Salicylic Acid and Disease Resistance in Plants

SA has been shown to play an important signaling role in the activation of various plant defense responses following pathogen attack. These responses include the induction of local and systemic disease resistance, the potentiation of host cell death, and the containment of pathogen spread. The mechanisms through which SA mediates these effects are varied and can involve alterations in the activity or synthesis of certain enzymes, increased defense gene expression, potentiation of several defense responses, and/or the generation of free radicals. Through the analysis of mutant plants exhibiting aberrant responses to pathogen infection, many genes encoding products involved in the SA-mediated defense pathway(s) have been isolated. In addition, mounting evidence suggests that certain defense responses can be activated via a SA-independent pathway(s). This review focuses primarily on recent discoveries pertaining to the SA signaling pathway(s) leading to disease resistance; however, a very brief discussion of the SA-independent pathway (s) and its ability to cross-talk with the SA pathway is also presented.     

Involvement of Reactive Oxygen Species, Glutathione Metabolism, and Lipid Peroxidation in the Cf-Gene-Dependent Defense Response of Tomato Cotyledons Induced by Race-Specific Elicitors of Cladosporium fulvum

The chronological order of responses to Cladosporium fulvum (Cooke) (Cf) race-specific elicitors was assessed in cotyledons of three near-isogenic tomato (Lycopersicon esculentum Mill.) lines carrying either Cf-9 or Cf-2 or no Cf gene. The responses observed were dependent on the presence of a Cf gene, Avr-gene product dose injected, and the relative humidity (RH) of the growth chamber. At ambient RH, superoxide formation and lipid peroxidation occurred after 2 h (Cf9) and 4 h (Cf2). At elevated RH (98%) and at lower avirulence elicitor dose, Cf-Avr-dependent lipid peroxidation was considerably attenuated. Significant electrolyte leakage occurred by 18 h but only at the lower RH. Total glutathione levels began to increase 2 to 4 h and 4 to 8 h after challenge of Cf9 and Cf2 cells, respectively, and by 48 h reached 665 and 570% of initial levels. A large proportion of this accumulation (87%) was as oxidized glutathione. When the RH was increased to 98%, increases in glutathione levels were strongly attenuated. Increased lipoxygenase enzyme activity was detected 8 h postchallenge in either incompatible interaction. These results indicate that the activation of the Cf-Avr-mediated defense response results in severe oxidative stress.     

Overexpression of the Tomato 13-Lipoxygenase Gene TomloxD Increases Generation of Endogenous Jasmonic Acid and Resistance to Cladosporium fulvum and High Temperature

Expression of the tomato gene encoding 13-lipoxygenase,TomloxD, is stimulated by wounding, pathogen infection, jasmonate, and systemin, but its role during growth and development of tomato (Lycopersicon Spp.) remains unclear. To assess the physiological role of TomloxD, we produced transgenic tomato plants with greatly increased TomloxD content using sense constructs under the control of the CaMV 35S promoter. Overexpression of TomloxD in transgenic tomatoes led to a marked increase in the levels of lipoxygenase activity and content of endogenous jasmonic acid (JA), which suggested that TomloxD can use α-linolenic acid as a substrate to produce (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT); the 13-HPOT produced appears to be metabolized further to synthesize JA. Real-time RT-PCR revealed that the expression levels of defense genes LeHSP90, LePR1, LePR6 and LeZAT in the transformants were higher than those in non-transformed plants. Assay for resistance to pathogenic fungus and high temperature stresses suggested that transgenic plants harboring TomloxD were more tolerant to Cladosporium fulvum and high temperature stress than non-transformed tomato plants. The data presented here indicate clearly that TomloxD is involved in endogenous JA synthesis and tolerance to biotic and abiotic stress. The tomloxD gene has potential applications in engineering cropping plants that are resistant to biotic and/or abiotic stress factors.     

转番茄几丁质酶基因西瓜植株的获得及其抗病性研究

构建了以CaMV35s启动子驱动的含有番茄几丁质酶基因（Chi3）的植物表达载体,通过冻融法导入根癌农杆菌（Agrobacterium tumefaciens）菌株EHAl05中。用叶盘法转化西瓜（Citrullus lanatus）栽培种“中育1号”,通过PCR、Southern blot和RT-PCR鉴定,表明外源基因已成功整合到西瓜基闪组中,并获得表达。利用尖孢镰刀菌西瓜专化型进行的转基因植株叶片粗提液抑菌效果检测,表明转基因植株的抗病性均有一定程度的增强。     

植物抗病基因克隆的进展

抗病基因是抗病分子生物学和抗病育种的基因,本文介绍了近几年植物抗病基因克隆的进展,抗病基因克隆的新策略以及抗病基因结构特点与抗病机制的研究进展,讨论了抗病基因克隆的应用前景。     

A High-Affinity Binding Site for the AVR9 Peptide Elicitor of Cladosporium fulvum Is Present on Plasma Membranes of Tomato and Other Solanaceous Plants

The race-specific Cladosporium fulvum peptide elicitor AVR9, which specifically induces a hypersensitive response in tomato genotypes carrying the Cf-9 resistance gene, was labeled with iodine-125 at the N-terminal tyrosine residue and used in binding studies. 125I-AVR9 showed specific, saturable, and reversible binding to plasma membranes isolated from leaves of tomato cultivar Moneymaker without Cf resistance genes (MM-Cf0) or from a near-isogenic genotype with the Cf-9 resistance gene (MM-Cf9). The dissociation constant was found to be 0.07 nM, and the receptor concentration was 0.8 pmol/mg microsomal protein. Binding was highly influenced by pH and the ionic strength of the binding buffer and by temperature, indicating the involvement of both electrostatic and hydrophobic interactions. Binding kinetics and binding capacity were similar for membranes of the MM-Cf0 and MM-Cf9 genotypes. In all solanaceous plant species tested, an AVR9 binding site was present, whereas in the nonsolanaceous species that were analyzed, such a binding site could not be identified. The ability of membranes isolated from different solanaceous plant species to bind AVR9 seems to correlate with the presence of members of the Cf-9 gene family, but whether this correlation is functional remains to be determined.     

Expression of Pokeweed Antiviral Protein in Transgenic Plants Induces Virus Resistance in Grafted Wild-Type Plants Independently of Salicylic Acid Accumulation and Pathogenesis-Related Protein Synthesis

Pokeweed antiviral protein (PAP), a 29-kD protein isolated from Phytolacca americana, inhibits translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. Transgenic tobacco (Nicotiana tabacum) plants expressing PAP or a variant (PAP-v) were shown to be resistant to a broad spectrum of plant viruses. Expression of PAP-v in transgenic plants induces synthesis of pathogenesis-related proteins and a very weak (<2-fold) increase in salicylic acid levels. Using reciprocal grafting experiments, we demonstrate here that transgenic tobacco rootstocks expressing PAP-v induce resistance to tobacco mosaic virus infection in both N. tabacum NN and nn scions. Increased resistance to potato virus X was also observed in N. tabacum nn scions grafted on transgenic rootstocks. PAP expression was not detected in the wild-type scions or rootstocks that showed virus resistance, nor was there any increase in salicylic acid levels or pathogenesis-related protein synthesis. Grafting experiments with transgenic plants expressing an inactive PAP mutant demonstrated that an intact active site of PAP is necessary for induction of virus resistance in wild-type scions. These results indicate that enzymatic activity of PAP is responsible for generating a signal that renders wild-type scions resistant to virus infection in the absence of increased salicylic acid levels and pathogenesis-related protein synthesis.     

Studies on the Salicylic Acid Inducted Lipid Peroxidation and Defense Gene Expression in Tobacco Cell Culture

Salicylic acid (SA) could inhibit catalase activity, induce rapid lipid peroxidation and PR-1 gene expression of the tobacco ( Nicotiana tabacum L. ) cell culture which was incubated with exogenous SA. Ρ-ihydroxybenzene and H2O2 could also induce lipid peroxidation and PR-1 gene expression at different level, but they were not able to inhibit the catalase activity of tobacco cells. Inhi0itors of mRNA and protein-synthesis (a-amanitine and cycloheximide, respectively) could not induce both lipid peroxidation and PR-1 gene expression of tobacco cell culture. However, coordinated action with SA respectively, a-amanitine or cycloheximide was able to induce lipid peroxidation effectively, but strongly blocked the activation of PR-1 gene expression by SA in tobacco cell culture. These results suggested that the generation of reactive metabolites or free radicals, which were induced by SA or other inducers through reaction with catalase or other compounds, initiated lipid peroxidation, subsequently activated pathogen-resistance genes expression. Obviously the lipid peroxidation molecule played an important role in SA signal transduction in tobacco.     

Effect of Ethylene on Cell Division and Deoxyribonucleic Acid Synthesis in Pisum sativum

Ethylene and supraoptimal levels of 2,4-dichlorophenoxyacetic acid inhibit the growth of the apical hook region of etiolated Pisum sativum (var. Alaska) seedlings by stopping almost all cell divisions. Cells are prevented from entering prophase. The hormones also retard cell division in intact root tips and completely stop the process in lateral buds. The latter inhibition is reversed partially by benzyl adenine. In root tips and the stem plumular and subhook regions, ethylene inhibits DNA synthesis. The magnitude of this inhibition is correlated with the degree of repression of cell division in meristematic tissue, suggesting that the effect on cell division results from a lack of DNA synthesis. Ethylene inhibits cell division within a few hours with a dose-response curve similar to that for most other actions of the gas. Experiments with seedlings grown under hypobaric conditions suggest that the gas naturally controls plumular expansion and cell division in the apical region.     

青枯病抗性不同的番茄品种根际拮抗菌拮抗能力差异研究

以植物基因型影响根际微生物的多样性为依据,通过平板拮抗试验,从番茄的感病品种和抗病品种、感病品种的严重发病植株和轻微发病植株根际筛选到36株抬抗菌,不同来源的抬抗菌群落具有不同的特征。严重发病的感病品种植株根际的拮抗菌群落多样性低,仅筛选到1个拮抗菌株,而轻微发病的感病品种和未发病的抗病品种植株根际筛选到的拮抗菌株数较多;抗病品种根际桔抗菌表现出强拮抗能力,而感病品种根际拮抗菌的拮抗能力较弱。测定植株根面和根颈处导管内青枯菌的种群数量、3株拮抗菌的防病效果,发现导管内青枯茵数量与防病效果基本一致,探讨了导管内青枯菌数量作为拮抗菌防病效果指标的可能性。     

Changes in Protein Synthesis and Morphology during Acid Adaptation of Propionibacterium freudenreichii

Survival of bacteria in changing environments depends on their ability to adapt to abiotic stresses. Microorganisms used in food technology face acid stress during fermentation processes. Similarly, probiotic bacteria have to survive acid stress imposed within the stomach in order to reach the intestine and play a beneficial role. Propionibacteria are used both as cheese starters and as probiotics in human alimentation. Adaptation to low pH thus constitutes a limit to their efficacy. Acid stress adaptation in the probiotic SI41 strain of Propionibacterium freudenreichii was therefore investigated. The acid tolerance response (ATR) was evidenced in a chemically defined medium. Transient exposure to pH 5 afforded protection toward acid challenge at pH 2. Protein neosynthesis was shown to be required for optimal ATR, since chloramphenicol reduced the acquired acid tolerance. Important changes in genetic expression were observed with two-dimensional electrophoresis during adaptation. Among the up-regulated polypeptides, a biotin carboxyl carrier protein and enzymes involved in DNA synthesis and repair were identified during the early stress response, while the universal chaperonins GroEL and GroES corresponded to a later response. The beneficial effect of ATR was evident at both the physiological and morphological levels. This study constitutes a first step toward understanding the very efficient ATR described in P. freudenreichii.     

Changes in Salicylic and Abscisic Acid Contents during Heat Treatment and Their Effect on Thermotolerance of Grape Plants

Heat treatment (38°C) of young grape plants (Vitis vinifera L., cv. Jingxiu) or leaf spraying with 100 µM salicylic acid (SA) increased leaf thermotolerance. Both treatments led to an increase in ABA content and a decrease in lipoxygenase (LOX) activity. ABA content showed a drastic rise by one hour after treatments and then sharply declined while LOX activity continuously decreased. In the course of heat treatment of grape plants, endogenous SA level and phenylalanine ammonia-lyase activity rapidly increased during the first hour, then declined. These results showed that endogenous SA and ABA can be involved in grape plant response to heat treatment resulted in improved thermotolerance.From Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 578–583.Original English Text Copyright © 2005 by Wang, Huang, Liu, Zhan.The text was submitted by the authors in English.     

钙对水杨酸诱导番茄叶片灰霉病抗性及防御酶活性的影响

以番茄‘L402’品种幼苗为试材,经水杨酸(SA)诱导处理后接种灰霉病菌,再进行外源Ca2+、Ca2+螯合剂和Ca2+抑制剂处理,分析Ca2+和SA处理番茄叶片对灰霉病抗性和主要防御酶系活性的变化,探讨Ca2+和SA对番茄诱导抗病性的影响。结果显示:(1)外源SA可显著提高番茄诱导叶和非诱导叶抗灰霉病能力,Ca2+能进一步增强SA诱导的抗病能力;而Ca2+螯合剂EGTA和质膜钙通道抑制剂LaCl3则不同程度地抑制了SA诱导的番茄灰霉病抗性。(2)外源SA能提高番茄诱导叶和非诱导叶中苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)、过氧化物酶(POD)活性,外源Ca2+亦进一步增强了SA诱导的上述防御酶活性,但缺钙处理则不同程度降低这些防御酶活性。(3)外源补充Ca2+及不同缺钙处理对SA诱导的过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性未发现规律性影响。研究表明,钙对SA诱导番茄抗灰霉病性的增强效应,可能与其提高SA诱导番茄叶片中PAL、PPO和POD等防御酶活性有关。     

Systemic Resistance in Tomato Induced by Biocontrol Bacteria Against the Root-Knot Nematode, Meloidogyne javanica is Independent of Salicylic Acid Production

Salicylic acid (SA)‐mediated induction of systemic resistance by Pseudomonas aeruginosa strain 7NSK2 and P. fluorescens strain CHA0 against soil‐borne fungi and viruses have been reported. The role of SA biosynthesis in the enhancement of defence mechanism against plant‐parasitic nematodes by these bacterial strains in tomato is not known. To better understand the importance of SA in rhizobacteria‐mediated suppression of root‐knot nematodes, biocontrol potential of SA‐negative or SA‐overproducing mutants against Meloidogyne javanica was evaluated with their respective wild type counter parts. Culture supernatant of 7NSK2, CHA0 and their respective mutants caused significant mortality of M. javanica juveniles in vitro. SA deletion in 7NSK2 and SA overproduction in CHA0 did not influence bacterial efficacy to cause nematode deaths. Similarly, culture supernatants resulting from King's B liquid medium amended with FeCl₃ did not influence nematicidal activity of the bacterial strains. Strain CHA0 induced juvenile deaths more than 7NSK2 did. In pot experiments, the bacterial strains applied in unsterilized sandy loam soil markedly reduced final nematode population densities in roots and subsequent root‐knot infection in tomato seedlings. SA‐negative or overproducing derivatives prevented tomato roots in kinetics similar to those with their respective wild types. When soil iron concentration was lowered by the addition of ethylenediamine di(o‐hydroxyphenylacetic acid), nematode biocontrol by the bacterial strains (both wild type and mutants) remained unaltered. To understand the mechanism involved in rhizobacteria‐mediated suppression of root‐knot nematode in tomato, bacterial performance was assessed in a split root trial in which one‐half of the root system was treated with bacterium while the other inoculated with nematode. Compared with the controls, application of the bacterial cell suspension to one‐half of the root system lowered the populations of root‐knot nematode in non‐bacterized nematode‐treated sections indicating enhanced defence in the non‐bacterized half. With respect to nematode infection, mutants induced systemic resistance to a similar extent as that caused by the wild types in both wild type tomato and NahG tomato plants. It is concluded that fluorescent pseudomonads induce systemic resistance against root‐knot nematode via a signal transduction pathway, which is independent of SA accumulation in roots.     

Cell Surfaces in Plant-Microorganism Interactions : IV. Fungal Glycopeptides Which Elicit the Synthesis of Ethylene in Plants

The production of ethylene by melon (Cucumis melo cv Cantaloup charentais) tissues is stimulated during incubation in the presence of fungal glycopeptides extracted from Colletotrichum lagenarium, a pathogen of melon. These glycopeptides, called elicitors of ethylene, are found in the mycelium, the cell wall, and the culture filtrate. Elicitation of ethylene is a relatively early phenomenon and lasts for several hours. Upon purification of the crude elicitor extract by gel filtration and ion exchange chromatography, three elicitors were isolated. The three elicitors contained amino acid, sugar, and phosphate residues, and they have a decreased activity after partial chemical degradation of their sugar moiety.

Elicitation of ethylene is not fungal species specific. Elicitors of phytoalexins, obtained from three Phytophtora species, enhanced ethylene biosynthesis in melon tissues.