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Concept: Pseudomonas syringae


BACKGROUND: The gene encoding PAD4 (PHYTOALEXIN-DEFICIENT4) is required in Arabidopsis for expression of several genes involved in the defense response to Pseudomonas syringae pv. maculicola. AtPAD4 (Arabidopsis thaliana PAD4) encodes a lipase-like protein that plays a regulatory role mediating salicylic acid signaling. RESULTS: We expressed the gene encoding AtPAD4 in soybean roots of composite plants to test the ability of AtPAD4 to deter plant parasitic nematode development. The transformed roots were challenged with two different plant parasitic nematode genera represented by soybean cyst nematode (SCN; Heterodera glycines) and root-knot nematode (RKN; Meloidogyne incognita). Expression of AtPAD4 in soybean roots decreased the number of mature SCN females 35 days after inoculation by 68 percent. Similarly, soybean roots expressing AtPAD4 exhibited 77 percent fewer galls when challenged with RKN. CONCLUSIONS: Our experiments show that AtPAD4 can be used in an economically important crop, soybean, to provide a measure of resistance to two different genera of nematodes.

Concepts: Heterodera, Pseudomonas syringae, Model organism, Plant pathogens and diseases, Parasites, Root-knot nematode, Nematodes, Nematode


Environmental conditions profoundly affect plant disease development; however, the underlying molecular bases are not well understood. Here we show that elevated temperature significantly increases the susceptibility of Arabidopsis to Pseudomonas syringae pv. tomato (Pst) DC3000 independently of the phyB/PIF thermosensing pathway. Instead, elevated temperature promotes translocation of bacterial effector proteins into plant cells and causes a loss of ICS1-mediated salicylic acid (SA) biosynthesis. Global transcriptome analysis reveals a major temperature-sensitive node of SA signalling, impacting ~60% of benzothiadiazole (BTH)-regulated genes, including ICS1 and the canonical SA marker gene, PR1. Remarkably, BTH can effectively protect Arabidopsis against Pst DC3000 infection at elevated temperature despite the lack of ICS1 and PR1 expression. Our results highlight the broad impact of a major climate condition on the enigmatic molecular interplay between temperature, SA defence and function of a central bacterial virulence system in the context of a widely studied susceptible plant-pathogen interaction.

Concepts: Molecular biology, Bacteria, Genetics, Gene expression, Gene, Cell wall, DNA, Pseudomonas syringae


Autophagy and the ubiquitin-proteasome system (UPS) are two major protein degradation pathways implicated in the response to microbial infections in eukaryotes. In animals, the contribution of autophagy and the UPS to anti-bacterial immunity is well documented and several bacteria have evolved measures to target and exploit these systems to the benefit of infection. In plants, the UPS has been established as a hub for immune responses and is targeted by bacteria to enhance virulence. However, the role of autophagy during plant-bacterial interactions is less understood. Here, we have identified both pro- and anti-bacterial functions of autophagy mechanisms upon infection of Arabidopsis with virulent Pseudomonas syringae pv. tomato DC3000 (Pst). We show that Pst activates autophagy in a type-III effector (T3E)-dependent manner, and stimulates the autophagic removal of proteasomes (proteaphagy) to support bacterial proliferation. We further identify the T3E Hrp outer protein M1 (HopM1) as a principle mediator of autophagy-inducing activities during infection. In contrast to the pro-bacterial effects of Pst-induced proteaphagy, NEIGHBOR OF BRCA1 (NBR1)-dependent selective autophagy counteracts disease progression and limits the formation of HopM1-mediated water-soaked lesions. Together, we demonstrate that distinct autophagy pathways contribute to host immunity and bacterial pathogenesis during Pst infection, and provide evidence for an intimate crosstalk between proteasome and autophagy in plant-bacterial interactions.

Concepts: Antibody, Proteasome, Antiseptic, Pseudomonas syringae, Infectious disease, Infection, Bacteria, Immune system


The pathogen Pseudomonas syringae requires a type III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector-triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP-triggered immune responses compared to wild type plants. An N-terminal region of HopK1 shares similarity with the corresponding region in the well-studied type III effector AvrRps4, however, their C-terminal regions are dissimilar indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The processed forms of HopK1 and AvrRps4 are chloroplast-localized indicating that the shared N-terminal regions of these type III effectors represent a chloroplast transit peptide. HopK1’s virulence contribution and the ability of HopK1 and AvrRps4 to suppress immunity required their respective transit peptides, but the AvrRps4-induced HR did not. Our results suggest that a primary virulence target of these type III effectors resides in chloroplasts and that the recognition of AvrRps4 by the plant immune system occurs elsewhere. Moreover, our results reveal that distinct type III effectors utilize a cleavable transit peptide to localize to chloroplasts and that targets within this organelle are important for immunity. This article is protected by copyright. All rights reserved.

Concepts: Cell, Pseudomonas syringae, Innate immune system, Chloroplast, Eukaryote, Protein, Photosynthesis, Immune system


The Pseudomonas syringae effector AvrB interacts with four related soybean (Glycine max) proteins (GmRIN4a-d), three (GmRIN4b, c, d) of which also interact with the cognate resistance ® protein, Rpg1-b. Here, we investigated the specific requirements for the GmRIN4 proteins in R-mediated resistance and examined the mechanism of Rpg1-b activation. Using virus-induced gene silencing, we show that only GmRIN4a and b are required for Rpg1-b-mediated resistance. In planta binding assays show that GmRIN4a can associate with Rpg1-b indirectly via GmRIN4b. Pathogen-delivered AvrB induces the phosphorylation of GmRIN4b alone, and prevents interactions between GmRIN4b and Rpg1-b or GmRIN4a. Consistent with this result, a phosphomimic derivative of GmRIN4b (pm4b) fails to bind Rpg1-b and GmRIN4a. Conversely, a phosphodeficient derivative of GmRIN4b (pd4b) continues to bind the R protein and GmRIN4a, in the presence of AvrB. Coexpression of Rpg1-b with pm4b, but not GmRIN4b or pd4b, induces cell death and ion leakage in the heterologous Nicotiana benthamiana. Our data suggest that the AvrB-induced phosphorylation of GmRIN4b, and the subsequent inhibition of interaction among GmRIN4b, GmRIN4a and Rpg1-b, might activate the R protein. Furthermore, even though GmRIN4c and d are not required for Rpg1-b-derived resistance, they do function in resistance derived from other R loci.

Concepts: Pseudomonas syringae, Protein, Amino acid, Soybean, DNA, Gene, RNA, Cell


Several plant lipid transfer proteins (LTP) act positively in plant disease resistance. Here, we show that LTP3 (At5g59320), a pathogen and ABA-induced gene, negatively regulates plant immunity in Arabidopsis. Overexpression of LTP3 (LTP3-OX) led indeed to an enhanced susceptibility to virulent bacteria and compromised resistance to avirulent bacteria. Upon infection of LTP3-OX plants with Pseudomonas syringae pv. tomato, genes involved in abscisic acid (ABA) biosynthesis, NCED3 and AAO3 were highly induced whereas salicylic acid (SA) related genes, ICS1 and PR1 were down-regulated. Accordingly, in LTP3-OX plants we observed an increased ABA levels and lower SA levels compared to the wild type. We also showed that the LTP3-overexpression-mediated enhanced susceptibility was partially dependent of AAO3. Interestingly, loss of function of LTP3 (ltp3-1) did not affect ABA pathways, but resulted in PR1 gene induction and elevated SA levels, suggesting that LTP3 can negatively regulate SA in an ABA-independent manner. However, double mutant consisting of ltp3-1 and silent LTP4 (ltp3/ltp4) showed reduced susceptibility to Pseudomonas and down-regulation of ABA biosynthesis genes, suggesting that LTP3 acts in a redundant way with its closest homologue LTP4 by modulating ABA pathway. Taken together, our data show that LTP3 is a novel negative regulator of plant immunity that is acting through the manipulation of the ABA-SA balance.

Concepts: Cell wall, DNA, Amino acid, Protein, Bacteria, Plant pathogens and diseases, Gene, Pseudomonas syringae


Plants have evolved elaborate mechanisms to regulate pathogen defense. Imbalances in this regulation may result in autoimmune responses that are affecting plant growth and development. In Arabidopsis, SAUL1 encodes a plant U-box ubiquitin ligase and regulates senescence and cell death. Here, we show that saul1-1 plants exhibit characteristics of an autoimmune mutant. A decrease in relative humidity or temperature resulted in reduced growth and systemic lesioning of saul1-1 rosettes. These physiological changes are associated with increased expression of salicylic acid-dependent and Pathogenesis-Related (PR) genes. Consistently, resistance of saul1-1 plants against Pseudomonas syringae pv maculicola (P.s.m.) ES4326, Pseudomonas syringae pv tomato (P.s.t.) DC3000, or Hyaloperonospora arabidopsidis (H.a.) Noco2 was enhanced. Transmission electron microscopy revealed alterations in saul1-1 chloroplast ultrastructure and cell wall depositions. Confocal analysis on aniline blue-stained leaf sections and cellular universal micro spectrophotometry further showed that these cell wall depositions contain callose and lignin. To analyze signaling downstream of SAUL1, we performed epistasis analyses between saul1-1 and mutants in the EDS1/PAD4/SAG101 hub. All phenotypes observed in saul1-1 plants at low temperature were dependent on EDS1 and PAD4, but not SAG101.Taken together, SAUL1 negatively regulates immunity upstream of EDS1/PAD4, likely through the degradation of an unknown activator of the pathway.

Concepts: Gene expression, Photosynthesis, Gene, Bacteria, Plant, Cell wall, Immune system, Pseudomonas syringae


The circadian clock integrates temporal information with environmental cues in regulating plant development and physiology. Recently, the circadian clock has been shown to affect plant responses to biotic cues. To further examine this role of the circadian clock, we tested disease resistance in mutants disrupted in CCA1 and LHY, which act synergistically to regulate clock activity. We found that cca1 and lhy mutants also synergistically affect basal and resistance gene-mediated defense against Pseudomonas syringae and Hyaloperonospora arabidopsidis. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also resulted in severe susceptibility to P. syringae. We identified a downstream target of CCA1 and LHY, GRP7, a key constituent of a slave oscillator regulated by the circadian clock and previously shown to influence plant defense and stomatal activity. We show that the defense role of CCA1 and LHY against P. syringae is at least partially through circadian control of stomatal aperture but is independent of defense mediated by salicylic acid. Furthermore, we found defense activation by P. syringae infection and treatment with the elicitor flg22 can feedback-regulate clock activity. Together this data strongly supports a direct role of the circadian clock in defense control and reveal for the first time crosstalk between the circadian clock and plant innate immunity.

Concepts: Immune system, Plant pathogens and diseases, Pseudomonas syringae, Plant physiology, Innate immune system, Regulation


Exosomes are extracellular vesicles (EVs) that play a central role in intercellular signaling in mammals by transporting proteins and small RNAs. Plants are also known to produce EVs, particularly in response to pathogen infection. The contents of plant EVs have not been analyzed, however, and their function is unknown. Here we describe a method for purifying EVs from the apoplastic fluids of Arabidopsis leaves. Proteomic analyses of these EVs revealed that they are highly enriched in proteins involved in biotic and abiotic stress responses. Consistent with this finding, EV secretion was enhanced in plants infected with Pseudomonas syringae and in response to treatment with salicylic acid. These findings suggest that EVs may represent an important component of plant immune responses.

Concepts: Pseudomonas syringae, Chlorophyll, Plant morphology, Protein, Fern, Photosynthesis, Bacteria, Immune system


Vesicular trafficking has emerged as an important means by which eukaryotes modulate responses to microbial pathogens, likely by contributing to the correct localization and levels of host components necessary for effective immunity. However, considering the complexity of membrane trafficking in plants, relatively few vesicular trafficking components with functions in plant immunity are known. Here we demonstrate that Arabidopsis thaliana Dynamin-Related Protein 2B (DRP2B), which has been previously implicated in constitutive clathrin-mediated endocytosis (CME), functions in responses to flg22 (the active peptide derivative of bacterial flagellin) and immunity against flagellated bacteria Pseudomonas syringae pv. tomato (Pto) DC3000. Consistent with a role of DRP2B in Pattern-Triggered Immunity (PTI), drp2b null mutant plants also showed increased susceptibility to Pto DC3000 hrcC-, which lacks a functional Type 3 Secretion System, thus is unable to deliver effectors into host cells to suppress PTI. Importantly, analysis of drp2b mutant plants revealed three distinct branches of the flg22-signaling network that differed in their requirement for RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD), the NADPH oxidase responsible for flg22-induced apoplastic reactive oxygen species production. Furthermore, in drp2b, normal MAPK signaling and increased immune responses via the RbohD/Ca2+-branch were not sufficient for promoting robust PR1 mRNA expression nor immunity against Pto DC3000 and Pto DC3000 hrcC-. Based on live-cell imaging studies, flg22-elicited internalization of the plant flagellin-receptor, FLAGELLIN SENSING 2 (FLS2), was found to be partially dependent on DRP2B, but not the closely related protein DRP2A, thus providing genetic evidence for a component, implicated in CME, in ligand-induced endocytosis of FLS2. Reduced trafficking of FLS2 in response to flg22 may contribute in part to the non-canonical combination of immune signaling defects observed in drp2b. In conclusion, this study adds DRP2B to the relatively short list of known vesicular trafficking proteins with roles in flg22-signaling and PTI in plants.

Concepts: NADPH oxidase, Cell, Antibody, Oxygen, Innate immune system, Pseudomonas syringae, Bacteria, Immune system