the Arabidopsis mutant med14 and wild type in response to infection of the avirulent bacterial pathogen Pst DC3000/avrRpt2. Results indicated that med14 exhibited slower kinetcis of transcriptional changes than the wild type after Pst DC3000/avrRpt2 infection, and the induction of a large group of defense genes was suppressed in the med14 mutant. Three biological replicates with leaves from 8 plants per sample were collected at 0, 4, 8, and 12 hours after inoculation with the avirulent bacterial pathogen Pst DC3000/avrRpt2. After extraction, RNA concentration was determined on a NanoDrop Spectrophotometer (Thermofisher Scientific, Waltham, MA) and sample quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Each replicate was used as one sample for microarray analysis.
Project description:The goal of the microarray experiment was to identify genes that are differentially expressed among the Arabidopsis mutant med14, med16, npr1, and wild type in response to infection of the avirulent bacterial pathogen Pst DC3000/avrRpt2. Results indicated that the induction of a large group of defense genes was suppressed in the med14 mutant compared with both med16 and the wild type. Three biological replicates with leaves from 8 plants per sample were collected at 0 and 4 hours after inoculation with the avirulent bacterial pathogen Pst DC3000/avrRpt2. After extraction, RNA concentration was determined on a NanoDrop Spectrophotometer (Thermofisher Scientific, Waltham, MA) and sample quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Each replicate was used as one sample for microarray analysis.