Functions of pipecolic acid on induced resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in tomato plants

Amino acid metabolic pathways are involved in the plant immune system. Pipecolic acid (Pip), a lysine-derived non-protein amino acid, acts as an important regulator of disease resistance. Here, we report the functions of Pip on tomato disease resistance. Tomato seedlings treated with 0.5 mM Pip showed increased resistance to Pst DC3000 and B. cinerea compared with the control. After pathogen infection, the expression of defence-related genes increased in plants pretreated with Pip, while reactive oxygen species (ROS) accumulation decreased. These data demonstrated that exogenous application of Pip induced resistance against Pst DC3000 and B. cinerea in tomatoes, possibly through the regulation of ROS accumulation and defence-related gene expression.     

Hexanoic acid is a resistance inducer that protects tomato plants against Pseudomonas syringae by priming the jasmonic acid and salicylic acid pathways

Hexanoic acid-induced resistance (Hx-IR) is effective against several pathogens in tomato plants. Our study of the mechanisms implicated in Hx-IR against Pseudomonas syringae pv. tomato DC3000 suggests that hexanoic acid (Hx) treatment counteracts the negative effect of coronatine (COR) and jasmonyl-isoleucine (JA-Ile) on the salicylic acid (SA) pathway. In Hx-treated plants, an increase in the expression of jasmonic acid carboxyl methyltransferase (JMT) and the SA marker genes PR1 and PR5 indicates a boost in this signalling pathway at the expense of a decrease in JA-Ile. Moreover, Hx treatment potentiates 12-oxo-phytodienoic acid accumulation, which suggests that this molecule might play a role per se in Hx-IR. These results support a positive relationship between the SA and JA pathways in Hx-primed plants. Furthermore, one of the mechanisms of virulence mediated by COR is stomatal re-opening on infection with P. syringae. In this work, we observed that Hx seems to inhibit stomatal opening in planta in the presence of COR, which suggests that, on infection in tomato, this treatment suppresses effector action to prevent bacterial entry into the mesophyll.     

Characterization of a new, nonpathogenic mutant of Botrytis cinerea with impaired plant colonization capacity

Botrytis cinerea is a necrotrophic pathogen that attacks more than 200 plant species. Here, the nonpathogenic mutant A336, obtained via insertional mutagenesis, was characterized. Mutant A336 was nonpathogenic on leaves and fruits, on intact and wounded tissue, while still able to penetrate the host plant. It grew normally in vitro on rich media but its conidiation pattern was altered. The mutant did not produce oxalic acid and exhibited a modified regulation of the production of some secreted proteins (acid protease 1 and endopolygalacturonase 1). Culture filtrates of the mutant triggered an important oxidative burst in grapevine (Vitis vinifera) suspension cells, and the mutant-plant interaction resulted in the formation of hypersensitive response-like necrosis. Genetic segregation analyses revealed that the pathogenicity phenotype was linked to a single locus, but showed that the mutated gene was not tagged by the plasmid pAN7-1. Mutant A336 is the first oxalate-deficient mutant to be described in B. cinerea and it differs from all the nonpathogenic B. cinerea mutants described to date.     

Overexpressing the N-terminus of CATALASE2 enhances plant jasmonic acid biosynthesis and resistance to necrotrophic pathogen Botrytis cinerea B05.10

Salicylic acid (SA) acts antagonistically to jasmonic acid (JA) in plant immunity. We previously reported that CATALASE2 (CAT2) promotes JA-biosynthetic acyl-CoA oxidase (ACX) activity to enhance plant resistance to necrotrophic Botrytis cinerea, and SA represses JA biosynthesis through inhibiting CAT2 activity, while the underlying mechanism remains to be further elucidated. Here, we report that the truncated CAT2 N-terminus (CAT2-N) interacts with and promotes ACX2/3, and CAT2-N-overexpressing plants have increased JA accumulation and enhanced resistance to B. cinerea B05.10, but compromised antagonism of SA on JA. Catalase inhibitor treatment or mutating CAT2 active amino acids abolished CAT2 H₂O₂-decomposing activity but did not affect its promotion of ACX2/3 activity via interaction. CAT2-N, a truncated protein with no catalase activity, interacted with and promoted ACX2/3. Overexpressing CAT2-N in Arabidopsis plants resulted in increased ACX activity, higher JA accumulation, and stronger resistance to B. cinerea B05.10 infection. Additionally, SA dramatically repressed JA biosynthesis and resistance to B. cinerea in the wild type but not in the CAT2-N-overexpressing plants. Together, our study reveals that CAT2-N can be utilized as an accelerator for JA biosynthesis during plant resistance to B. cinerea B05.10, and this truncated protein partly relieves SA repression of JA biosynthesis in plant defence responses.     

The response regulator BcSkn7 is required for vegetative differentiation and adaptation to oxidative and osmotic stresses in Botrytis cinerea

The high‐osmolarity glycerol pathway plays an important role in the responses of fungi to various environmental stresses. Saccharomyces cerevisiae Skn7 is a response regulator in the high‐osmolarity glycerol pathway, which regulates the oxidative stress response, cell cycle and cell wall biosynthesis. In this study, we characterized an Skn7 orthologue BcSkn7 in Botrytis cinerea. BcSKN7 can partly restore the growth defects of S. cerevisiae SKN7 mutant and vice versa. The BcSKN7 mutant (ΔBcSkn7‐1) revealed increased sensitivity to ionic osmotic and oxidative stresses and to ergosterol biosynthesis inhibitors. In addition, ΔBcSkn7‐1 was also impaired dramatically in conidiation and sclerotial formation. Western blot analysis showed that BcSkn7 positively regulated the phosphorylation of BcSak1 (the orthologue of S. cerevisiae Hog1) under osmotic stress, indicating that BcSkn7 is associated with the high‐osmolarity glycerol pathway in B. cinerea. In contrast with BcSak1, BcSkn7 is not involved in the regulation of B. cinerea virulence. All of the phenotypic defects of ΔBcSkn7‐1 are restored by genetic complementation of the mutant with the wild‐type BcSKN7. The results of this study indicate that BcSkn7 plays an important role in the regulation of vegetative differentiation and in the response to various stresses in B. cinerea.     

Suppression of the Defence-Related Oxidative Burst in Bean Leaf Tissue and Bean Suspension Cells by the Necrotrophic Pathogen Botrytis cinerea

The interaction of two selected isolates of Botrytis cinerea with bean suspension cells and bean leaf discs was compared in relation to levels of reactive oxygen intermediates (ROI). Isolate B 1.7 was arrested by a hypersensitive‐like necrosis of bean leaf tissue. According to its inability to spread and produce conidia on the bean leaf tissue it was classified as non‐aggressive. The second isolate induced a fast expanding light brownish necrosis of the leaf tissue. It was able to produce conidia on bean leaf discs and was classified as aggressive. The generation of superoxide was followed biochemically in inoculated bean cell suspensions. Both isolates induced a similar early superoxide peak approximately 18‐h post inoculation (hpi). While the non‐aggressive isolate induced a much stronger secondary superoxide burst at 33 hpi, the level of superoxide of suspension cells inoculated with the aggressive isolate was below the control level. This is the first report on the occurrence of a biphasic oxidative burst in plant cells induced by a fungal pathogen. Such a suppression of superoxide generation was also observed in bean leaf discs inoculated with the aggressive isolate. An oxidative burst‐suppressing agent was extracted from inoculated cell culture medium and determined as 2‐methyl‐succinate (2‐MS) by GC/MS analysis. The compound was detected approximately 20 hpi in the aggressive fungus–plant interaction. 2‐MS was able to suppress the hypersensitive response‐like necrosis on leaf discs as well as the second superoxide burst in suspension cells when inoculated with the non‐aggressive isolate. The early superoxide burst at 18 hpi was not affected. The results confirm the important role of enhanced production of ROI in plant resistance reactions, also for a necrotrophlike B. cinerea.     

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.     

Priming for enhanced defence responses by specific inhibition of the Arabidopsis response to coronatine

The priming agent β-aminobutyric acid (BABA) is known to enhance Arabidopsis resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 by potentiating salicylic acid (SA) defence signalling, notably PR1 expression. The molecular mechanisms underlying this phenomenon remain unknown. A genome-wide microarray analysis of BABA priming during Pst DC3000 infection revealed direct and primed up-regulation of genes that are responsive to SA, the SA analogue benzothiadiazole and pathogens. In addition, BABA was found to inhibit the Arabidopsis response to the bacterial effector coronatine (COR). COR is known to promote bacterial virulence by inducing the jasmonic acid (JA) response to antagonize SA signalling activation. BABA specifically repressed the JA response induced by COR without affecting other plant JA responses. This repression was largely SA-independent, suggesting that it is not caused by negative cross-talk between SA and JA signalling cascades. Treatment with relatively high concentrations of purified COR counteracted BABA inhibition. Under these conditions, BABA failed to protect Arabidopsis against Pst DC3000. BABA did not induce priming and resistance in plants inoculated with a COR-deficient strain of Pst DC3000 or in the COR-insensitive mutant coi1-16. In addition, BABA blocked the COR-dependent re-opening of stomata during Pst DC3000 infection. Our data suggest that BABA primes for enhanced resistance to Pst DC3000 by interfering with the bacterial suppression of Arabidopsis SA-dependent defences. This study also suggests the existence of a signalling node that distinguishes COR from other JA responses.     

Markers for oxidative stress associated with soft rots in French beans (Phaseolus vulgaris) infected by Botrytis cinerea

The role of active oxygen species has been studied in spreading soft-rot lesions caused by the necrotrophic fungal pathogen Botrytis cinerea Pers.:Fr. in leaves of four genotypes of French bean (Phaseolus vulgaris L.). Large increases were observed for the aldehydic end-products of oxidative damage, malondialdehyde and 4-hydroxy-2-nonenal, as a result of infection in each of the genotypes studied. Similar increases were found in a stable free radical and g=4.27 Fe(III) signals, but not Mn(II) signals, in electron paramagnetic resonance spectra. These changes were accompanied by large decreases in ascorbic acid levels, with changes in the antioxidant glutathione being genotype dependent. Received: 3 May 2000 / Accepted: 13 July 2000     

Melatonin protects against taurolithocholic‐induced oxidative stress in rat liver

Cholestasis, encountered in a variety of clinical disorders, is characterized by intracellular accumulation of toxic bile acids in the liver. Furthermore, oxidative stress plays an important role in the pathogenesis of bile acids. Taurolithocholic acid (TLC) was revealed in previous studies as the most pro‐oxidative bile acid. Melatonin, a well‐known antioxidant, is a safe and widely used therapeutic agent. Herein, we investigated the hepatoprotective role of melatonin on lipid and protein oxidation induced by TLC alone and in combination with FeCl₃ and ascorbic acid in rat liver homogenates and hepatic membranes. The lipid peroxidation products, malondialdehyde and 4‐hydroxyalkenals (MDA + 4‐HDA), and carbonyl levels were quantified as indices of oxidative damage to hepatic lipids and proteins, respectively. In the current study, the rise in MDA + 4‐HDA levels induced by TLC was inhibited by melatonin in a concentration‐dependent manner in both liver homogenates and in hepatic membranes. Melatonin also had protective effects against structural damage to proteins induced by TLC in membranes. These results suggest that the indoleamine melatonin may potentially act as a protective agent in the therapy of those diseases that involve bile acid toxicity. J. Cell. Biochem. 110: 1219–1225, 2010. Published 2010 Wiley‐Liss, Inc.     

Release of lipoxygenase products and monoterpenes by tomato plants as an indicator of Botrytis cinerea-induced stress

Changes in emission of volatile organic compounds (VOCs) from tomato induced by the fungus Botrytis cinerea were studied in plants inoculated by spraying with suspensions containing B. cinerea spores. VOC emissions were analysed using on-line gas chromatography–mass spectrometry, with a time resolution of about 1 h, for up to 2 days after spraying. Four phases were delimited according to the starting point and the applied day/night rhythm of the experiments. These phases were used to demonstrate changes in VOC flux caused by B. cinerea infestation. Tomato plants inoculated with B. cinerea emitted a different number and amount of VOCs after inoculation compared to control plants that had been sprayed with a suspension without B. cinerea spores. The changes in emissions were dependent on time after inoculation as well as on the severity of infection. The predominant VOCs emitted after inoculation were volatile products from the lipoxygenase pathway (LOX products). The increased emission of LOX products proved to be a strong indicator of a stress response, indicating that VOC emissions can be used to detect plant stress at an early stage. Besides emission of LOX products, there were also increases in monoterpene emissions. However, neither increased emission of LOX products nor of monoterpenes is specific for B. cinerea attack. The emission of LOX products is also induced by other stresses, and increased emission of monoterpenes seems to be the result of mechanical damage induced by secondary stress impacts on leaves.