The GH3 acyl adenylase family member PBS3 regulates salicylic acid-dependent defense responses in Arabidopsis

The pbs3-1 mutant, identified in a screen for Arabidopsis (Arabidopsis thaliana) mutants exhibiting enhanced susceptibility to the avirulent Pseudomonas syringae pathogen DC3000 (avrPphB), also exhibits enhanced susceptibility to virulent P. syringae strains, suggesting it may impact basal disease resistance. Because induced salicylic acid (SA) is a critical mediator of basal resistance responses, free and glucose-conjugated SA levels were measured and expression of the SA-dependent pathogenesis-related (PR) marker, PR1, was assessed. Surprisingly, whereas accumulation of the SA glucoside and expression of PR1 were dramatically reduced in the pbs3-1 mutant in response to P. syringae (avrRpt2) infection, free SA was elevated. However, in response to exogenous SA, the conversion of free SA to SA glucoside and the induced expression of PR1 were similar in pbs3-1 and wild-type plants. Through positional cloning, complementation, and sequencing, we determined that the pbs3-1 mutant contains two point mutations in the C-terminal region of the protein encoded by At5g13320, resulting in nonconserved amino acid changes in highly conserved residues. Additional analyses with Arabidopsis containing T-DNA insertion (pbs3-2) and transposon insertion (pbs3-3) mutations in At5g13320 confirmed our findings with pbs3-1. PBS3 (also referred to as GH3.12) is a member of the GH3 family of acyl-adenylate/thioester-forming enzymes. Characterized GH3 family members, such as JAR1, act as phytohormone-amino acid synthetases. Thus, our results suggest that amino acid conjugation plays a critical role in SA metabolism and induced defense responses, with PBS3 acting upstream of SA, directly on SA, or on a competitive inhibitor of SA.     

Induction of tomato stress protein mRNAs by ethephon, 2,6-dichloroisonicotinic acid and salicylate

To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, we investigated the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid (INA) and salicylic acid (SA) by northern blot analysis. Ethephon slightly induced some but not all mRNAs encoding intra- and extracellular PR proteins. INA induced all PR protein mRNAs analysed, except for intracellular chitinase and extracellular PR-4. SA induced all PR protein mRNAs analyzed, except for intracellular chitinase and osmotin. None of the inducers affected the expression of ACC synthase mRNA, whereas all three induced ethylene-forming enzyme (EFE) mRNA.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - HR hypersensitive response - INA 2,6-dichloroisonicotinic acid - PR pathogenesis-related - SA salicylic acid - SAR systemic acquired resistance     

Proteomic analysis of salicylic acid-induced resistance to Magnaporthe oryzae in susceptible and resistant rice

To probe salicylic acid (SA)-induced sequential events at translational level and factors associated with SA response, we conducted virulence assays and proteomic profiling analysis on rice resistant and susceptible cultivars against Magnaporthe oryzae at various time points after SA treatment. The results showed that SA significantly enhanced rice resistance against M. oryzae. Proteomic analysis of SA-treated leaves unveiled 36 differentially expressed proteins implicated in various functions, including defense, antioxidative enzymes, and signal transduction. Majority of these proteins were induced except three antioxidative enzymes, which were negatively regulated by SA. Consistent with the above findings, SA increased the level of reactive oxygen species (ROS) with resistant cultivar C101LAC showing faster response to SA and producing higher level of ROS than susceptible cultivar CO39. Furthermore, we showed that nucleoside diphosphate kinase 1, which is implicated in regulation of ROS production, was strongly induced in C101LAC but not in CO39. Taken together, the findings suggest that resistant rice cultivar might possess a more sensitive SA signaling system or effective pathway than susceptible cultivar. In addition, our results indicate that SA also coordinates other cellular activities such as photosynthesis and metabolism to facilitate defense response and recovery, highlighting the complexity of SA-induced resistance mechanisms.     

Effects of a biocontrol agent and methyl jasmonate on postharvest diseases of peach fruit and the possible mechanisms involved

AIMS: To investigate effects of application of 200 micromol l(-1) methyl jasmonate [MeJA (200)] and Cryptococcus laurentii alone or in combination against postharvest diseases (Monilinia fructicola and Penicillium expansum) in peach fruit stored at 25 and 0 degrees C, and to evaluate the possible mechanisms involved. METHODS AND RESULTS: The efficacy of controlling postharvest diseases by resistance induced in peach fruit treated with MeJA (200) and C. laurentii alone or in combination and the relationship between activities of defence-related enzymes in peach fruit and lesions caused by M. fructicola and P. expansum were examined. At the same time, the effects of MeJA (200) on the population of C. laurentii in the peach wounds and on the mycelial growth of M. fructicola and P. expansumin vitro were investigated. The results indicated that treatment of peach fruit with C. laurentii at 1 x 10(8) CFU ml(-1) alone, or combining C. laurentii at 5 x 10(7) CFU ml(-1) with MeJA (200) all resulted in a lower lesion diameter of brown rot and blue mould caused by M. fructicola and P. expansum compared with the controls in peach fruit. MeJA (200) enhanced the population of C. laurentii, and inhibited mycelial growth of P. expansum. However, it had a little effect on M. fructicolain vitro. MeJA and C. laurentii alone or in combination induced higher activities of Chitinase, beta-1,3-glucanase, phenylalanine ammonia-lyase and peroxidase (POD) than applying the yeast alone at both 25 and 0 degrees C. CONCLUSIONS: MeJA (200) not only directly inhibited mycelial spread of postharvest pathogens, but also increased population of C. laurentii, which induced stronger disease resistance in fruit than MeJA or yeast alone, and resulted in a lower lesion diameter of brown rot and blue mould caused by M. fructicola and P. expansum. SIGNIFICANCE AND IMPACT OF THE STUDY: MeJA (200) in combination with C. laurentii was beneficial for controlling brown rot and blue mould caused by M. fructicola and P. expansum in peach fruit. The inhibitory mechanism was mainly because of resistance induced in peach fruit by MeJA and C. laurentii. In addition, direct inhibition of MeJA on P. expansum also played a role in controlling blue mould.     

Benzothiadiazole affects the leaf proteome in arctic bramble (Rubus arcticus)

Benzothiadiazole (BTH) induces resistance to the downy mildew pathogen, Peronospora sparsa, in arctic bramble, but the basis for the BTH‐induced resistance is unknown. Arctic bramble cv. Mespi was treated with BTH to study the changes in leaf proteome and to identify proteins with a putative role in disease resistance. First, BTH induced strong expression of one PR‐1 protein isoform, which was also induced by salicylic acid (SA). The PR‐1 was responsive to BTH and exogenous SA despite a high endogenous SA content (20–25 µg/g fresh weight), which increased to an even higher level after treatment with BTH. Secondly, a total of 792 protein spots were detected in two‐dimensional gel electrophoresis, eight proteins being detected solely in the BTH‐treated plants. BTH caused up‐ or down‐regulation of 72 and 31 proteins, respectively, of which 18 were tentatively identified by mass spectrometry. The up‐regulation of flavanone‐3‐hydroxylase, alanine aminotransferase, 1‐aminocyclopropane‐1‐carboxylate oxidase, PR‐1 and PR‐10 proteins may partly explain the BTH‐induced resistance against P. sparsa. Other proteins with changes in intensity appear to be involved in, for example, energy metabolism and protein processing. The decline in ATP synthase, triosephosphate isomerase, fructose bisphosphate aldolase and glutamine synthetase suggests that BTH causes significant changes in primary metabolism, which provides one possible explanation for the decreased vegetative growth of foliage and rhizome observed in BTH‐treated plants.     

LSD1 regulates salicylic acid induction of copper zinc superoxide dismutase in Arabidopsis thaliana.

We characterized the accumulation patterns of Arabidopsis thaliana proteins, two CuZnSODs, FeSOD, MnSOD, PR1, PR5, and GST1, in response to various pathogen-associated treatments. These treatments included inoculation with virulent and avirulent Pseudomonas syringae strains, spontaneous lesion formation in the lsd1 mutant, and treatment with the salicylic acid (SA) analogs INA (2,6-dichloroisonicotinic acid) and BTH (benzothiadiazole). The PR1, PR5, and GST1 proteins were inducible by all treatments tested, as expected from previous mRNA blot analysis. The two CuZnSOD proteins were induced by SA analogs and in conjunction with lsd1-mediated spreading cell death. Additionally, LSD1 is a part of a signaling pathway for the induction of the CuZnSOD proteins in response to SA but not in lsd1-mediated cell death. We suggest that the spreading lesion phenotype of lsd1 results from a lack of up-regulation of a CuZnSOD responsible for detoxification of accumulating superoxide before the reactive oxygen species can trigger a cell death cascade.     

Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance

After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in triggering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants.     

Modifications of antioxidant activity and protein composition of bean leaf due to <Emphasis Type="Italic">Bean yellow mosaic virus</Emphasis> infection and salicylic acid treatments

The antioxidant status as well as protein composition of faba bean leaves infected with Bean yellow mosaic virus (BYMV) and the effect of salicylic acid application was examined in this paper. Some modifications in the antioxidant status were observed by changing some antioxidant enzymes activities and contents of antioxidant metabolites. BYMV-infected leaves revealed POD, CAT, APX and SOD induced activities while SA treatments could inhibit POD, CAT activities but induced SOD activity. The enzyme activities seemed to be SA concentration dependant. Higher H₂O₂ and MDA concentrations were recorded in virus-infected leaves than those of the corresponding controls while treatment with SA followed by virus inoculation caused lowering of MDA concentration and reducing the damage due to lipid peroxidation. Moreover, because of virus infection and/or SA treatments, an increase in the amounts of phenolics and flavonoids was noticed. As compared to the control, BYMV infection or SA application caused pronounced increase in the antioxidant activity of leaf extracts detected by DPPH assay, indicating an increase in the amounts of antioxidant compounds occurred. To test the protein composition, the contents of each protein fractions (soluble, insoluble and total) were analyzed and the change in protein patterns was visualized using SDS-PAGE. The BYMV-infected bean leaves had protein contents higher than the control indicating accumulation of pathogenesis-related proteins. Moreover, spraying SA with or without virus inoculation could accumulate soluble, insoluble and total proteins and the pattern of increase was in accordance with SA concentration. Alterations in protein patterns were observed in faba bean leaves (Vicia faba cv Giza 461) in response to BYMV infection and SA treatments. Because of BYMV infection and SA treatments, the protein profiles showed new bands in comparison to the control. Some polypeptides were highly accumulated in treatments of SA followed by virus inoculation. Changing antioxidant status and accumulation of some antioxidant metabolites as well as the pronounced alterations in the protein composition indicate a kind of plant response against pathogen invasion and in case of SA treatment it is considered a way by which a defence response was initiated and/or activated.     

Salicylic acid and the plant pathogen Erwinia carotovora induce defense genes via antagonistic pathways

Infection of tobacco plants with the plant pathogenic bacterium Erwinia carotovora subsp. carotovora or treatment of plants with Erwinia -derived elicitor preparations leads to the induction of a number of genes thought to play a role in plant defense response to pathogens. In order to determine the role of salicylic acid (SA) in the induction of the Erwinia responsive genes, the accumulation of mRNAs for these and other genes encoding pathogenesis-related proteins (PR genes) in response to both Erwinia elicitors and SA was determined. PR genes were identified which were preferentially induced by Erwinia elicitor preparations, one gene was induced by SA but not by Erwinia , and another gene was induced by both type of treatments. The differential expression of these genes and the timing of induction suggest that SA is not the signal molecule leading to the early response of plants to Erwinia . This was demonstrated by experiments using transgenic NahG plants that overproduce a salicylate hydroxylase inactivating SA. The elicitation of PR genes by Erwinia was similar in NahG and wild-type plants. Therefore, induction of plant defense genes by Erwinia and SA seems to be by two distinct pathways leading to expression of separate sets of genes. Furthermore, we could demonstrate that Erwinia elicitors antagonize the SA-mediated induction of PR genes. Similarly, SA appeared to inhibit the induction of PR genes elicited by Erwinia . The observed antagonism between the two signal transduction pathways indicates the presence of a common regulatory element in both pathways that acts downstream of SA in the SA-mediated response.     

Response of Jujube Fruits to Exogenous Oxalic Acid Treatment Based on Proteomic Analysis

In this study, we found that oxalic acid (OA) at the concentrationof 5 mM could delay jujube fruit sene-scence by reducing ethyleneproduction, repressing fruit reddening and reducing alcoholcontent, which consequently increased fruit resistance againstblue mold caused by Penicillium expansum. In order to gain afurther understanding of the mechanism by which OA delays senescenceand increases disease resistance of jujube fruit, we used aproteomics approach to compare soluble proteome of jujube fruitstreated with water or 5 mM OA for 10 min. A total of 25 differentiallyexpressed proteins were identified by using electrospray ionizationquadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS).Among these proteins, alcohol dehydrogenase 1, which plays adirect role in ethanol metabolism, was repressed, and the abundancesof three photosynthesis-related proteins was enhanced in jujubefruit after OA treatment. The protein identified as a cystathionineβ-synthase domain-containing protein, which can regulateethylene precursors, was also induced by OA treatment. The activityof 1-aminocyclopropane-1-carboxylic acid synthase was significantlysuppressed in OA-treated jujube fruit. In addition, three proteinsrelated to the defense/stress response were up-regulated byOA, and contributed to the establishment of systemic resistanceinduced by OA in jujube fruits. These results indicated thatOA treatment might affect ethanol and ethylene metabolism, resultingin delaying senescence, and increase resistance of jujube fruitsagainst fungal pathogens.     

水杨酸诱导果实采后抗病性机制研究进展

水杨酸(Salicylic acid, SA)是一种重要的内源信号分子,具有激活果实抗性防卫反应的能力。本文综述SA对多种果实采后病害的抑制效果及其诱导果实采后抗病性机制(如诱导果实抗氧化反应、诱导果实防御反应、调控果实呼吸代谢、诱导果实抗病基因表达和抗性相关蛋白表达),并展望该领域SA未来研究趋势与方向,对推动果实采后抗病理论的发展和指导生产实践具有重要意义。     

Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco

Salicylic acid (SA) has been shown to act as a signal molecule that is produced by many plants subsequent to the recognition of potentially pathogenic microbes. Increases in levels of SA often trigger the activation of plant defenses and can result in increased resistance to subsequent challenge by pathogens. We observed that the polyketide 6-methylsalicylic acid (6-MeSA), a compound that apparently is not endogenous to tobacco, can mimic SA. Tobacco leaves treated with 6-MeSA show enhanced accumulation of the pathogenesis-related (PR) proteins PR1, beta-1,3-glucanase, and chitinase and also develop increased resistance to tobacco mosaic virus. We transformed tobacco with 6msas, the 6-methylsalicylic acid synthase (6MSAS) gene from Penicillium patulum, to generate plants that constitutively accumulate 6-MeSA. Analysis of primary transformants and the first generation progeny of 6MSAS tobacco revealed that plants can be engineered to accumulate significant amounts of 6-MeSA as a conjugate. Levels of total 6-MeSA increased with plant age. Increased 6-MeSA accumulation correlated with increased levels of PR1 and chitinase proteins and resulted in enhanced resistance of NN genotype 6MSAS tobacco to tobacco mosaic virus. Our results demonstrate that a multistep biosynthetic pathway can be engineered into plants using a single fungal polyketide synthase gene. The functional expression of 6msas can be used to activate disease resistance pathways that normally are induced by SA.     

Accumulation of Pathogenesis-Related Proteins in Tobacco Leaves Irradiated with UV-B

Nicotiana tabacum L. (cv. Petit Havana SR1) were grown under ultraviolet-B (UV-B, 290–320 nm) irradiation, and soluble proteins were extracted from the leaves. Two-dimensional electrophoresis revealed that a minimum of 12 polypeptides were induced by UV-B. Polypeptides which were so abundant as to be detectable by Coomassie brilliant blue staining were then subjected to N-terminal amino acid sequence analyses. Two of the polypeptides were identified as a 23 kDa protein of PS II and 6 as a pathogenesis-related protein 5 (PR-5). Immunoblotting demonstrated that other PR proteins, PR-1 and PR-3 were also induced by UV-B. Salicylic acid (SA), which is an important component of signal transduction that leads to the expression of PR proteins and exhibition of acquired resistance to pathogens, increased in response to exposure to UV-B. In addition, the activity of phenylalanine ammonialyase, which catalyzes the synthesis from phenylalanine of trans-cinnamic acid, the endogenous precursor of SA, was transiently increased by UV-B irradiation. These results suggest that UV-B activates the signal transduction pathway, which is a common step in pathogen infection. Received 8 May 2000/ Accepted in revised form 29 August 2000     

Induction of pathogen resistance and pathogenesis-related genes in tobacco by a heat-stable Trichoderma mycelial extract and plant signal messengers

Heat-stable mycelial extracts of the nonpathogenic fungus Trichoderma longibrachiatum induced resistance in tobacco seedlings ( Nicotiana tabacum L. cv. Wisconsin 38) to the pathogen Phytophthora parasitica var. nicotianae (race 0), which did not involve a hypersensitive response. Resistance could not be induced with mycelial extract prepared in the same manner from P. parasitica . The nonpathogenic mycelial extract induced expression of PR-1b and osmotin (PR-5) genes to a higher level than did mycelial extract from the pathogenic fungus. The tissue-specific pattern of PR gene induction by the nonpathogenic mycelial extract was different from that of the pathogenic mycelial extract and was consistent with the ability of the former to cause disease resistance. The expression patterns of these two PR genes and the accumulations of their encoded proteins also were affected by salicylic acid (SA), methyl jasmonate (MeJA), ethylene (E) and combinations of these plant signal messengers. However, only combined SA and MeJA treatment mimicked the pattern of PR gene mRNA and protein accumulation induced by the nonpathogenic mycelial extract. E inhibitors blocked both mycelial extract-induced and SA/MeJA-induced PR gene expression, and the cis pattern of responsiveness on the osmotin promoter was the same for the mycelial extract, SA, E, or E/MeJA. Seedlings treated with P. parasitica spores in the presence of SA/MeJA were protected from pathogen colonization. However, these seedlings exhibited symptoms of cell death (disease symptoms) both in the absence and presence of P. parasitica spores, in contrast to seedlings treated with nonpathogenic mycelial extract, which remained healthy. These results suggest that the signal transduction pathways for elicitation of defense responses by exogenously applied heat-stable nonpathogenic mycelial extract and SA/MeJA overlap at the point of PR protein induction but are not identical.     

Sweating treatment enhances citrus fruit disease resistance by inducing the accumulation of amino acids and salicylic acid‐induced resistance pathway

To clarify the mechanism of fruit disease resistance activated by sweating treatment, ‘Guoqing NO.1’ Satsuma mandarin (Citrus unshiu Marc.) fruits were treated by sweating, which is a traditional prestorage treatment in China. Subsequently, we performed inoculation and physiological characterization, two‐dimensional gel electrophoresis (2‐DE) proteomics analysis and metabonomics analysis based on gas chromatography coupled to mass spectrometry (GC‐MS) and high‐performance liquid chromatography/electrospray ionization‐time of flight‐mass spectrometry (HPLC‐qTOF‐MS). The results showed that sweating treatment significantly inhibited pathogen infection without negatively affecting the fruit commercial quality. In addition, sweating treatment rapidly promoted the accumulation of amino acids (such as proline and serine). Meanwhile, hydrogen peroxide (H₂O₂) and salicylic acid (SA) were significantly accumulated in the sweating‐treated fruit. Thereafter, some stress‐response proteins and metabolites [such as ascorbate peroxidase (APX), β‐1,3‐glucanase, vanillic acid and rutin] which can be induced by SA were also significantly increased in the sweating‐treated fruit. Taken together, the disease resistance induced by sweating treatment might be attributed to: (1) the induction of the accumulation of amino acids; and (2) the accumulation of SA and subsequent activation of SA‐induced resistance pathway, which can induce the stress‐response proteins and metabolites that can directly inhibit pathogen development.