Probenazole induces systemic acquired resistance in Arabidopsis with a novel type of action

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide), which is the active ingredient in Oryzemate, has been used widely in Asia to protect rice plants against the rice blast fungus Magnaporthe grisea. To study PBZ's mode of action, we analyzed its ability, as well as that of its active metabolite 1, 2-benzisothiazol-3 (2H)-one 1,1-dioxide (BIT) to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with PBZ or BIT exhibited increased expression of several pathogenesis-related genes, increased levels of total salicylic acid (SA), and enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC 3000 and the oomycete pathogen Peronospora parasitica Emco5. The role of several defense signaling hormones, such as SA, ethylene and jasmonic acid (JA), in activating resistance following PBZ or BIT treatment was analyzed using NahG transgenic plants and etr1-1 and coi1-1 mutant plants, respectively. In addition, the involvement of NPR1, a key component in the SA signaling pathway leading to defense responses, was assessed. PBZ or BIT treatment did not induce disease resistance or PR-1 expression in NahG transgenic or npr1 mutant plants, but it did activate these phenomena in etr1-1 and coi 1-1 mutant plants. Thus SA and NPR1 appear to be required for PBZ- and BIT-mediated activation of defense responses, while ethylene and JA are not. Furthermore, our data suggest that PBZ and BIT comprise a novel class of defense activators that stimulate the SA/NPR1-mediated defense signaling pathway upstream of SA.     

An Extracellular Subtilase Switch for Immune Priming in Arabidopsis

In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA)-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity.     

N-cyanomethyl-2-chloroisonicotinamide induces systemic acquired resistance in arabidopsis without salicylic acid accumulation

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid-mediated pathway. N-cyanomethyl-2-chloroisonicotinamide (NCI) is able to induce a broad range of disease resistance in tobacco and rice and induces SAR marker gene expression without SA accumulation in tobacco. To clarify the detailed mode of action of NCI, we analyzed its ability to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with NCI exhibited increased expression of several pathogenesis-related genes and enhanced resistance to the bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. NCI induced disease resistance and PR gene expression in NahG transgenic plants, but not in the npr1 mutant. NCI could induce PR gene expression in the etr1-1, ein2-1 and jar1-1 mutants. Thus, NCI activates SAR, independently from ethylene and jasmonic acid, by stimulating the site between SA and NPR1.     

Evidence for an Important Role of WRKY DNA Binding Proteins in the Regulation of NPR1 Gene Expression

The Arabidopsis NPR1 gene is a positive regulator of inducible plant disease resistance. Expression of NPR1 is induced by pathogen infection or treatment with defense-inducing compounds such as salicylic acid (SA). Transgenic plants overexpressing NPR1 exhibit enhanced resistance to a broad spectrum of microbial pathogens, whereas plants underexpressing the gene are more susceptible to pathogen infection. These results suggest that regulation of NPR1 gene expression is important for the activation of plant defense responses. In the present study, we report the identification of W-box sequences in the promoter region of the NPR1 gene that are recognized specifically by SA-induced WRKY DNA binding proteins from Arabidopsis. Mutations in these W-box sequences abolished their recognition by WRKY DNA binding proteins, rendered the promoter unable to activate a downstream reporter gene, and compromised the ability of NPR1 to complement npr1 mutants for SA-induced defense gene expression and disease resistance. These results provide strong evidence that certain WRKY genes act upstream of NPR1 and positively regulate its expression during the activation of plant defense responses. Consistent with this model, we found that SA-induced expression of a number of WRKY genes was independent of NPR1.     

The Arabidopsis AtNPR1 inversely modulates defense responses against fungal, bacterial, or viral pathogens while conferring hypersensitivity to abiotic stresses in transgenic rice

The nonexpressor of pathogenesis-related (PR) genes (NPR1) protein plays an important role in mediating defense responses activated by pathogens in Arabidopsis. In rice, a disease-resistance pathway similar to the Arabidopsis NPR1-mediated signaling pathway one has been described. Here, we show that constitutive expression of the Arabidopsis NPR1 (AtNPR1) gene in rice confers resistance against fungal and bacterial pathogens. AtNPR1 exerts its protective effects against fungal pathogens by priming the expression of salicylic acid (SA)-responsive endogenous genes, such as the PR1b, TLP (PR5), PR10, and PBZ1. However, expression of AtNPR1 in rice has negative effects on viral infections. The AtNPR1-expressing rice plants showed a higher susceptibility to infection by the Rice yellow mottle virus (RYMV) which correlated well with a misregulation of RYMV-responsive genes, including expression of the SA-regulated RNA-dependent RNA polymerase 1 gene (OsRDR1). Moreover, AtNPR1 negatively regulates the expression of genes playing a role in the plant response to salt and drought stress (rab21, salT, and dip1), which results in a higher sensitivity of AtNPR1 rice to the two types of abiotic stress. These observations suggest that AtNPR1 has both positive and negative regulatory roles in mediating defense responses against biotic and abiotic stresses.     

Ethylene and jasmonic acid signaling affect the NPR1-independent expression of defense genes without impacting resistance to Pseudomonas syringae and Peronospora parasitica in the Arabidopsis ssi1 mutant

Salicylic acid (SA), ethylene, and jasmonic acid (JA) are important signaling molecules in plant defense to biotic stress. An intricate signaling network involving SA, ethylene, and JA fine tunes plant defense responses. SA-dependent defense responses in Arabidopsis thaliana are mediated through NPR1-dependent and -independent mechanisms. We have previously shown that activation of an NPR1-independent defense mechanism confers enhanced disease resistance and constitutive expression of the pathogenesis-related (PR) genes in the Arabidopsis ssi1 mutant. In addition, the ssi1 mutant constitutively expresses the defensin gene PDF1.2. Moreover, SA is required for the ssi1-conferred constitutive expression of PDF1.2 in addition to PR genes. Hence, the ssi1 mutant appears to target a step common to SA- and ethylene- or JA-regulated defense pathways. In the present study, we show that, in addition to SA, ethylene and JA signaling also are required for the ssi1-conferred constitutive expression of PDF1.2 and the NPR1-independent expression of PR-1. Furthermore, the ethylene-insensitive ein2 and JA-insensitive jar1 mutants enhance susceptibility of ssi1 plants to the necrotrophic fungus Botrytis cinerea. However, defects in either the ethylene- or JA-signaling pathways do not compromise ssi1-conferred resistance to the bacterial pathogen Pseudomonas synringae pv. maculicola and the oomycete pathogen Peronospora parasitica. Interestingly, ssi1 exhibits a marginal increase in the levels of ethylene and JA, suggesting that low endogenous levels of these phytohormones are sufficient to activate expression of defense genes. Taken together, our results indicate that although cross talk in ssi1 renders expression of ethylene- or JA-responsive defense genes sensitive to SA and vice versa, it does not affect downstream signaling leading to resistance.     

Non-host defense response in a novel Arabidopsis-Xanthomonas citri subsp. citri pathosystem

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. Progress of breeding citrus canker-resistant varieties is modest due to limited resistant germplasm resources and lack of candidate genes for genetic manipulation. The objective of this study is to establish a novel heterologous pathosystem between Xcc and the well-established model plant Arabidopsis thaliana for defense mechanism dissection and resistance gene identification. Our results indicate that Xcc bacteria neither grow nor decline in Arabidopsis, but induce multiple defense responses including callose deposition, reactive oxygen species and salicylic aicd (SA) production, and defense gene expression, indicating that Xcc activates non-host resistance in Arabidopsis. Moreover, Xcc-induced defense gene expression is suppressed or attenuated in several well-characterized SA signaling mutants including eds1, pad4, eds5, sid2, and npr1. Interestingly, resistance to Xcc is compromised only in eds1, pad4, and eds5, but not in sid2 and npr1. However, combining sid2 and npr1 in the sid2npr1 double mutant compromises resistance to Xcc, suggesting genetic interactions likely exist between SID2 and NPR1 in the non-host resistance against Xcc in Arabidopsis. These results demonstrate that the SA signaling pathway plays a critical role in regulating non-host defense against Xcc in Arabidopsis and suggest that the SA signaling pathway genes may hold great potential for breeding citrus canker-resistant varieties through modern gene transfer technology.     

Ectopic expression of the cotton non-symbiotic hemoglobin gene GhHbd1 triggers defense responses and increases disease tolerance in Arabidopsis

Plant non-symbiotic hemoglobins (nsHbs) play important roles in a variety of cellular processes. Previous evidence from this laboratory indicates that the expression of a class 1 nsHb gene (GhHb1) from cotton is induced in cotton roots challenged with the Verticillium wilt fungus. The present study examined further the expression patterns of the GhHb1 gene in cotton plants and characterized its in vivo function through ectopic overexpression of the gene in Arabidopsis thaliana. Expression of GhHb1 in cotton plants was induced by exogenously applied salicylic acid, methyl jasmonic acid, ethylene, hydrogen peroxide (H(2)O(2)) and nitric oxide (NO). Ectopic overproduction of GhHb1 in Arabidopsis led to constitutive expression of the defense genes PR-1 and PDF1.2, and conferred enhanced disease resistance to Pseudomonas syringae and tolerance to V. dahliae. GhHb1-transgenic Arabidopsis seedlings were more tolerant to exogenous NO and contained lower levels of cellular NO than the wild-type control. Moreover, transgenic plants with relatively high levels of expression of the GhHb1 gene developed spontaneous hypersensitive lesions on the leaves in the absence of pathogen inoculation. Our results indicate that GhHb1 proteins play a role in the defense responses against pathogen invasions, possibly by modulating the NO level and the ratio of H(2)O(2)/NO in the defense process.     

Diverse Roles of the Salicylic Acid Receptors NPR1 and NPR3/NPR4 in Plant Immunity

The plant defense hormone salicylic acid (SA) is perceived by two classes of receptors, NPR1 and NPR3/NPR4. They function in two parallel pathways to regulate SA-induced defense gene expression. To better understand the roles of the SA receptors in plant defense, we systematically analyzed their contributions to different aspects of Arabidopsis (Arabidopsis thaliana) plant immunity using the SA-insensitive npr1-1 npr4-4D double mutant. We found that perception of SA by NPR1 and NPR4 is required for activation of N-hydroxypipecolic acid biosynthesis, which is essential for inducing systemic acquired resistance. In addition, both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are severely compromised in the npr1-1 npr4-4D double mutant. Interestingly, the PTI and ETI attenuation in npr1-1 npr4-4D is more dramatic compared with the SA-induction deficient2-1 (sid2-1) mutant, suggesting that the perception of residual levels of SA in sid2-1 also contributes to immunity. Furthermore, NPR1 and NPR4 are involved in positive feedback amplification of SA biosynthesis and regulation of SA homeostasis through modifications including 5-hydroxylation and glycosylation. Thus, the SA receptors NPR1 and NPR4 play broad roles in plant immunity.     

Multiple roles of WIN3 in regulating disease resistance, cell death, and flowering time in Arabidopsis

The salicylic acid (SA) regulatory gene HOPW1-1-INTERACTING3 (WIN3) was previously shown to confer resistance to the biotrophic pathogen Pseudomonas syringae. Here, we report that WIN3 controls broad-spectrum disease resistance to the necrotrophic pathogen Botrytis cinerea and contributes to basal defense induced by flg22, a 22-amino acid peptide derived from the conserved region of bacterial flagellin proteins. Genetic analysis indicates that WIN3 acts additively with several known SA regulators, including PHYTOALEXIN DEFICIENT4, NONEXPRESSOR OF PR GENES1 (NPR1), and SA INDUCTION-DEFICIENT2, in regulating SA accumulation, cell death, and/or disease resistance in the Arabidopsis (Arabidopsis thaliana) mutant acd6-1. Interestingly, expression of WIN3 is also dependent on these SA regulators and can be activated by cell death, suggesting that WIN3-mediated signaling is interconnected with those derived from other SA regulators and cell death. Surprisingly, we found that WIN3 and NPR1 synergistically affect flowering time via influencing the expression of flowering regulatory genes FLOWERING LOCUS C and FLOWERING LOCUS T. Taken together, our data reveal that WIN3 represents a novel node in the SA signaling networks to regulate plant defense and flowering time. They also highlight that plant innate immunity and development are closely connected processes, precise regulation of which should be important for the fitness of plants.     

NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) play key roles. In this study, we investigated the molecular mechanism of the antagonistic effect of SA on JA signaling. Arabidopsis plants unable to accumulate SA produced 25-fold higher levels of JA and showed enhanced expression of the JA-responsive genes LOX2, PDF1.2, and VSP in response to infection by Pseudomonas syringae pv tomato DC3000, indicating that in wild-type plants, pathogen-induced SA accumulation is associated with the suppression of JA signaling. Analysis of the Arabidopsis mutant npr1, which is impaired in SA signal transduction, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for the suppression of JA signaling, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.     

Constitutive expression of mammalian nitric oxide synthase in tobacco plants triggers disease resistance to pathogens

Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H₂O₂, with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance.     

拟南芥NPR1基因的克隆与表达载体的构建

NPR1基因为植物抗病基因表达和系统获得性抗性中的一个关键基因。该文以DNA PCR扩增的方法,从拟南芥基因组DNA中克隆出NPR1基因,通过序列分析,所克隆的 NPR1 基因与报道的基因序列完全一致。将其构建成植物表达载体,为今后植物抗病基因工程的开展奠定了基础。     

Genetic elucidation of nitric oxide signaling in incompatible plant-pathogen interactions

Recent experiments indicate that nitric oxide (NO) plays a pivotal role in disease resistance and several other physiological processes in plants. However, most of the current information about the function of NO in plants is based on pharmacological studies, and additional approaches are therefore required to ascertain the role of NO as an important signaling molecule in plants. We have expressed a bacterial nitric oxide dioxygenase (NOD) in Arabidopsis plants and/or avirulent Pseudomonas syringae pv tomato to study incompatible plant-pathogen interactions impaired in NO signaling. NOD expression in transgenic Arabidopsis resulted in decreased NO levels in planta and attenuated a pathogen-induced NO burst. Moreover, NOD expression in plant cells had very similar effects on plant defenses compared to NOD expression in avirulent Pseudomonas. The defense responses most affected by NO reduction during the incompatible interaction were decreased H(2)O(2) levels during the oxidative burst and a blockage of Phe ammonia lyase expression, the key enzyme in the general phenylpropanoid pathway. Expression of the NOD furthermore blocked UV light-induced Phe ammonia lyase and chalcone synthase gene expression, indicating a general signaling function of NO in the activation of the phenylpropanoid pathway. NO possibly functions in incompatible plant-pathogen interactions by inhibiting the plant antioxidative machinery, and thereby ensuring locally prolonged H(2)O(2) levels. Additionally, albeit to a lesser extent, we observed decreases in salicylic acid production, a diminished development of hypersensitive cell death, and a delay in pathogenesis-related protein 1 expression during these NO-deficient plant-pathogen interactions. Therefore, this genetic approach confirms that NO is an important regulatory component in the signaling network of plant defense responses.     

A recessive mutation in the Arabidopsis SSI2 gene confers SA- and NPR1-independent expression of PR genes and resistance against bacterial and oomycete pathogens

The Arabidopsis thaliana NPR1 gene is required for salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance. However, loss-of-function mutations in NPR1 do not confer complete loss of PR gene expression or disease resistance. Thus these responses also can be activated via an NPR1-independent pathway that currently remain to be elucidated. The ssi2-1 mutant, identified in a genetic screen for suppressors of npr1-5, affects signaling through the NPR1-independent defense pathway(s). In comparison with the wild-type (SSI2 NPR1) plants and the npr1-5 mutant (SSI2 npr1-5), the ssi2-1 npr1-5 double mutant and the ssi2-1 NPR1 single mutant constitutively express PR genes [PR-1, BGL2 (PR-2) and PR-5]; accumulate elevated levels of SA; spontaneously develop lesions; and possess enhanced resistance to a virulent strain of Peronospora parasitica. The ssi2-1 mutation also confers enhanced resistance to Pseudomonas syringae pv. tomato (Pst); however, this is accomplished primarily via an NPR1-dependent pathway. Analysis of ssi2-1 NPR1 nahG and ssi2-1 npr1-5 nahG plants revealed that elevated SA levels were not essential for the ssi2-1-conferred phenotypes. However, expression of the nahG transgene did reduce the intensity of some ssi2-1-conferred phenotypes, including PR-1 expression, and disease resistance. Based on these results, SSI2 or an SSI2-generated signal appears to modulate signaling of an SA-dependent, NPR1-independent defense pathway, or an SA- and NPR1-independent defense pathway.