Abscisic acid determines arbuscule development and functionality in the tomato arbuscular mycorrhiza

The role of abscisic acid (ABA) during the establishment of the arbuscular mycorrhiza (AM) was studied using ABA sitiens tomato (Lycopersicon esculentum) mutants with reduced ABA concentrations. Sitiens plants and wild-type (WT) plants were colonized by Glomus intraradices. Trypan blue and alkaline phosphatase histochemical staining procedures were used to determine both root colonization and fungal efficiency. Exogenous ABA and silver thiosulfate (STS) were applied to establish the role of ABA and putative antagonistic cross-talk between ABA and ethylene during AM formation, respectively. Sitiens plants were less susceptible to the AM fungus than WT plants. Microscopic observations and arbuscule quantification showed differences in arbuscule morphology between WT and sitiens plants. Both ABA and STS increased susceptibility to the AM fungus in WT and sitiens plants. Fungal alkaline phosphate activity in sitiens mutants was completely restored by ABA application. * The results demonstrate that ABA contributes to the susceptibility of tomato to infection by AM fungi, and that it seems to play an important role in the development of the complete arbuscule and its functionality. Ethylene perception is crucial to AM regulation, and the impairment of mycorrhiza development in ABA-deficient plants is at least partly attributable to ethylene.     

Antagonistic interaction between systemic acquired resistance and the abscisic acid-mediated abiotic stress response in Arabidopsis

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR development in dicotyledonous plants, such as tobacco (Nicotiana tabacum) and Arabidopsis thaliana, is mediated by salicylic acid (SA). Here, using two types of SAR-inducing chemicals, 1,2-benzisothiazol-3(2H)-one1,1-dioxide and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester, which act upstream and downstream of SA in the SAR signaling pathway, respectively, we show that treatment with abscisic acid (ABA) suppresses the induction of SAR in Arabidopsis. In an analysis using several mutants in combination with these chemicals, treatment with ABA suppressed SAR induction by inhibiting the pathway both upstream and downstream of SA, independently of the jasmonic acid/ethylene-mediated signaling pathway. Suppression of SAR induction by the NaCl-activated environmental stress response proved to be ABA dependent. Conversely, the activation of SAR suppressed the expression of ABA biosynthesis-related and ABA-responsive genes, in which the NPR1 protein or signaling downstream of NPR1 appears to contribute. Therefore, our data have revealed that antagonistic crosstalk occurs at multiple steps between the SA-mediated signaling of SAR induction and the ABA-mediated signaling of environmental stress responses.     

Benzothiadiazole inhibits mitochondrial NADH:ubiquinone oxidoreductase in tobacco

An inducer of acquired disease resistance in plants, benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester, exhibited direct, concentration-dependent inhibition of the NADH:ubiquinone oxidoreductase activity of complex I of the mitochondrial electron transport chain of cultured tobacco cells. The complex I activity was less sensitive to inhibition by salicylic acid, an endogenous activator of acquired disease resistance. Using a dichlorodihydrofluorescein assay, it was found that benzothiadiazole, salicylic acid and the complex I inhibitor rotenone, increased reactive oxygen species production within cells in a concentration-dependent manner. The results indicate that both benzothiadiazole and salicylic acid affect the mitochondria of treated plant cells and result in increased production of reactive oxygen species. The biochemical basis of this response could be related to the inhibition of the NADH:ubiquinone oxidoreductase activity of complex I that results in channelling of electrons via complex II, with concomitant higher levels of superoxide production.     

beta-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophic fungus Botrytis cinerea

The non-protein amino acid beta-aminobutyric acid (BABA) protects numerous plants against various pathogens. Protection of Arabidopsis plants against virulent pathogens involves the potentiation of pathogen-specific defense responses. To extend the analysis of the mode of action of BABA to necrotrophs we evaluated the effect of this chemical on Arabidopsis plants infected with the gray mold fungus Botrytis cinerea. BABA-treated Arabidopsis were found to be less sensitive to two different strains of this pathogen. BABA protected mutants defective in the jasmonate and ethylene pathways, but was inactive in plants impaired in the systemic acquired resistance transduction pathway. Treatments with benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester, a functional analog of salicylic acid (SA), also markedly reduced the level of infection. Moreover, BABA potentiated mRNA accumulation of the SA-associated PR-1, but not the jasmonate/ethylene-dependent PDF1.2 gene. Thus, besides jasmonate/ethylene-dependent defense responses, SA-dependent signaling also contributes to restrict B. cinerea infection in Arabidopsis. Our results also suggest that SA-dependent signaling is down-regulated after infection by B. cinerea. The observed up-regulation of the PDF1.2 gene in mutants defective in the SA-dependent signaling pathway points to a cross-talk between SA- and jasmonate/ethylene-dependent signaling pathways during pathogen ingress.     

beta-Aminobutyric acid-induced resistance against downy mildew in grapevine acts through the potentiation of callose formation and jasmonic acid signaling

beta-Aminobutyric acid (BABA) was used to induce resistance in grapevine (Vitis vinifera) against downy mildew (Plasmopara viticola). This led to a strong reduction of mycelial growth and sporulation in the susceptible cv. Chasselas. Comparing different inducers, the best protection was achieved with BABA followed by jasmonic acid (JA), whereas benzo (1,2,3)-thiadiazole-7-carbothionic acid-S-methyl ester (a salicylic acid [SA] analog) and abscisic acid (ABA) treatment did not increase the resistance significantly. Marker genes for the SA and JA pathways showed potentiated expression patterns in BABA-treated plants following infection. The callose synthesis inhibitor 2-deoxy-D-glucose partially suppressed BABA- and JA-induced resistance against P viticola in Chasselas. Application of the phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid and the lipoxygenase (LOX) inhibitor 5, 8, 11, 14-eicosatetraynoic acid (ETYA) also led to a reduction of BABA-induced resistance (BABA-IR), suggesting that callose deposition as well as defense mechanisms depending on phenylpropanoids and the JA pathways all contribute to BABA-IR. The similar phenotype of BABA- and JA-induced resistance, the potentiated expression pattern of JA-regulated genes (LOX-9 and PR-4) following BABA treatment, and the suppression of BABA-IR with ETYA suggest an involvement of the JA pathway in BABA-IR of grapevine leading to a primed deposition of callose and lignin around the infection sites.     

Chloroisonicotinamide derivative induces a broad range of disease resistance in rice and tobacco

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogens. SAR in dicotyledonous plants such as tobacco and Arabidopsis has been partially elucidated and is mediated by salicylic acid (SA). However, the SAR mechanism of monocotyledonous rice plants remains to be clarified, although some similarities between SAR mechanisms in both types have been reported. Here we have characterized N-cyanomethyl-2-chloroisonicotinamide (NCI) as an effective SAR inducer in both plant species. Soil drench application of NCI induces a broad range of disease resistance in tobacco and rice and, more specifically, PR gene expression in tobacco. Both SA measurements in wild-type NCI-treated tobacco and pathogenic infection studies using NahG transgenic tobacco plants indicate that NCI-induced resistance enhancement does not require SA. Therefore, it is suggested that NCI induces SAR by triggering signaling at the same level as or downstream of SA accumulation as do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and 2,6-dichloroisonicotinic acid. The fact that all of these chemicals are effective in rice and tobacco suggests that several common components function in disease resistance in both plant species.     

Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4

Salicylic acid (SA) is an important regulator of plant defense responses, and a variety of Arabidopsis mutants impaired in resistance against bacterial and fungal pathogens show defects in SA accumulation, perception, or signal transduction. Nevertheless, the role of SA-dependent defense responses against necrotrophic fungi is currently unclear. We determined the susceptibility of a set of previously identified Arabidopsis mutants impaired in defense responses to the necrotrophic fungal pathogen Botrytis cinerea. The rate of development of B. cinerea disease symptoms on primary infected leaves was affected by responses mediated by the genes EIN2, JAR1, EDS4, PAD2, and PAD3, but was largely independent of EDS5, SID2/ICS1, and PAD4. Furthermore, plants expressing a nahG transgene or treated with a phenylalanine ammonia lyase (PAL) inhibitor showed enhanced symptoms, suggesting that SA synthesized via PAL, and not via isochorismate synthase (ICS), mediates lesion development. In addition, the degree of lesion development did not correlate with defensin or PR1 expression, although it was partially dependent upon camalexin accumulation. Although npr1 mutant leaves were normally susceptible to B. cinerea infection, a double ein2 npr1 mutant was significantly more susceptible than ein2 plants, and exogenous application of SA decreased B. cinerea lesion size through an NPR1-dependent mechanism that could be mimicked by the cpr1 mutation. These data indicate that local resistance to B. cinerea requires ethylene-, jasmonate-, and SA-mediated signaling, that the SA affecting this resistance does not require ICS1 and is likely synthesized via PAL, and that camalexin limits lesion development.     

Pyrazolecarboxylic acid derivative induces systemic acquired resistance in tobacco

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through asalicylic acid (SA)-mediated pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid (CMPA) as an effective SAR inducer in tobacco. Soil drench application of CMPA induced PR gene expression and a broad range of disease resistance without antibacterial activity in tobacco. Both analysis of CMPA's effects on NahG transgenic tobacco plants and SA measurement in wild-type plants indicated that CMPA-induced resistance enhancement does not require SA. Therefore, it is suggested that CMPA induces SAR by triggering the signaling at the same level as or downstream of SA accumulation as do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and N-cyanomethyl-2-chloroisonicotinamide.     

Induction of systemic resistance to Botrytis cinerea in tomato by Pseudomonas aeruginosa 7NSK2: role of salicylic acid,pyochelin, and pyocyanin

The rhizobacterium Pseudomonas aeruginosa 7NSK2 produces secondary metabolites such as pyochelin (Pch), its precursor salicylic acid (SA), and the phenazine compound pyocyanin. Both 7NSK2 and mutant KMPCH (Pch-negative, SA-positive) induced resistance to Botrytis cinerea in wild-type but not in transgenic NahG tomato. SA-negative mutants of both strains lost the capacity to induce resistance. On tomato roots, KMPCH produced SA and induced phenylalanine ammonia lyase activity, while this was not the case for 7NSK2. In 7NSK2, SA is probably very efficiently converted to Pch. However, Pch alone appeared not to be sufficient to induce resistance. In mammalian cells, Fe-Pch and pyocyanin can act synergistically to generate highly reactive hydroxyl radicals that cause cell damage. Reactive oxygen species are known to play an important role in plant defense. To study the role of pyocyanin in induced resistance, a pyocyanin-negative mutant of 7NSK2, PHZ1, was generated. PHZ1 is mutated in the phzM gene encoding an O-methyltransferase. PHZ1 was unable to induce resistance to B. cinerea, whereas complementation for pyocyanin production or co-inoculation with mutant 7NSK2-562 (Pch-negative, SA-negative, pyocyanin-positive) restored induced resistance. These results suggest that pyocyanin and Pch, rather than SA, are the determinants for induced resistance in wild-type P. aeruginosa 7NSK2.     

葡萄幼苗高温锻炼过程中与水杨酸相关的信号转导的初步研究

对无性繁殖的‘京秀’葡萄（Vitis vinifera cv.Jingxiu）幼苗高温锻炼12 h或外施水杨酸（SA）后12 h发现,两种处理引起葡萄内源ABA含量的变化趋势相似,即在处理后1 h急剧升高,然后又迅速下降,并保持这种低水平到处理结束, 而脂氧合酶（LOX）活性并没有表现出和ABA相似的变化趋势。高温锻炼期间,葡萄叶片内自由态SA含量与ABA变化趋势相似,表现出一种典型的化学信号分子特征。而SA合成的限速酶苯丙氨酸解氨酶（PAL）也表现出和前两者相似的变化趋势。推测SA单独或通过ABA诱导葡萄抗热性。     

Mi-1-Mediated aphid resistance involves salicylic acid and mitogen-activated protein kinase signaling cascades

The tomato Mi-1 gene confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum eluphorbiae), and whiteflies (Bemisia tabaci and B. tabaci biotype B). Resistance to potato aphid is developmentally regulated and is not associated with induction of a hypersensitive response. The NahG transgene that eliminates endogenous salicylic acid (SA) was used to test the role of the SA signaling pathway in the resistance mediated by Mi-1 to potato aphids. Aphids survived longer on NahG tomato plants than on wild type. However, aphid reproduction was not affected on NahG tomato. Aphid resistance in Mi-1 NahG plants was completely abolished and the phenotype was successfully rescued by application of BTH (benzo(1,2,3)-thiaiazole-7-carbothioic acid S-methyl ester), indicating that the SA signaling pathway is an important component of Mi-1-mediated aphid resistance. Using virus-induced gene silencing, one or more mitogen-activated protein kinase (MAPK) cascades required for Mi-1-mediated aphid resistance were identified. Silencing plants for MAPK kinase (LeMKK2) and MAPKs (LeMPK2 and LeMPK1, or LeMPK3) resulted in attenuation of Mi-1-mediated aphid resistance. These results further demonstrate that resistance gene-mediated signaling events against piercing-sucking insects are similar to those against other plant pathogens.     

Benzothiadiazole induces local resistance to Bemisia tabaci (Hemiptera: Aleyrodidae) in tomato plants

Plant resistance to the B and Q biotypes of sweetpotato whitefly, Bemisa tabaci (Gennadius), induced by benzo [1,2,3] thiadiazole-7-carbothioic acid-S-methyl ester (BTH or acibenzolar-S-methyl) in tomato 'Marmande' plants was evaluated in free-choice and no-choice assays under different conditions. BTH is the active ingredient of the Syngenta plant activator Bion. BTH treatment affected host preference of B. tabaci (B and Q biotypes) adults on plants sprayed with Bion at 0.2 and 0.4 g/liter during the earlier days of free-choice assays. As a consequence, a decrease in the total number of eggs (although female fecundity was not affected) and in the final number of pupae and empty pupal cases was observed. The effect produced by BTH applied at 0.1 g/liter Bion was not significant. In no-choice assays, a reduction of the numbers of first-stage larvae and total individuals and a delay in insect development were observed when local treatment was restricted to one leaflet per plant, 5 d before B. tabaci (biotype B) infestation. This acquired resistance induced by BTH seemed to be locally expressed because of the differences between treated and nontreated leaflets in the same plants, whereas no differences in nontreated leaflets were observed between BTH-treated and control plants.     

The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth.

We isolated a dominant gain-of-function Arabidopsis mutant, accelerated cell death 6 (acd6), with elevated defenses, patches of dead and enlarged cells, reduced stature, and increased resistance to Pseudomonas syringae. The acd6-conferred phenotypes are suppressed by removing a key signaling molecule, salicylic acid (SA), by using the nahG transgene, which encodes SA hydroxylase. This suppression includes phenotypes that are not induced by application of SA to wild-type plants, indicating that SA acts with a second signal to cause many acd6-conferred phenotypes. acd6-nahG plants show hyperactivation of all acd6-conferred phenotypes after treatment with a synthetic inducer of the SA pathway, benzo(1,2, 3)thiadiazole-7-carbothioic acid (BTH), suggesting that SA acts with and also modulates the levels and/or activity of the second defense signal. acd6 acts partially through a NONEXPRESSOR OF PR 1 (NPR1) gene-independent pathway that activates defenses and confers resistance to P. syringae. Surprisingly, BTH-treated acd6-nahG plants develop many tumor-like abnormal growths, indicating a possible role for SA in modulating cell growth.     

外源Ca2+对水杨酸诱导番茄抗灰霉病的增效机制

探讨了外源Ca²⁺对水杨酸(SA)诱导番茄抗灰霉病的增效机制.以番茄灰霉病敏感型品种‘L402’幼苗为材料,分别进行H₂O(对照)、SA、SA+Ca和SA+EGTA(Ca²⁺螯合剂)处理,期间(1～5 d)分析各处理植株叶片活性氧(ROS)含量,苯丙氨酸解氨酶、几丁质酶和β-1,3-葡聚糖酶活性,以及病程相关蛋白编码基因PR1、PR2和PR3表达水平的变化,并调查处理3 d后灰霉病情指数.结果表明： 与对照(病情指数为74.8)相比,SA、SA+Ca和SA+EGTA处理的植株叶片灰霉病的病情指数分别为46.9、38.5和70.3;SA处理明显提高叶片ROS含量以及苯丙氨酸解氨酶、几丁质酶和β-1,3-葡聚糖酶活性,这些参数在SA+Ca处理的植株中被进一步提高,但在SA+EGTA处理的植株中则被降低;SA处理明显提高了PR1、PR2a和PR3b的表达水平,Ca²⁺进一步加强了这一效果,而EGTA则起抑制作用.SA或SA+Ca处理期间的PR2b和PR3a表达较未处理的对照上调了1～2倍,而PR1、PR2a和PR3b上调了2～5倍.表明Ca²⁺对SA诱导番茄抗灰霉病具有增效作用,其机理至少与Ca²⁺和SA协同作用促进ROS形成有关,而ROS作为信号分子增加植株抗病相关酶活性以及PR1、PR2a和PR3b等防卫基因的表达.     

外源Ca2+对水杨酸诱导番茄抗灰霉病的增效机制

探讨了外源Ca²⁺对水杨酸(SA)诱导番茄抗灰霉病的增效机制.以番茄灰霉病敏感型品种‘L402’幼苗为材料,分别进行H₂O(对照)、SA、SA+Ca和SA+EGTA(Ca²⁺螯合剂)处理,期间(1～5 d)分析各处理植株叶片活性氧(ROS)含量,苯丙氨酸解氨酶、几丁质酶和β-1,3-葡聚糖酶活性,以及病程相关蛋白编码基因PR1、PR2和PR3表达水平的变化,并调查处理3 d后灰霉病情指数.结果表明： 与对照(病情指数为74.8)相比,SA、SA+Ca和SA+EGTA处理的植株叶片灰霉病的病情指数分别为46.9、38.5和70.3;SA处理明显提高叶片ROS含量以及苯丙氨酸解氨酶、几丁质酶和β-1,3-葡聚糖酶活性,这些参数在SA+Ca处理的植株中被进一步提高,但在SA+EGTA处理的植株中则被降低;SA处理明显提高了PR1、PR2a和PR3b的表达水平,Ca²⁺进一步加强了这一效果,而EGTA则起抑制作用.SA或SA+Ca处理期间的PR2b和PR3a表达较未处理的对照上调了1～2倍,而PR1、PR2a和PR3b上调了2～5倍.表明Ca²⁺对SA诱导番茄抗灰霉病具有增效作用,其机理至少与Ca²⁺和SA协同作用促进ROS形成有关,而ROS作为信号分子增加植株抗病相关酶活性以及PR1、PR2a和PR3b等防卫基因的表达.     

Abscisic acid deficiency leads to rapid activation of tomato defence responses upon infection with Erwinia chrysanthemi

In addition to the important role of abscisic acid (ABA) in abiotic stress signalling, basal and high ABA levels appear to have a negative effect on disease resistance. Using the ABA-deficient sitiens tomato ( Solanum lycopersicum ) mutant and different application methods of exogenous ABA, we demonstrated the influence of this plant hormone on disease progression of Erwinia chrysanthemi . This necrotrophic plant pathogenic bacterium is responsible for soft rot disease on many plant species, causing maceration symptoms mainly due to the production and secretion of pectinolytic enzymes. On wild-type (WT) tomato cv. Moneymaker E. chrysanthemi leaf inoculation resulted in maceration both within and beyond the infiltrated zone of the leaf, but sitiens showed a very low occurrence of tissue maceration, which never extended the infiltrated zone. A single ABA treatment prior to infection eliminated the effect of pathogen restriction in sitiens , while repeated ABA spraying during plant development rendered both WT and sitiens very susceptible. Quantification of E. chrysanthemi populations inside the leaf did not reveal differences in bacterial growth between sitiens and WT. Sitiens was not more resistant to pectinolytic cell-wall degradation, but upon infection it showed a faster and stronger activation of defence responses than WT, such as hydrogen peroxide accumulation, peroxidase activation and cell-wall fortifications. Moreover, the rapid activation of sitiens peroxidases was also observed after application of bacteria-free culture filtrate containing E. chrysanthemi cell-wall-degrading enzymes and was absent during infection with an out E. chrysanthemi mutant impaired in secretion of these extracellular enzymes.     

Salicylic acid-mediated cell death in the Arabidopsis len3 mutant

The Arabidopsis lesion initiation 3 (len3) mutant develops lesions on leaves without pathogen attack. len3 plants exhibit stunted growth, constitutively express pathogenesis-related (PR) genes, PR-1, PR-2, and PR-5, and accumulate elevated levels of salicylic acid (SA). Furthermore, len3 is a semidominant, male gametophytic lethal mutation with partial defects in female gametophytic development. To determine the signaling pathway activated in len3 plants, we crossed the len3 plants with nahG, npr1-1, and pad4-1 plants and analyzed the phenotypes of the double mutants. The len3-conferred phenotypes, including cell death and PR-1 expressions, were suppressed in the double mutants. Thus SA, NPR1, and PAD4 are required for the phenotypes. However, none of these double mutants could completely suppress the len3-conferred stunted growth. This result suggests that an SA-, NPR1-, and PAD4-independent pathway is also involved in the phenotype. Treatment with BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid), an SA analog, induced cell death in len3 nahG plants but not in len3 npr1 or len3 pad4 plants, suggesting the involvement of the PAD4-dependent but SA-independent second signal pathway in cell death in len3 plants.