The Arabidopsis genes RPW8.1 and RPW8.2 confer induced resistance to powdery mildew diseases in tobacco

Plant disease resistance (R) gene products recognize pathogen avirulence (Avr) gene products and induce defense responses. It is not known if an R gene can function in different plant families, however. The Arabidopsis thaliana R genes RPW8.1 and RPW8.2 confer resistance to the powdery mildew pathogens Erysiphe orontii, E. cichoracearum, and Oidium lycopersici, which also infect plants from other families. We produced transgenic Nicotiana tabacum, N. benthamiana, and Lycopersicon esculentum plants containing RPW8.1 and RPW8.2. Transgenic N. tabacum plants had increased resistance to E. orontii and O. lycopersici, transgenic N. benthamiana plants had increased resistance to E. cichoracearum, but transgenic L. esculentum plants remained susceptible to these pathogens. The defense responses induced in transgenic N. tabacum and N. benthamiana were similar to those mediated by RPW8.1 and RPW8.2 in Arabidopsis. Apparently, RPW8.1 and RPW8.2 could be used to control powdery mildew diseases of plants from other families.     

Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the Arabidopsis mutant acd5

Salicylic acid (SA) is required for resistance to many diseases in higher plants. SA-dependent cell death and defense-related responses have been correlated with disease resistance. The accelerated cell death 5 mutant of Arabidopsis provides additional genetic evidence that SA regulates cell death and defense-related responses. However, in acd5, these events are uncoupled from disease resistance. acd5 plants are more susceptible to Pseudomonas syringae early in development and show spontaneous SA accumulation, cell death, and defense-related markers later in development. In acd5 plants, cell death and defense-related responses are SA dependent but they do not confer disease resistance. Double mutants with acd5 and nonexpressor of PR1, in which SA signaling is partially blocked, show greatly attenuated cell death, indicating a role for NPR1 in controlling cell death. The hormone ethylene potentiates the effects of SA and is important for disease symptom development in Arabidopsis. Double mutants of acd5 and ethylene insensitive 2, in which ethylene signaling is blocked, show decreased cell death, supporting a role for ethylene in cell death control. We propose that acd5 plants mimic P. syringae-infected wild-type plants and that both SA and ethylene are normally involved in regulating cell death during some susceptible pathogen infections.     

Positive and negative regulation of salicylic acid-dependent cell death and pathogen resistance in Arabidopsis lsd6 and ssi1 mutants

Salicylic acid (SA) is a key defense molecule in higher plants that is required for resistance to diverse pathogens. A number of mutants of Arabidopsis with elevated resistance to pathogens and constitutive activation of defense-related genes and cell death have been shown to require SA for all of their phenotypes. These mutants potentially identify interesting regulatory genes that control diverse SA responses. When dominant mutations confer SA-dependent phenotypes, it is important to know the genetic basis of dominance in order to draw conclusions on the possible mechanisms of action of the genes identified. Here I characterize the basis of the dominant phenotypes conferred by the ssi1 and lsd6 mutations. I show that ssi1 is haploinsufficient, while lsd6 is a gain-of-function mutation. Thus, SA-dependent responses are under both negative and positive regulation.     

The pepper E3 ubiquitin ligase RING1 gene, CaRING1, is required for cell death and the salicylic acid-dependent defense response

Ubiquitination is essential for ubiquitin/proteasome-mediated protein degradation in plant development and defense. Here, we identified a novel E3 ubiquitin ligase RING1 gene, CaRING1, from pepper (Capsicum annuum). In pepper, CaRING1 expression is induced by avirulent Xanthomonas campestris pv vesicatoria infection. CaRING1 contains an amino-terminal transmembrane domain and a carboxyl-terminal RING domain. In addition, it displays in vitro E3 ubiquitin ligase activity, and the RING domain is essential for E3 ubiquitin ligase activity in CaRING1. CaRING1 also localizes to the plasma membrane. In pepper plants, virus-induced gene silencing of CaRING1 confers enhanced susceptibility to avirulent X. campestris pv vesicatoria infection, which is accompanied by compromised hypersensitive cell death, reduced expression of PATHOGENESIS-RELATED1, and lowered salicylic acid levels in leaves. Transient expression of CaRING1 in pepper leaves induces cell death and the defense response that requires the E3 ubiquitin ligase activity of CaRING1. By contrast, overexpression of CaRING1 in Arabidopsis (Arabidopsis thaliana) confers enhanced resistance to hemibiotrophic Pseudomonas syringae pv tomato and biotrophic Hyaloperonospora arabidopsidis infections. Taken together, these results suggest that CaRING1 is involved in the induction of cell death and the regulation of ubiquitination during the defense response to microbial pathogens.     

Salicylic acid in the machinery of hypersensitive cell death and disease resistance

Although extensive data has described the key role of salicylic acid (SA) in signaling pathogen-induced disease resistance, its function in physiological processes related to cell death is still poorly understood. Recent studies have explored the requirement of SA for mounting the hypersensitive response (HR) against an invading pathogen, where a particular cell death process is activated at the site of attempted infection causing a confined lesion. Biochemical data suggest that SA potentiates the signal pathway for HR by affecting an early phosphorylation-sensitive step preceding the generation of pro-death signals, including those derived from the oxidative burst. Accordingly, the epistatic relationship between cell death and SA accumulation, analyzed in crosses between lesion-mimic mutants (spontaneous lesion formation) and the transgenic nahG line (depleted in SA) places the SA activity in a feedback loop downstream and upstream of cell death. Exciting advances have been made in the identification of cellular protective functions and cell death suppressors that might operate in HR. Moreover, the spatio-temporal patterns of the SA accumulation (non-homogeneous distribution, biphasic kinetics) described in some HR lesions, may also reveal important clues for unraveling the complex cellular network that tightly balances pro- and anti-death functions in the hypersensitive cell death.     

Role of autophagy in disease resistance and hypersensitive response-associated cell death

Ancient autophagy pathways are emerging as key defense modules in host eukaryotic cells against microbial pathogens. Apart from actively eliminating intracellular intruders, autophagy is also responsible for cell survival, for example by reducing the deleterious effects of endoplasmic reticulum stress. At the same time, autophagy can contribute to cellular suicide. The concurrent engagement of autophagy in these processes during infection may sometimes mask its contribution to differing pro-survival and pro-death decisions. The importance of autophagy in innate immunity in mammals is well documented, but how autophagy contributes to plant innate immunity and cell death is not that clear. A few research reports have appeared recently to shed light on the roles of autophagy in plant-pathogen interactions and in disease-associated host cell death. We present a first attempt to reconcile the results of this research.     

The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae

Many plant pathogens suppress antimicrobial defenses using virulence factors that modulate endogenous host defenses. The Pseudomonas syringae phytotoxin coronatine (COR) is believed to promote virulence by acting as a jasmonate analog, because COR-insensitive 1 (coil) Arabidopsis thaliana and tomato mutants are impaired in jasmonate signaling and exhibit reduced susceptibility to P. syringae. To further investigate the role of jasmonate signaling in disease development, we analyzed several jasmonate-insensitive A. thaliana mutants for susceptibility to P. syringae pv. tomato strain DC3000 and sensitivity to COR. Jasmonate-insensitive 1 (jin1) mutants exhibit both reduced susceptibility to P. syringae pv. tomato DC3000 and reduced sensitivity to COR, whereas jasmonate-resistant 1 (jar1) plants exhibit wild-type responses to both COR and P. syringae pv. tomato DC3000. A jin1 jar1 double mutant does not exhibit enhanced jasmonate insensitivity, suggesting that JIN1 functions downstream of jasmonic acid-amino acid conjugates synthesized by JAR1. Reduced disease susceptibility in jin1 mutants is correlated with elevated expression of pathogenesis-related 1 (PR-1) and is dependent on accumulation of salicylic acid (SA). We also show that JIN1 is required for normal P. syringae pv. tomato DC3000 symptom development through an SA-independent mechanism. Thus, P. syringae pv. tomato DC3000 appears to utilize COR to manipulate JIN1-dependent jasmonate signaling both to suppress SA-mediated defenses and to promote symptom development.     

Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance

The RPW8 locus from Arabidopsis thaliana Ms-0 includes two functional paralogous genes ( RPW8.1 and RPW8.2 ) and confers broad-spectrum resistance via the salicylic acid-dependent signaling pathway to the biotrophic fungal pathogens Golovinomyces spp. that cause powdery mildew diseases on multiple plant species. To identify proteins involved in regulation of the RPW8 protein function, a yeast two-hybrid screen was performed using RPW8.2 as bait. The 14-3-3 isoform lambda (designated GF14λ) was identified as a potential RPW8.2 interactor. The RPW8.2–GF14λ interaction was specific and engaged the C-terminal domain of RPW8.2, which was confirmed by pulldown assays. The physiological impact of the interaction was revealed by knocking down GF14λ by T-DNA insertion, which compromised basal and RPW8-mediated resistance to powdery mildew. In addition, over-expression of GF14λ resulted in hypersensitive response-like cell death and enhanced resistance to powdery mildew via the salicylic acid-dependent signaling pathway. The results from this study suggest that GF14λ may positively regulate the RPW8.2 resistance function and play a role in enhancing basal resistance in Arabidopsis.     

Identification of genes required for Cf-dependent hypersensitive cell death by combined proteomic and RNA interfering analyses

Identification of hypersensitive cell death (HCD) regulators is essential to dissect the molecular mechanisms underlying plant disease resistance. In this study, combined proteomic and RNA interfering (RNAi) analyses were employed to identify genes required for the HCD conferred by the tomato resistance gene Cf-4 and the Cladosporium fulvum avirulence gene Avr4. Forty-nine proteins differentially expressed in the tomato seedlings mounting and those not mounting Cf-4/Avr4-dependent HCD were identified through proteomic analysis. Among them were a variety of defence-related proteins including a cysteine protease, Pip1, an operative target of another C. fulvum effector, Avr2. Additionally, glutathione-mediated antioxidation is a major response to Cf-4/Avr4-dependent HCD. Functional analysis through tobacco rattle virus-induced gene silencing and transient RNAi assays of the chosen 16 differentially expressed proteins revealed that seven genes, which encode Pip1 homologue NbPip1, a SIPK type MAP kinase Nbf4, an asparagine synthetase NbAsn, a trypsin inhibitor LeMir-like protein NbMir, a small GTP-binding protein, a late embryogenesis-like protein, and an ASR4-like protein, were required for Cf-4/Avr4-dependent HCD. Furthermore, the former four genes were essential for Cf-9/Avr9-dependent HCD; NbPip1, NbAsn, and NbMir, but not Nbf4, affected a nonadaptive bacterial pathogen Xanthomonas oryzae pv. oryzae-induced HCD in Nicotiana benthamiana. These data demonstrate that Pip1 and LeMir may play a general role in HCD and plant immunity and that the application of combined proteomic and RNA interfering analyses is an efficient strategy to identify genes required for HCD, disease resistance, and probably other biological processes in plants.     

Accurate and adequate spatiotemporal expression and localization of RPW8.2 is key to activation of resistance at the host-pathogen interface

Numerous fungal and oomycete pathogens penetrate the plant cell wall and extract nutrition from the host cells by a feeding structure called the haustorium. We recently revealed that the Arabidopsis resistance protein RPW8.2 is specifically targeted to the extrahaustorial membrane (EHM) for activation of haustorium-targeted resistance to powdery mildew pathogens. Consistent with its EHM-localization, RPW8.2 contains a putative transmembrane (TM) domain at its N-terminus. Here, we show that translational fusion of YFP to the N-terminus of RPW8.2 results in localization of YFP-RPW8.2 to both the plasma membrane and the EHM, and loss of RPW8.2''s defense function. We also show that deletion of the TM domain results in mis-localization of the RPW8.2-YFP fusion protein and extremely low levels of accumulation. These results indicate that an intact N-terminal TM domain is necessary for EHM-specific localization and defense function of RPW8.2. In addition, we show that when expressed from the strong constitutive 35S viral promoter, RPW8.2 accumulates at low levels in the EHM insufficient to activate resistance, highlighting the importance of strong spatiotemporal expression of RPW8.2 from its native promoter. Taken together, our results indicate that accurate and adequate spatiotemporal expression and localization of RPW8.2 is key to activation of resistance at the host-pathogen interface.Key words: Arabidopsis, RPW8.2, resistance, powdery mildew, haustorium, extrahaustorial membrane, host-pathogen interface, protein localizationIn order to establish successful colonization on plant hosts, a haustorium-forming fungus such as powdery mildew must conquer two spatio-temporally interconnected layers of host resistance: pre-invasion (penetration) resistance and post-invasion resistance.¹ Pre-invasion resistance protects plants from non-adapted pathogens by blocking their entry into the host cell.^2–4 One common induced cellular defense response at this resistance level is the deposition of defense chemicals, including callose (β-1,3-glucan) at the site of penetration, resulting in cell wall apposition, a subcellular structure also known as a papilla.^5–7 It has been reported that a syntaxin encoded by PENETRATION 1 (PEN1) is required for the timely assembly of the papilla,⁸ which is consistent with PEN1''s role in pre-invasion resistance.² Once the fungus penetrates the plant cell wall, it will have to overcome the second layer of resistance, i.e., post-invasion resistance, to develop a functional haustorium in close contact with the host cell cytoplasm for successful colonization. Hypersensitive response (HR) manifested as rapid collapse of the invaded cell is often associated with post-invasion resistance.^9–11 Another cellular defense response to haustorial invasion is the formation of an encasement of the haustorial complex (EHC).^12–16 Like the papilla, the EHC is also enriched for callose and thought to be formed via extension from the papilla by rim-growth.¹⁷We have recently reported that RPW8.2-mediated broad-spectrum powdery mildew resistance is associated with both HR and an enhancement of EHC formation.¹⁸ Most strikingly, we found that the RPW8.2-YFP fusion protein expressed from its native promoter (NP) is specifically targeted to the extrahaustorial membrane (EHM), suggesting that RPW8.2 functions at the host-pathogen interface to activate post-invasion resistance. How RPW8.2 is targeted to the EHM and directs host defense to the host-pathogen interface remains to be an open question.     

Ozone-induced cell death occurs via two distinct mechanisms in Arabidopsis: the role of salicylic acid

Previous studies suggest that salicylic acid (SA) plays an important role in influencing plant resistance to ozone (O3). To further define the role of SA in O3-induced responses, we compared the responses of two Arabidopsis genotypes that accumulate different amounts of SA in response to O3 and a SA-deficient transgenic Col-0 line expressing salicylate hydroxylase (NahG). The differences observed in O3-induced changes in SA levels, the accumulation of active oxygen species, defense gene expression, and the kinetics and severity of lesion formation indicate that SA influences O3 tolerance via two distinct mechanisms. Detailed analyses indicated that features associated with a hypersensitive response (HR) were significantly greater in O3-exposed Cvi-0 than in Col-0, and that NahG plants failed to exhibit these HR-like responses. Furthermore, O3-induced antioxidant defenses, including the redox state of glutathione, were greatly reduced in NahG plants compared to Col-0 and Cvi-0. This suggests that O3-induced cell death in NahG plants is due to the loss of SA-mediated potentiation of antioxidant defenses, while O3-induced cell death in Cvi-0 is due to activation of a HR. This hypothesis is supported by the observation that inhibition of NADPH-oxidases reduced O3-induced H2O2 levels and the O3-induced cell death in Cvi-0, while no major changes were observed in NahG plants. We conclude that although SA is required to maintain the cellular redox state and potentiate defense responses in O3 exposed plants, high levels of SA also potentiate activation of an oxidative burst and a cell death pathway that results in apparent O3 sensitivity.     

The Arabidopsis aberrant growth and death2 mutant shows resistance to Pseudomonas syringae and reveals a role for NPR1 in suppressing hypersensitive cell death

A novel Arabidopsis mutant has been identified with constitutive expression of GST1-GUS using plants with a pathogen-responsive reporter transgene containing the beta-glucuronidase (GUS) coding region driven by the GST1 promoter. The recessive mutant, called agd2 (aberrant growth and death2), has salicylic acid (SA)-dependent increased resistance to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae, elevated SA levels, a low level of spontaneous cell death, callose deposition, and enlarged cells in leaves. The enhanced resistance of agd2 to virulent P. syringae requires the SA signaling component NONEXPRESSOR OF PR1 (NPR1). However, agd2 renders the resistance response to P. syringae carrying avrRpt2 NPR1-independent. Thus agd2 affects both an SA- and NPR1-dependent general defense pathway and an SA-dependent, NPR1-independent pathway that is active during the recognition of avirulent P. syringae. agd2 plants also fail to show a hypersensitive cell death response (HR) unless NPR1 is removed. This novel function for NPR1 is also apparent in otherwise wild-type plants: npr1 mutants show a stronger HR, while NPR1-overproducing plants show a weaker HR when infected with P. syringae carrying the avrRpm1 gene. Spontaneous cell death in agd2 is partially suppressed by npr1, indicating that NPR1 can suppress or enhance cell death depending on the cellular context. agd2 plants depleted of SA show a dramatic exacerbation of the cell-growth phenotype and increased callose deposition, suggesting a role for SA in regulating growth and this cell-wall modification. AGD2 may function in cell death and/or growth control as well as the defense response, similarly to what has been described in animals for the functions of NFkappaB.