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.     

Role of the penetration‐resistance genes PEN1, PEN2 and PEN3 in the hypersensitive response and race‐specific resistance in Arabidopsis thaliana

Plants are highly capable of recognizing and defending themselves against invading microbes. Adapted plant pathogens secrete effector molecules to suppress the host's immune system. These molecules may be recognized by host‐encoded resistance proteins, which then trigger defense in the form of the hypersensitive response (HR) leading to programmed cell death of the host tissue at the infection site. The three proteins PEN1, PEN2 and PEN3 have been found to act as central components in cell wall‐based defense against the non‐adapted powdery mildew Blumeria graminis fsp. hordei (Bgh). We found that loss of function mutations in any of the three PEN genes cause decreased hypersensitive cell death triggered by recognition of effectors from oomycete and bacterial pathogens in Arabidopsis. There were considerable additive effects of the mutations. The HR induced by recognition of AvrRpm1 was almost completely abolished in the pen2 pen3 and pen1 pen3 double mutants and the loss of cell death could be linked to indole glucosinolate breakdown products. However, the loss of the HR in pen double mutants did not affect the plants' ability to restrict bacterial growth, whereas resistance to avirulent isolates of the oomycete Hyaloperonospora arabidopsidis was strongly compromised. In contrast, the double and triple mutants demonstrated varying degrees of run‐away cell death in response to Bgh. Taken together, our results indicate that the three genes PEN1, PEN2 and PEN3 extend in functionality beyond their previously recognized functions in cell wall‐based defense against non‐host pathogens.     

Arabidopsis CURLY LEAF functions in leaf immunity against fungal pathogens by concomitantly repressing SEPALLATA3 and activating ORA59

Arabidopsis non-host resistance against non-adapted fungal pathogens including Colletotrichum fungi consists of pre-invasive and post-invasive immune responses. Here we report that non-host resistance against non-adapted Colletotrichum spp. in Arabidopsis leaves requires CURLY LEAF (CLF), which is critical for leaf development, flowering and growth. Microscopic analysis of pathogen behavior revealed a requirement for CLF in both pre- and post-invasive non-host resistance. The loss of a functional SEPALLATA3 (SEP3) gene, ectopically expressed in clf mutant leaves, suppressed not only the defect of the clf plants in growth and leaf development but also a defect in non-host resistance against the non-adapted Colletotrichum tropicale. However, the ectopic overexpression of SEP3 in Arabidopsis wild-type leaves did not disrupt the non-host resistance. The expression of multiple plant defensin (PDF) genes that are involved in non-host resistance against C. tropicale was repressed in clf leaves. Moreover, the Octadecanoid-responsive Arabidopsis 59 (ORA59) gene, which is required for PDF expression, was also repressed in clf leaves. Notably, when SEP3 was overexpressed in the ora59 mutant background, C. tropicale produced clear lesions in the inoculated leaves, indicating an impairment in non-host resistance. Furthermore, ora59 plants overexpressing SEP3 exhibited a defect in leaf immunity to the adapted Colletotrichum higginsianum. Since the ora59 plants overexpressing SEP3 did not display obvious leaf curling or reduced growth, in contrast to the clf mutants, these results strongly suggest that concomitant SEP3 repression and ORA59 induction via CLF are required for Arabidopsis leaf immunity to Colletotrichum fungi, uncoupled from CLF’s function in growth and leaf development.     

The tolerance of the Arabidopsis defense hormone receptor mutant coi1 against the vascular pathogen Verticillium longisporum is not due to increased levels of the active hormone jasmonoyl-isoleucine

Verticillium longisporum is a soil-borne vascular pathogen found primarily on oilseed rape in Northern Europe. Infection of the model plant Arabidopsis thaliana can be achieved under laboratory conditions. In the article related to this addendum, we have shown that Arabidopsis dde2–2 mutants that are compromised in their ability to synthesize the defense hormone jasmonoyl-isoleucine (JA-Ile) are slightly more susceptible than wild-type. Contrary to the expectation that hormone biosynthesis mutants and their respective receptor mutants should have the same phenotype, we found that plants that lack the JA-Ile receptor CORONATINE INSENSITIVE1 (COI1) are more tolerant to the disease. This addendum addressed the question whether the increased JA-Ile levels found in coi1 are responsible for its tolerance phenotype. Based on the evidence that the JA-Ile-deficient dde2–2 coi1-t double mutant is as tolerant as coi1-t, we conclude that increased JA-Ile levels do not protect Arabidopsis against the fungus in the absence of COI1.     

Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species

Programmed cell death (PCD) is of fundamental importance to development and defense in animals and plants. In plants, a well-recognized form of PCD is hypersensitive response (HR) triggered by pathogens, which involves the generation of reactive oxygen species (ROS) and other signaling molecules. While the mitochondrion is a master regulator of PCD in animals, the chloroplast is known to regulate PCD in plants. Arabidopsis Mosaic Death 1 (MOD1), an enoyl-acyl carrier protein (ACP) reductase essential for fatty acid biosynthesis in chloroplasts, negatively regulates PCD in Arabidopsis. Here we report that PCD in mod1 results from accumulated ROS and can be suppressed by mutations in mitochondrial complex I components, and that the suppression is confirmed by pharmaceutical inhibition of the complex I-generated ROS. We further show that intact mitochondria are required for full HR and optimum disease resistance to the Pseudomonas syringae bacteria. These findings strongly indicate that the ROS generated in the electron transport chain in mitochondria plays a key role in triggering plant PCD and highlight an important role of the communication between chloroplast and mitochondrion in the control of PCD in plants.     

Presence of LYM2 dependent but CERK1 independent disease resistance in Arabidopsis

Plants have the ability to detect invading fungi through the perception of chitin fragments released from the fungal cell walls. Plant chitin receptor consists of two types of plasma membrane proteins, CEBiP and CERK1. However, the contribution of these proteins to chitin signaling is different between Arabidopsis and rice. In Arabidopsis, it seems CERK1 receptor kinase is enough for both ligand perception and signaling, whereas both CEBiP and OsCERK1 are required for chitin signaling in rice. Here we report that Arabidopsis CEBiP homolog, LYM2, is not involved in chitin signaling but contributes to resistance against a fungal pathogen, Alternaria brassicicola, indicating the presence of a novel disease resistance mechanism in Arabidopsis.     

Penetration and Post-Penetration Resistance to Magnaporthe oryzae in Arabidopsis

The rate of entry of Magnaporthe oryzae into the Arabidopsis pen2 quintuple (pen2 NahG pmr5 agb1 mlo2) mutant was significantly higher than those into the pen2 quadruple (pen2 NahG pmr5 agb1 and pen2 NahG pmr5 mlo2) mutants. The lengths of the infection hyphae in the pen2 quintuple mutant were intermediate between the pen2 quadruple mutants. These results suggest that different genetic networks, consisting of PEN2, PMR5, AGB1, and MLO2, control penetration and post-penetration resistance to M. oryzae in Arabidopsis.     

ERECTA contributes to non-host resistance to Magnaporthe oryzae in Arabidopsis

ERECTA controls both developmental processes and disease resistance in Arabidopsis. We investigated the function of ERECTA in non-host resistance to Magnaporthe oryzae in Arabidopsis. In the pen2 er mutant, penetration resistance and post-penetration resistance to M. oryzae were compromised. These results suggest that ERECTA is involved in both penetration and post-penetration resistance to M. oryzae in Arabidopsis.     

Arabidopsis non-host resistance PSS30 gene enhances broad-spectrum disease resistance in the soybean cultivar Williams 82

Non-host resistance (NHR), which protects all members of a plant species from non-adapted or non-host plant pathogens, is the most common form of plant immunity. NHR provides the most durable and robust form of broad-spectrum immunity against non-adaptive pathogens pathogenic to other crop species. In a mutant screen for loss of Arabidopsis (Arabidopsis thaliana) NHR against the soybean (Glycine max (L.) Merr.) pathogen Phytophthora sojae, the Phytophthora sojae-susceptible 30 (pss30) mutant was identified. The pss30 mutant is also susceptible to the soybean pathogen Fusarium virguliforme. PSS30 encodes a folate transporter, AtFOLT1, which was previously localized to chloroplasts and implicated in the transport of folate from the cytosol to plastids. We show that two Arabidopsis folate biosynthesis mutants with reduced folate levels exhibit a loss of non-host immunity against P. sojae. As compared to the wild-type Col-0 ecotype, the steady-state folate levels are reduced in the pss1, atfolt1 and two folate biosynthesis mutants, suggesting that folate is required for non-host immunity. Overexpression of AtFOLT1 enhances immunity of transgenic soybean lines against two serious soybean pathogens, the fungal pathogen F. virguliforme and the soybean cyst nematode (SCN) Heterodera glycines. Transgenic lines showing enhanced SCN resistance also showed increased levels of folate accumulation. This study thus suggests that folate contributes to non-host plant immunity and that overexpression of a non-host resistance gene could be a suitable strategy for generating broad-spectrum disease resistance in crop plants.     

Foliar aphid feeding recruits rhizosphere bacteria and primes plant immunity against pathogenic and non-pathogenic bacteria in pepper

Background and Aims

Plants modulate defence signalling networks in response to different biotic stresses. The present study evaluated the effect of a phloem-sucking aphid on plant defence mechanisms in pepper (Capsicum annuum) during subsequent pathogen attacks on leaves and rhizosphere bacteria on roots.

Methods

Plants were pretreated with aphids and/or the chemical trigger benzothiadiazol (BTH) 7 d before being challenged with two pathogenic bacteria, Xanthomonas axonopodis pv. vesicatoria (Xav) as a compatible pathogen and X. axonopodis pv. glycines (Xag) as an incompatible (non-host) pathogen.

Key Results

Disease severity was noticeably lower in aphid- and BTH + aphid-treated plants than in controls. Although treatment with BTH or aphids alone did not affect the hypersensitive response (HR) against Xag strain 8ra, the combination treatment had a synergistic effect on the HR. The aphid population was reduced by BTH pretreatment and by combination treatment with BTH and bacterial pathogens in a synergistic manner. Analysis of the expression of the defence-related genes Capsicum annum pathogenesis-related gene 9 (CaPR9), chitinase 2 (CaCHI2), SAR8·2 and Lipoxygenase1 (CaLOX1) revealed that aphid infestation resulted in the priming of the systemic defence responses against compatible and incompatible pathogens. Conversely, pre-challenge with the compatible pathogen Xav on pepper leaves significantly reduced aphid numbers. Aphid infestation increased the population of the beneficial Bacillus subtilis GB03 but reduced that of the pathogenic Ralstonia solanacearum SL1931. The expression of defence-related genes in the root and leaf after aphid feeding indicated that the above-ground aphid infestation elicited salicylic acid and jasmonic acid signalling throughout the whole plant.

Conclusions

The findings of this study show that aphid feeding elicits plant resistance responses and attracts beneficial bacterial populations to help the plant cope with subsequent pathogen attacks.     

Dysfunction of Arabidopsis MACPF domain protein activates programmed cell death via tryptophan metabolism in MAMP‐triggered immunity

Plant immune responses triggered upon recognition of microbe‐associated molecular patterns (MAMPs) typically restrict pathogen growth without a host cell death response. We isolated two Arabidopsis mutants, derived from accession Col‐0, that activated cell death upon inoculation with nonadapted fungal pathogens. Notably, the mutants triggered cell death also when treated with bacterial MAMPs such as flg22. Positional cloning identified NSL1 (Necrotic Spotted Lesion 1) as a responsible gene for the phenotype of the two mutants, whereas nsl1 mutations of the accession No‐0 resulted in necrotic lesion formation without pathogen inoculation. NSL1 encodes a protein of unknown function containing a putative membrane‐attack complex/perforin (MACPF) domain. The application of flg22 increased salicylic acid (SA) accumulation in the nsl1 plants derived from Col‐0, while depletion of isochorismate synthase 1 repressed flg22‐inducible lesion formation, indicating that elevated SA is needed for the cell death response. nsl1 plants of Col‐0 responded to flg22 treatment with an RBOHD‐dependent oxidative burst, but this response was dispensable for the nsl1‐dependent cell death. Surprisingly, loss‐of‐function mutations in PEN2, involved in the metabolism of tryptophan (Trp)‐derived indole glucosinolates, suppressed the flg22‐induced and nsl1‐dependent cell death. Moreover, the increased accumulation of SA in the nsl1 plants was abrogated by blocking Trp‐derived secondary metabolite biosynthesis, whereas the nsl1‐dependent hyperaccumulation of PEN2‐dependent compounds was unaffected when the SA biosynthesis pathway was blocked. Collectively, these findings suggest that MAMP‐triggered immunity activates a genetically programmed cell death in the absence of the functional MACPF domain protein NSL1 via Trp‐derived secondary metabolite‐mediated activation of the SA pathway.     

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca²⁺-permeable channel currents by ABA or oscillation of the cytosolic free Ca²⁺ concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca²⁺ oscillation in ABA signal pathway of Arabidopsis guard cells.     

ENHANCED DISEASE RESISTANCE4 Associates with CLATHRIN HEAVY CHAIN2 and Modulates Plant Immunity by Regulating Relocation of EDR1 in Arabidopsis

Obligate biotrophs, such as the powdery mildew pathogens, deliver effectors to the host cell and obtain nutrients from the infection site. The interface between the plant host and the biotrophic pathogen thus represents a major battleground for plant-pathogen interactions. Increasing evidence shows that cellular trafficking plays an important role in plant immunity. Here, we report that Arabidopsis thaliana ENHANCED DISEASE RESISTANCE4 (EDR4) plays a negative role in resistance to powdery mildew and that the enhanced disease resistance in edr4 mutants requires salicylic acid signaling. EDR4 mainly localizes to the plasma membrane and endosomal compartments. Genetic analyses show that EDR4 and EDR1 function in the same genetic pathway. EDR1 and EDR4 accumulate at the penetration site of powdery mildew infection, and EDR4 physically interacts with EDR1, recruiting EDR1 to the fungal penetration site. In addition, EDR4 interacts with CLATHRIN HEAVY CHAIN2 (CHC2), and edr4 mutants show reduced endocytosis rates. Taken together, our data indicate that EDR4 associates with CHC2 and modulates plant immunity by regulating the relocation of EDR1 in Arabidopsis.     

Disease resistance to Pectobacterium carotovorum is negatively modulated by the Arabidopsis Lectin Receptor Kinase LecRK-V.5

Plant stomata function in disease resistance by restricting bacteria entry inside leaves. During plant-bacteria interactions, stomatal closure is initiated by the recognition of Microbe-Associated Molecular Patterns (MAMPs). Recently, we have shown that the Lectin Receptor Kinase V.5 (LecRK-V.5) negatively regulates bacterium- and MAMP-induced stomatal closure upstream of Reactive Oxygen Species (ROS) production mediated by abscisic acid signaling. Closed stomata in lecrk-V.5 mutants are correlated with constitutive high level of ROS in guard cells. Consequently, lecrk-V.5 mutants are more resistant to hemi-biotrophic pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000). In this report, we further investigate the role of LecRK-V.5 in resistance against necrotrophic bacteria Pectobacterium carotovorum ssp. carotovorum (Pcc). Upon surface-inoculation lecrk-V.5 mutants exhibited enhanced resistance against Pcc whereas a wild-type level of resistance was observed using infiltration-inoculation, an inoculation method that bypasses the epidermal barrier. Enhanced resistance of dip-inoculated lecrk-V.5 mutants against necrotrophic bacteria, that induce different defense responses than hemi-biotrophic bacteria, further suggests a possible role for LecRK-V.5 in stomatal immunity.     

Warriors at the gate that never sleep: non-host resistance in plants

The native resistance of most plant species against a wide variety of pathogens is known as non-host resistance (NHR), which confers durable protection to plant species. Only a few pathogens or parasites can successfully cause diseases. NHR is polygenic and appears to be linked with basal plant resistance, a form of elicited protection. Sensing of pathogens by plants is brought about through the recognition of invariant pathogen-associated molecular patterns (PAMPs) that trigger downstream defense signaling pathways. Race-specific resistance, (R)-gene mediated resistance, has been extensively studied and reviewed, while our knowledge of NHR has advanced only recently due to the improved access to excellent model systems. The continuum of the cell wall (CW) and the CW-plasma membrane (PM)-cytoskeleton plays a crucial role in perceiving external cues and activating defense signaling cascades during NHR. Based on the type of hypersensitive reaction (HR) triggered, NHR was classified into two types, namely type-I and type-II. Genetic analysis of Arabidopsis mutants has revealed important roles for a number of specific molecules in NHR, including the role of SNARE-complex mediated exocytosis, lipid rafts and vesicle trafficking. As might be expected, R-gene mediated resistance is found to overlap with NHR, but the extent to which the genes/pathways are common between these two forms of disease resistance is unknown. The present review focuses on the various components involved in the known mechanisms of NHR in plants with special reference to the role of CW-PM components.     

Negative Regulation of Methyl Jasmonate-Induced Stomatal Closure by Glutathione in Arabidopsis

Glutathione (GSH) has been shown to negatively regulate methyl jasmonate (MeJA)-induced stomatal closure. We investigated the roles of GSH in MeJA signaling in guard cells using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase. MeJA-induced stomatal closure and decreased GSH contents in guard cells. Decreasing GSH by the cad2-1 mutation enhanced MeJA-induced stomatal closure. Depletion of GSH by the cad2-1 mutation or increment of GSH by GSH monoethyl ester did not affect either MeJA-induced production of reactive oxygen species (ROS) or MeJA-induced cytosolic alkalization in guard cells. MeJA and abscisic acid (ABA) induced stomatal closure and GSH depletion in atrbohD and atrbohF single mutants but not in the atrbohD atrbohF double mutant. Moreover, exogenous hydrogen peroxide induced stomatal closure but did not deplete GSH in guard cells. These results indicate that GSH affects MeJA signaling as well as ABA signaling and that GSH negatively regulates a signal component other than ROS production and cytosolic alkalization in MeJA signal pathway of Arabidopsis guard cells.