The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate- and jasmonate/ethylene-dependent signaling pathways

Bacillus cereus AR156 is a plant growth-promoting rhizobacterium that induces resistance against a broad spectrum of pathogens including Pseudomonas syringae pv. tomato DC3000. This study analyzed AR156-induced systemic resistance (ISR) to DC3000 in Arabidopsis ecotype Col-0 plants. Compared with mock-treated plants, AR156-treated ones showed an increase in biomass and reductions in disease severity and pathogen density in the leaves. The defense-related genes PR1, PR2, PR5, and PDF1.2 were concurrently expressed in the leaves of AR156-treated plants, suggesting simultaneous activation of the salicylic acid (SA)- and the jasmonic acid (JA)- and ethylene (ET)-dependent signaling pathways by AR156. The above gene expression was faster and stronger in plants treated with AR156 and inoculated with DC3000 than that in plants only inoculated with DC3000. Moreover, the cellular defense responses hydrogen peroxide accumulation and callose deposition were induced upon challenge inoculation in the leaves of Col-0 plants primed by AR156. Also, pretreatment with AR156 led to a higher level of induced protection against DC3000 in Col-0 than that in the transgenic NahG, the mutant jar1 or etr1, but the protection was absent in the mutant npr1. Therefore, AR156 triggers ISR in Arabidopsis by simultaneously activating the SA- and JA/ET-signaling pathways in an NPR1-dependent manner that leads to an additive effect on the level of induced protection.     

Systemic Responses in Arabidopsis thaliana Infected and Challenged with Pseudomonas syringae pv syringae

Attack of plants by necrotizing pathogens leads to acquired resistance to the same or other pathogens in tissues adjacent to or remotely located from the site of initial attack. We have used Arabidopsis thaliana inoculated with the incompatible pathogen Pseudomonas syringae pv syringae on the lower leaves to test the induction of systemic reactions. When plants were challenged with Pseudomonas syringae pv syringae in the upper leaves, bacterial titers remained stable in those preinfected on the lower leaves. However, there was a distinct decrease in symptoms that correlated with a local and systemic increase in salicylic acid (SA) and in chitinase activity. Peroxidase activity only increased at the site of infection. No changes in catalase activity were observed, either at the local or at the systemic level. No inhibition of catalase could be detected in tissue in which the endogenous levels of SA were elevated either naturally (after infection) or artificially (after feeding SA to the roots). The activity of catalase in homogenates of A. thaliana leaves could not be inhibited in vitro by SA. SA accumulation was induced by H2O2 in leaves, suggesting a link between H2O2 from the oxidative burst commonly observed during the hypersensitive reaction and the induction of a putative signaling molecule leading to system acquired resistance.     

Pathogen-associated molecular pattern recognition rather than development of tissue necrosis contributes to bacterial induction of systemic acquired resistance in Arabidopsis

Systemic acquired resistance (SAR) is usually described as a phenomenon whereby localized inoculation with a necrotizing pathogen renders a plant more resistant to subsequent pathogen infection. Here we show that Pseudomonas syringae strains for which Arabidopsis thaliana represents a non-host plant systemically elevate resistance although the underlying interactions neither trigger a hypersensitive response nor cause necrotic disease symptoms. A similar enhancement of systemic resistance was observed when elicitor-active preparations of two typical bacterial pathogen-associated molecular patterns (PAMPs), flagellin and lipopolysaccharides (LPS), were applied in a localized manner. Several lines of evidence indicate that the observed systemic resistance responses are identical to SAR. Localized applications of non-adapted bacteria, flagellin or LPS elevate levels of the SAR regulatory metabolite salicylic acid (SA) and pathogenesis-related (PR) gene expression not only in treated but also in distant leaves. All treatments also systemically increase expression of the SAR marker gene FLAVIN-DEPENDENT MONOOXYGENASE 1. Further, a whole set of SAR-deficient Arabidopsis lines, including mutants in SA biosynthesis and signalling, are impaired in establishing the systemic resistance response triggered by non-host bacteria or PAMPs. We also show that the magnitude of defence reactions such as SA accumulation, PR gene expression or camalexin accumulation induced at sites of virulent or avirulent P. syringae inoculation but not the extent of tissue necrosis during these interactions determines the extent of SAR in distant leaves. Our data indicate that PAMPs significantly contribute to SAR initiation in Arabidopsis and that tissue necroses at inoculation sites are dispensable for SAR activation.     

Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation.

In Arabidopsis, systemic acquired resistance against pathogens has been associated with the accumulation of salicylic acid (SA) and the expression of the pathogenesis-related proteins PR-1, PR-2, and PR-5. We report here the isolation of two nonallelic mutants impaired in the pathway leading to SA biosynthesis. These SA induction-deficient (sid) mutants do not accumulate SA after pathogen inoculation and are more susceptible to both virulent and avirulent forms of Pseudomonas syringae and Peronospora parasitica. However, sid mutants are not as susceptible to these pathogens as are transgenic plants expressing the nahG gene encoding an SA hydroxylase that degrades SA to catechol. In contrast to NahG plants, only the expression of PR-1 is strongly reduced in sid mutants, whereas PR-2 and PR-5 are still expressed after pathogen attack. Furthermore, the accumulation of the phytoalexin camalexin is normal. These results indicate that SA-independent compensation pathways that do not operate in NahG plants are active in sid mutants. One of the mutants is allelic to eds5 (for enhanced disease susceptibility), whereas the other mutant has not been described previously.     

Role of salicylic acid and NIM1/NPR1 in race-specific resistance in arabidopsis

Salicylic acid (SA) and the NIM1/NPR1 protein have both been demonstrated to be required for systemic acquired resistance (SAR) and implicated in expression of race-specific resistance. In this work, we analyzed the role that each of these molecules play in the resistance response triggered by members of two subclasses of resistance (R) genes, members of which recognize unrelated pathogens. We tested the ability of TIR and coiled-coil-class (also known as leucine-zipper-class) R genes to confer resistance to Pseudomonas syringae pv. tomato or Peronospora parasitica in SA-depleted (NahG) and nim1/npr1 plants. We found that all of the P. syringae pv. tomato-specific R genes tested were dependent upon SA accumulation, while none showed strong dependence upon NIM1/NPR1 activity. A similar SA dependence was observed for the P. parasitica TIR and CC-class R genes RPP5 and RPP8, respectively. However, the P. parasitica-specific R genes differed in their requirement for NIM1/NPR1, with just RPP5 depending upon NIM1/NPR1 activity for effectiveness. These data are consistent with the hypothesis that at least in Arabidopsis, SA accumulation is necessary for the majority of R-gene-triggered resistance, while the role of NIM1/NPR in race-specific resistance is limited to resistance to P. parasitica mediated by TIR-class R genes.     

The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance

Upon localized attack by necrotizing pathogens, plants gradually develop increased resistance against subsequent infections at the whole-plant level, a phenomenon known as systemic acquired resistance (SAR). To identify genes involved in the establishment of SAR, we pursued a strategy that combined gene expression information from microarray data with pathological characterization of selected Arabidopsis (Arabidopsis thaliana) T-DNA insertion lines. A gene that is up-regulated in Arabidopsis leaves inoculated with avirulent or virulent strains of the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) showed homology to flavin-dependent monooxygenases (FMO) and was designated as FMO1. An Arabidopsis knockout line of FMO1 proved to be fully impaired in the establishment of SAR triggered by avirulent (Psm avrRpm1) or virulent (Psm) bacteria. Loss of SAR in the fmo1 mutants was accompanied by the inability to initiate systemic accumulation of salicylic acid (SA) and systemic expression of diverse defense-related genes. In contrast, responses at the site of pathogen attack, including increases in the levels of the defense signals SA and jasmonic acid, camalexin accumulation, and expression of various defense genes, were induced in a similar manner in both fmo1 mutant and wild-type plants. Consistently, the fmo1 mutation did not significantly affect local disease resistance toward virulent or avirulent bacteria in naive plants. Induction of FMO1 expression at the site of pathogen inoculation is independent of SA signaling, but attenuated in the Arabidopsis eds1 and pad4 defense mutants. Importantly, FMO1 expression is also systemically induced upon localized P. syringae infection. This systemic up-regulation is missing in the SAR-defective SA pathway mutants sid2 and npr1, as well as in the defense mutant ndr1, indicating a close correlation between systemic FMO1 expression and SAR establishment. Our findings suggest that the presence of the FMO1 gene product in systemic tissue is critical for the development of SAR, possibly by synthesis of a metabolite required for the transduction or amplification of a signal during the early phases of SAR establishment in systemic leaves.     

细菌性角斑病菌诱导黄瓜叶片水杨酸的积累

对黄瓜(Cucumis sativa L.)幼苗第一片真叶接种细菌性角斑病菌(Pseudomonas syringae pv.lachy-mans)后,利用TLC和HPLC方法分离测定了不同时间内游离态和结合态水杨酸(SA)的含量。结果表明,接种显著提高了第一片真叶游离态SA含量,增加了30％～197％,结合态SA的含量变化与游离态相似,提高了79％～240％,但是接种后第三天游离态SA和结合态SA含量都下降。接种还诱导了非接种第二片真叶中SA的积累:游离态SA含量在接种初期增加了29％～46％,接种后第三天达到峰值,第五天回复到正常水平,结合态SA含量在接种初期没有变化,接种后第四至六天也显著增加,提高了62％～107％。     

不结球白菜PR4蛋白基因的克隆与诱导表达分析

从不结球白菜抗病品种‘苏州青’中克隆到一个受SA和病原菌诱导的病程相关蛋白4(PR4)基因,命名为BcPR4(DDBJ登录号:AB325873),该基因核苷酸序列全长593 bp,编码140个氨基酸,与其它植物的PR4蛋白基因具有较高的相似性。系统进化树分析表明,该基因在不同物种之间具有保守性。基因组DNA杂交表明BcPR4属于多基因家族。实时定量PCR(qPCR)检测表明,SA和Peronospora parasitica均能诱导不结球白菜BcPR4转录表达,BcPR4在不结球白菜叶片中的表达特征说明它可能参与寄主对病原菌的抗性。     

Role of polyisoprenoids in tobacco resistance against biotic stresses

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria ( P.  syringae ) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.     

HSR203 antisense suppression in tobacco accelerates development of hypersensitive cell death

Activation of the tobacco gene hsr203 is rapid, highly localized, specific for incompatible plant-pathogen interactions, and strongly correlated with programmed cell death occurring in response to diverse pathogens. Functional characterization of hsr203 gene product has shown that HSR203 is a serine hydrolase that displays esterase activity. We show here that transgenic tobacco plants deficient in HSR203 protein exhibit an accelerated hypersensitive response when inoculated with an avirulent strain of Ralstonia solanacearum. This response was accompanied by a maximal level of cell death and a drastic inhibition of in planta bacterial growth. Transgenic plants deficient in HSR203 were also found to show increased resistance in a dosage-dependent manner to Pseudomonas syringae pv. pisi, another avirulent bacterial pathogen, and to virulent and avirulent races of Phytophthora parasitica, a fungal pathogen of tobacco, but not to different virulent bacteria. Surprisingly, expression of another hsr gene, hsr515, and that of the defence genes PR1-a and PR5, was strongly reduced in the transgenic lines. Our results suggest that hsr203 antisense suppression in tobacco can have pleiotropic effects on HR cell death and defence mechanisms, and induces increased resistance to different pathogens.     

Overexpression of Arabidopsis MAP kinase kinase 7 leads to activation of plant basal and systemic acquired resistance

There is a growing body of evidence indicating that mitogen-activated protein kinase (MAPK) cascades are involved in plant defense responses. Analysis of the completed Arabidopsis thaliana genome sequence has revealed the existence of 20 MAPKs, 10 MAPKKs and 60 MAPKKKs, implying a high level of complexity in MAPK signaling pathways, and making the assignment of gene functions difficult. The MAP kinase kinase 7 (MKK7) gene of Arabidopsis has previously been shown to negatively regulate polar auxin transport. Here we provide evidence that MKK7 positively regulates plant basal and systemic acquired resistance (SAR). The activation-tagged bud1 mutant, in which the expression of MKK7 is increased, accumulates elevated levels of salicylic acid (SA), exhibits constitutive pathogenesis-related (PR) gene expression, and displays enhanced resistance to both Pseudomonas syringae pv. maculicola (Psm) ES4326 and Hyaloperonospora parasitica Noco2. Both PR gene expression and disease resistance of the bud1 plants depend on SA, and partially depend on NPR1. We demonstrate that the constitutive defense response in bud1 plants is a result of the increased expression of MKK7, and requires the kinase activity of the MKK7 protein. We found that expression of the MKK7 gene in wild-type plants is induced by pathogen infection. Reducing mRNA levels of MKK7 by antisense RNA expression not only compromises basal resistance, but also blocks the induction of SAR. Intriguingly, ectopic expression of MKK7 in local tissues induces PR gene expression and resistance to Psm ES4326 in systemic tissues, indicating that activation of MKK7 is sufficient for generating the mobile signal of SAR.     

A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance.

Systemic acquired resistance (SAR) is a nonspecific defense response in plants that is associated with an increase in the endogenous level of salicylic acid (SA) and elevated expression of pathogenesis-related (PR) genes. To identify mutants involved in the regulation of PR genes and the onset of SAR, we transformed Arabidopsis with a reporter gene containing the promoter of a beta-1,3-glucanase-encoding PR gene (BGL2) and the coding region of beta-glucuronidase (GUS). The resulting transgenic line (BGL2-GUS) was mutagenized, and the M2 progeny were scored for constitutive GUS activity. We report the characterization of one mutant, cpr1 (constitutive expressor of PR genes), that was identified in this screen and shown by RNA gel blot analysis also to have elevated expression of the endogenous PR genes BGL2, PR-1, and PR-5. Genetic analyses indicated that the phenotype conferred by cpr1 is caused by a single, recessive nuclear mutation and is suppressed in plants producing a bacterial salicylate hydroxylase, which inactivates SA. Furthermore, biochemical analysis showed that the endogenous level of SA is elevated in the mutant. Finally, the cpr1 plants were found to be resistant to the fungal pathogen Peronospora parasitica NOCO2 and the bacterial pathogen Pseudomonas syringae pv maculicola ES4326, which are virulent in wild-type BGL2-GUS plants. Because the cpr1 mutation is recessive and associated with an elevated endogenous level of SA, we propose that the CPR1 gene product acts upstream of SA as a negative regulator of SAR.     

Induced expression of sarcotoxin IA enhanced host resistance against both bacterial and fungal pathogens in transgenic tobacco

We demonstrate here that induced expression of sarcotoxin IA, a bactericidal peptide from Sarcophaga peregrina, enhanced the resistance of transgenic tobacco plants to both bacterial and fungal pathogens. The peptide was produced with a modified PR1a promoter, which is further activated by salicylic acid treatment and necrotic lesion formation by pathogen infection. Host resistance to infection of bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tabaci was shown to be dependent on the amounts of sarcotoxin IA expressed. Since we found antifungal activity of the peptide in vitro, transgenic seedlings were also inoculated with fungal pathogens Rhizoctonia solani and Pythium aphanidermatum. Transgenic plants expressing higher levels of sarcotoxin were able to withstand fungal infection and remained healthy even after 4 weeks, while control plants were dead by fungal infection after 2 weeks.     

Characterization of a novel,defense-related Arabidopsis mutant,cir1, isolated by luciferase imaging

In order to identify components of the defense signaling network engaged following attempted pathogen invasion, we generated a novel PR-1::luciferase (LUC) transgenic line that was deployed in an imaging-based screen to uncover defense-related mutants. The recessive mutant designated cir1 exhibited constitutive expression of salicylic acid (SA), jasmonic acid (JA)/ethylene, and reactive oxygen intermediate-dependent genes. Moreover, this mutation conferred resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and a virulent oomycete pathogen Peronospora parasitica Noco2. Epistasis analyses were undertaken between cir1 and mutants that disrupt the SA (nprl, nahG), JA (jar1), and ethylene (ET) (ein2) signaling pathways. While resistance against both P. syringae pv. tomato DC3000 and Peronospora parasitica Noco2 was partially reduced by npr1, resistance against both of these pathogens was lost in an nahG genetic background. Hence, cirl-mediated resistance is established via NPR1-dependent and -independent signaling pathways and SA accumulation is essential for the function of both pathways. While jar1 and ein2 reduced resistance against P. syringae pv. tomato DC3000, these mutations appeared not to impact cir1-mediated resistance against Peronospora parasitica Noco2. Thus, JA and ET sensitivity are required for cir1-mediated resistance against P. syringae pv. tomato DC3000 but not Peronospora parasitica Noco2. Therefore, the cir1 mutation may define a negative regulator of disease resistance that operates upstream of SA, JA, and ET accumulation.