Endophytic Pseudomonas fluorescens Endo2 and Endo35 induce resistance in black gram (Vigna mungo L. Hepper) to the pathogen Macrophomina phaseolina

Abstract

The effect of endophytic Pseudomonas fluorescens isolates Endo2 and Endo35 on induced systemic disease protection against dry root rot of black gram (Vigna mungo L. Hepper) caused by Macrophomina phaseolina was investigated under glasshouse conditions. When the bacterized black gram plants were inoculated with dry root rot pathogen, the activities of peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL) were stimulated in addition to accumulation of phenolics and lignin. Activity of phenylalanine ammonia-lyase (PAL) reached the maximum 24 h after pathogen challenge inoculation, whereas the activities of PO and PPO reached the maximum at 72 h and 48 h, respectively. Isoform analysis revealed that a unique PPO3 isozyme was induced in bacterized black gram tissues inoculated with the pathogen. Phenolics were found to accumulate in bacterized black gram tissues challenged with M. phaseolina one day after pathogen challenge. The accumulation of phenolics reached maximum at the third day after pathogen inoculation. Similar observation was found in the lignin content of black gram plants. In untreated control plants, the accumulation of defence enzymes and chemicals started at the first day and drastically decreased 3 days after pathogen inoculation. These results suggest that induction of defense enzymes involved in phenylpropanoid pathway and accumulation of phenolics and PR-proteins might have contributed to restricting invasion of Macrophomina phaseolina in black gram roots.     

An early salicylic acid-, pathogen- and elicitor-inducible tobacco glucosyltransferase: role in compartmentalization of phenolics and H2O2 metabolism.

Treatment of tobacco cell suspension cultures with a fungal elicitor of defense responses resulted in an early accumulation of the phenylpropanoid glucosyltransferase TOGT, along with the rapid synthesis and secretion of scopolin, the glucoside of scopoletin. Elicitor-triggered extracellular accumulation of the aglycone scopoletin and of free caffeic and ferulic acids could only be revealed in the presence of diphenylene iodonium, an inhibitor of extracellular H2O2 production. Our results strongly support a role for TOGT in the elicitor-stimulated production of transportable phenylpropanoid glucosides, followed by the release of free antioxidant phenolics into the extracellular medium and subsequent H2O2 scavenging.     

Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to bacterial pathogens

Reactive oxygen species (ROS) are responsible for mediating cellular defense responses in plants. Controversy has existed over the origin of ROS in plant defense. We have isolated a novel extracellular peroxidase gene, CaPO2, from pepper (Capsicum annuum). Local or systemic expression of CaPO2 is induced in pepper by avirulent Xanthomonas campestris pv vesicatoria (Xcv) infection. We examined the function of the CaPO2 gene in plant defense using the virus-induced gene silencing technique and gain-of-function transgenic plants. CaPO2-silenced pepper plants were highly susceptible to Xcv infection. Virus-induced gene silencing of the CaPO2 gene also compromised hydrogen peroxide (H(2)O(2)) accumulation and hypersensitive cell death in leaves, both locally and systemically, during avirulent Xcv infection. In contrast, overexpression of CaPO2 in Arabidopsis (Arabidopsis thaliana) conferred enhanced disease resistance accompanied by cell death, H(2)O(2) accumulation, and PR gene induction. In CaPO2-overexpression Arabidopsis leaves infected by Pseudomonas syringae pv tomato, H(2)O(2) generation was sensitive to potassium cyanide (a peroxidase inhibitor) but insensitive to diphenylene iodonium (an NADPH oxidase inhibitor), suggesting that H(2)O(2) generation depends on peroxidase in Arabidopsis. Together, these results indicate that the CaPO2 peroxidase is involved in ROS generation, both locally and systemically, to activate cell death and PR gene induction during the defense response to pathogen invasion.     

Phenolic acids and peroxidase activity in alfalfa (Medicago sativa) embryogenic cultures after ethephon treatment

Treatment with ethephon increased the concentration of exogenous ethylene in Medicago sativa L. embryogenic cell suspension cultures (consisting of single cells, small cellular clumps and globular somatic embryos) and induced changes in the metabolism of phenolic substances, activities of peroxidase (EC 1.11.1.7) and caused significant suppression of suspension culture growth. Treatment with the ethylene-releasing substance, ethephon, resulted in a several-fold increase in phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity above the basal level and was accompanied by an elevated accumulation of phenolic acids (significant increase of methoxy-substituted acids). The majority of newly synthesised phenolic acids was incorporated into the fractions of glycosides and esters bound to the cell wall. Phenolic glycosides seemed to serve as a metabolic pool from which the phenolics were utilised during further culture. The increased activity of wall-bound ionic peroxidase after ethephon application correlated with the pronounced incorporation of ferulic acid in the cell walls. In contrast, the increased level of exogenous ethylene did not influence the growth of culture of more advanced embryos nor did it significantly alter phenylpropanoid metabolism.     

核黄素和接种番茄黄化曲叶病毒对番茄叶片防御酶活性的影响

以番茄品种‘1479’为材料,研究了喷施核黄素(Riboflavin)和接种番茄黄化曲叶病毒(TYLCV)对幼苗叶片中过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)3种防御酶活性的影响。结果显示:(1)核黄素能显著降低番茄植株的番茄黄化曲叶病毒病的病情指数,并以2 mmol/L时诱导效果最佳,诱抗效果最高可达41.91%。(2)2mmol/L核黄素处理后96h内,番茄叶片的POD、PPO和PAL活性显著高于对照。(3)接种TYLCV后,核黄素处理和接种TYLCV处理均可诱导番茄叶片中防御酶活性显著增强。研究表明,核黄素处理可诱导POD、PPO和PAL活性的系统增强与番茄对TYLCV的诱导抗性密切相关。     

Accumulation of phenylpropanoid derivatives in chitosan-induced cell suspension culture of Cocos nucifera

Chitosan-induced elicitation responses of dark-incubated Cocos nucifera (coconut) endosperm cell suspension cultures led to the rapid formation of phenylpropanoid derivatives, which essentially mimics the defense-induced biochemical changes in coconut palm as observed under in vivo conditions. An enhanced accumulation of p-hydroxybenzoic acid as the major wall-bound phenolics was evident. This was followed by p-coumaric acid and ferulic acid. Along with enhanced peroxidases activities in elicited lines, the increase in activities of the early phenylpropanoid pathway enzymes such as, phenylalanine ammonia lyase (PAL), p-coumaroyl-CoA ligase (4CL) and p-hydroxybenzaldehyde dehydrogenase (HBD) in elicited cell cultures were also observed. Furthermore, supplementation of specific inhibitors of PAL, C4H and 4CL in elicited cell cultures led to suppressed accumulation of p-hydroxybenzoic acid, which opens up interesting questions regarding the probable route of the biosynthesis of this phenolic acid in C. nucifera.     

Down-regulation of free riboflavin content induces hydrogen peroxide and a pathogen defense in Arabidopsis

Riboflavin mediates many bioprocesses associated with the generation of hydrogen peroxide (H?O?), a cellular signal that regulates defense responses in plants. Although plants can synthesize riboflavin, the levels vary widely in different organs and during different stages of development, indicating that changes in riboflavin levels may have physiological effects. Here, we show that changing riboflavin content affects H?O? accumulation and a pathogen defense in Arabidopsis thaliana. Leaf content of free riboflavin was modulated by ectopic expression of the turtle gene encoding riboflavin-binding protein (RfBP). The RfBP-expressing Arabidopsis thaliana (REAT) plants produced the RfBP protein that possessed riboflavin-binding activity. Compared with the wild-type plant, several tested REAT lines had >70% less flavins of free form. This change accompanied an elevation in the level of H?O? and an enhancement of plant resistance to a bacterial pathogen. All the observed REAT characters were eliminated due to RfBP silencing (RfBPi) under REAT background. When an H?O? scavenger was applied, H?O? level declined in all the plants, and REAT no longer exhibited the phenotype of resistance enhancement. However, treatment with an NADPH oxidase inhibitor diminished H?O? content and pathogen defense in wild-type and RfBPi but not in REAT. Our results suggest that the intrinsic down-regulation of free flavins is responsible for NADPH oxidase-independent H?O? accumulation and the pathogen defense.     

Riboflavin-induced Priming for Pathogen Defense in Arabidopsis thaliana

Riboflavin (vitamin B2) participates in a variety of redox processes that affect plant defense responses. Previously we have shown that riboflavin induces pathogen resistance in the absence of hypersensitive cell death (HCD) in plants. Herein, we report that riboflavin induces priming of defense responses in Arabidopsis thaliana toward infection by virulent Pseudomonas syringae pv. Tomato DC3000 (Pst). Induced resistance was mechanistically connected with the expression of defense response genes and cellular defense events, including H2O2 burst, HCD, and callose deposition in the plant. Riboflavin treatment and inoculation of plants with Pst were neither active but both synergized to induce defense responses. The priming process needed NPR1 (essential regulator of systemic acquired resistance) and maintenance of H2O2 burst but was independent of salicylic acid, jasmonic acid, ethylene, and abscisic acid. Our results suggest that the role of riboflavin in priming defenses is subject to a signaling process distinct from the known pathways of hormone signal transduction.     

Influence of phosphate on the formation of the indole alkaloids and phenolic compounds in cell suspension cultures of Catharanthus roseus I. Comparison of enzyme activities and product accumulation

In cell suspension cultures of Catharanthus roseus a rapid accumulation of secondary compounds (tryptamine, indole alkaloids, phenolics) was observed after transfer of the cells into special ‘induction’-media devoid of phosphate and other essential growth factors [11, 14]. The increase of product levels was suppressed in the presence of phosphate which was almost completely taken up from the medium and accumulated by the cells within 48 h after inoculation. The activities of tryptophan decarboxylase (TDC), the first enzyme in indole alkaloid biosynthesis, and of phenyl-alanine ammonia-lyase (PAL), the key enzyme of phenylpropanoid biosynthesis, were influenced differently by phosphate. Whereas the accumulation of phenolics and PAL activity were similarly inhibited by low concentration of phosphate, the medium-induced enhanced activity of TDC was not affected although the product pools were considerably reduced. Some consequences for the regulation of secondary metabolism will be discussed.     

Nitric oxide is involved in methyl jasmonate-induced defense responses and secondary metabolism activities of Taxus cells

Methyl jasmonate (MeJA), a methyl ester of jasmonic acid (JA), is a well-established signal molecule in plant defense responses and an effective inducer of secondary metabolite accumulation in plant cell cultures such as the valuable anticancer diterpenoid taxol (paclitaxel) in Taxus spp. This work examines the involvement of nitric oxide (NO) in MeJA-induced plant defense responses and secondary metabolism in Taxus chinensis cell cultures. Exogenously supplied MeJA at 100 microM induced rapid production of NO in the Taxus cell cultures, reaching a maximum within 6 h of MeJA supply. Several other responses occurred concomitantly, including the production of hydrogen peroxide (H2O2), and the increases in intracellular malondialdehyde (MDA) content, lipoxygenase (LOX) and phenylalanine ammonium-lyase (PAL) activities. The MeJA-induced H2O2 production was suppressed by an NO donor, sodium nitroprusside (SNP), but enhanced by NO inhibitors, N (omega)-nitro-L-arginine (L-NNA) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO). In contrast, the MeJA-induced MDA, LOX and PAL were all enhanced by the NO donor but suppressed by the NO inhibitors. The NO inhibitors also suppressed MeJA-induced taxol accumulation. These results are suggestive of a role for NO as a signal element for activating the MeJA-induced defense responses and secondary metabolism activities of plant cells.     

叶片涂施茉莉酸对玉米幼苗防御反应的时间和浓度效应

茉莉酸是环境胁迫下植物产生防御反应的重要信号物质, 但它发挥生理作用的时间和浓度效应以及该效应在叶片和根系中差异性并不清楚。该文以‘高油115’玉米(Zea mays)为材料, 采用4种浓度(1、2.5、5和10 mmol·L^-1)的外源茉莉酸溶液涂施玉米幼苗叶片, 在3~48 h的不同时间内跟踪测定叶片和根系中的直接防御物质(丁布(DIMBOA)和总酚)含量及其合成调控基因(Bx1、Bx9和PAL)、直接防御蛋白调控基因(PR-1、PR-2a和MPI)和间接防御物质挥发物调控基因(FPS和TPS)表达的动态变化。结果表明, 外源茉莉酸处理对玉米叶和根系的化学防御反应具有显著的时间和浓度效应。茉莉酸处理玉米叶片后3~6 h就能诱导叶片中Bx9和PAL基因的表达, 使得丁布和总酚的含量显著增加, 且与处理浓度有呈正比的趋势, 随后诱导作用逐渐减弱; 茉莉酸处理还能明显诱导叶片中PR-2a和MPI基因的表达, 诱导作用分别持续到24和48 h; 在处理后3~6 h内, 高浓度茉莉酸处理对挥发物调控基因FPS表达起诱导作用, 而低浓度茉莉酸则对TPS基因的表达起诱导作用。此外, 茉莉酸处理玉米叶片还能间接影响到根系的防御反应, 但大部分检测指标表明间接诱导作用主要出现在处理后期(24~48 h)。例如, 在处理后48 h, 茉莉酸能系统增加根系中直接防御物质丁布和总酚的含量, 增强根系中防御相关基因PR-2a、MPI、FPS和TPS的表达, 并有随茉莉酸处理浓度的增加而增强的趋势。可见, 外源茉莉酸叶片涂施玉米幼苗对根系的间接诱导作用不如对叶片的直接诱导作用强; 叶片启动防御反应的时间较根系早; 随着处理浓度的增加, 茉莉酸对叶片和根系中防御反应的诱导作用有增强的趋势。     

质膜NAD(P)H氧化酶参予金瓜炭疽(Colletotrichum lagerarium)细胞壁激发子诱导人参细胞产生激发反应(英文)

在人参(Panax ginseng C.A.Meyer)悬浮细胞质膜上测出了NAD(P)H氧化酶活性。这类NAD(P)H氧化酶活性可以被金瓜炭疽细胞壁激发子(Cle)诱导。Cle处理还能诱导人参悬浮细胞的氧进发、促进人参悬浮细胞的皂苷合成、提高苯丙氨酸解氨酶(PAL)的活力、以及诱导查尔式酮酶(CHS)的累积和细胞壁上抗性相关蛋白基因脯氨酸富裕蛋白基因hrgp(Hydroxyprolin-rich glycoproleins)的表达。当用哺乳动物白细胞质膜NADPH氧化酶的特异性抑制剂二亚苯基碘(Diphenylene iodonium,DPI)与奎吖因(quinacrine)预处理人参悬浮细胞30 min 后,Cle诱导的H2O2释放与Cle激活的质膜NAD(P)H氧化酶活性被抑制,同时Cle诱导的PAL活性及CHS的积累下降,皂苷合成与hrgp的表达被抑制。由此推测:人参细胞质膜NAD(P)H氧化酶与哺乳动物白细胞质膜NADPH氧化酶有很大的相似性。在Cle激发人参悬浮细胞产生氧进发的过程中,NAD(P)H氧化酶活性被诱导从而导致H2O2的产生,H2O2作为第二信使,激活苯丙氨酸途径,诱发人参皂苷的合成及hrgp防御基因的表达。这一过程中还涉及到Ca2+内流,胞内Ca2+浓度的升高,蛋白磷酸化与去磷酸化。人参细胞质膜NAD(P)H氧化酶在人参细胞对Cle的反应过程中起一种介导作用。因此可能存在由Cle刺激,NAD(P)H氧化酶被诱导,H2O2释放,到人     

植物抗逆性与水杨酸介导的信号传导途径的关系

基因表达既受发育过程的调控又受外界环境的影响。无论是内因还是外因诱发一组基因表达时都涉及信号传导(signal transduction)问题。局部器官和组织所发生的生理变化的信息要传递到远处的组织,引起基因表达时间和空间上的协调。信号传导途径的研究是当今分子生物学的研究热点之一。 作为信号传递的分子主要是小分子物质,属于次生代谢产物。也发现某些小肽具有信号分子的功能。信号分子可以在胞间扩散,亦可通过输导组织传送到远处的器官。近年来研究甚多的一种信号     

Signaling role of nitric oxide in the induction of plant defense by exogenous application of abiotic inducers

The objective of this work was to search out the probable molecule behind the activation of broad spectrum resistance during abiotic elicitors such as arachidonic acid, cupric chloride, chitosan, isonicotinic acid and salicylic acid mediated induced systemic resistance (ISR) in Raphanus sativus L. The elicitor compounds were sprayed on the radish leaves of healthy plant and after 24 h incubation a significant increase of β-1,3 glucanase, peroxidase, polyphenol oxidase and phenolics as well as a remarkable increase of nitric oxide (NO), a probable potent defense-signaling molecule in plant, was observed. Furthermore, treatment of the host with NO donor, sodium nitroprusside, also induced the same defense molecules. The results suggests that NO might be the signaling molecule during abiotic elicitor mediated ISR induction in the host system.     

亚精胺诱导黄瓜幼苗对白粉病抗性的研究

以黄瓜品种‘长春密刺’幼苗为材料,研究了亚精氨(Spd)诱导黄瓜幼苗对白粉病的抗性,并测定Spd处理和白粉菌接种对黄瓜叶片4种防御酶活性及3种防卫基因表达的影响。结果显示:(1)0.2～1.0mmol.L-1 Spd对黄瓜幼苗白粉病抗性均有不同程度的诱抗效果,并以0.8mmol.L-1 Spd处理效果最明显,诱导效率可达55.3%。(2)喷施Spd或接种白粉菌均可提高黄瓜叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶和β-1,3-葡聚糖酶的活性,且诱导并接种处理的植株叶片上述酶活性均比只诱导不接种处理的上升速度更快;同时,Spd处理和接种白粉菌可以提高植株叶片中POX、PAL、PR-1a基因的表达量。研究表明,Spd处理可以诱导防卫基因表达的增强,提高防御酶活性,显著降低病情指数,增强黄瓜幼苗对白粉病的抗性。     

Hydrogen Peroxide from the Oxidative Burst Is Neither Necessary Nor Sufficient for Hypersensitive Cell Death Induction, Phenylalanine Ammonia Lyase Stimulation, Salicylic Acid Accumulation, or Scopoletin Consumption in Cultured Tobacco Cells Treated with Elicitin

H(2)O(2) from the oxidative burst, cell death, and defense responses such as the production of phenylalanine ammonia lyase (PAL), salicylic acid (SA), and scopoletin were analyzed in cultured tobacco (Nicotiana tabacum) cells treated with three proteinaceous elicitors: two elicitins (alpha-megaspermin and beta-megaspermin) and one glycoprotein. These three proteins have been isolated from Phytophthora megasperma H20 and have been previously shown to be equally efficient in inducing a hypersensitive response (HR) upon infiltration into tobacco leaves. However, in cultured tobacco cells these elicitors exhibited strikingly different biological activities. beta-Megaspermin was the only elicitor that caused cell death and induced a strong, biphasic H(2)O(2) burst. Both elicitins stimulated PAL activity similarly and strongly, while the glycoprotein caused only a slight increase. Only elicitins induced SA accumulation and scopoletin consumption, and beta-megaspermin was more efficient. To assess the role of H(2)O(2) in HR cell death and defense response expression in elicitin-treated cells, a gain and loss of function strategy was used. Our results indicated that H(2)O(2) was neither necessary nor sufficient for HR cell death, PAL activation, or SA accumulation, and that extracellular H(2)O(2) was not a direct cause of intracellular scopoletin consumption.     

Quantitative resistance of potato to Pectobacterium atrosepticum and Phytophthora infestans: integrating PAMP-triggered response and pathogen growth

While the mechanisms underlying quantitative resistance of plants to pathogens are still not fully elucidated, the Pathogen-Associated Molecular Patterns (PAMPs)-triggered response model suggests that such resistance depends on a dynamic interplay between the plant and the pathogen. In this model, the pathogens themselves or elicitors they produce would induce general defense pathways, which in turn limit pathogen growth and host colonisation. It therefore suggests that quantitative resistance is directly linked to a common set of general host defense mechanisms, but experimental evidence is still inconclusive. We tested the PAMP-triggered model using two pathogens (Pectobacterium atrosepticum and Phytophthora infestans) differing by their infectious processes and five potato cultivars spanning a range of resistance levels to each pathogen. Phenylalanine ammonia-lyase (PAL) activity, used as a defense marker, and accumulation of phenolics were measured in tuber slices challenged with lipopolysaccharides from P. atrosepticum or a concentrated culture filtrate from P. infestans. PAL activity increased following treatment with the filtrate but not with lipopolysaccharides, and varied among cultivars. It was positively related to tuber resistance to P. atrosepticum, but negatively related to tuber resistance to P. infestans. It was also positively related to the accumulation of total phenolics. Chlorogenic acid, the main phenolic accumulated, inhibited growth of both pathogens in vitro, showing that PAL induction caused active defense against each of them. Tuber slices in which PAL activity had been induced before inoculation showed increased resistance to P. atrosepticum, but not to P. infestans. Our results show that inducing a general defense mechanism does not necessarily result in quantitative resistance. As such, they invalidate the hypothesis that the PAMP-triggered model alone can explain quantitative resistance. We thus designed a more complex model integrating physiological host response and a key pathogen life history trait, pathogen growth, to explain the differences between the two pathosystems.