Endogenous enzyme reactions closely related to photon emission in the plant defense response

Lipoxygenase (LOX) and peroxidase (POD) reactions, which are involved in the production of reactive oxygen and radical species, are shown to be associated with ultraweak photon emission in plant defense mechanisms. These enzyme reactions induced high-level ultraweak photon emission in an in vitro reaction system. The application of LOX to sweet potato slices caused photon emission directly in plants. LOX substrate promoted photon emission in chitosan-treated sweet potato, and LOX inhibitor markedly suppressed this emission. Therefore, a LOX-related pathway, including LOX and other downstream reactions, is principally associated with photon emission in plant defense mechanisms.     

Highly sensitive determination of transient generation of biophotons during hypersensitive response to cucumber mosaic virus in cowpea

The hypersensitive response (HR) is one mechanism of the resistance of plants to pathogen infection. It involves the generation of reactive oxygen species (ROS) which have crucial roles in signal transduction or as toxic agents leading to cell death. Often, ROS generation is accompanied by an ultraweak photon emission resulting from radical reactions that are initiated by ROS through the oxidation of living materials such as lipids, proteins, and DNA. This photon emission, referred to as 'biophotons', is extremely weak, but, based on the technique of photon counting imaging, a system has been developed to analyse the spatiotemporal properties of photon emission. Using this system, the dynamics of photon emission which might be associated with the oxidative burst, which promotes the HR, have been determined. Here, the transient generation of biophotons is demonstrated during the HR process in cowpea elicited by cucumber mosaic virus. The distinctive dynamics in spatiotemporal properties of biophoton emission during the HR expression on macroscopic and microscopic levels are also described. This study reveals the involvement of ROS generation in biophoton emission in the process of HR through the determination of the inhibitory effect of an antioxidant (Tiron) on biophoton emission.     

Identification of an essential component of the elicitation active site of the EIX protein elicitor

Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene biosynthesis, expression of pathogen-related proteins and hypersensitive responses (HR). Pathogen-derived elicitors trigger these defense responses. The Elicitor Ethylene-inducing Xylanase (EIX) elicits HR and other plant defense responses in some tobacco and tomato cultivars independently of its xylan degradation activity. The elicitation epitope on the EIX protein responsible for inducing the HR response has been elucidated. Through the generation of EIX-specific polyclonal antibodies and screening of combinatorial phage display peptide libraries an essential sequence of the EIX elicitation activity has been identified. This sequence consists of the pentapeptide TKLGE mapped to an exposed beta-strand of the EIX protein. Substitution of the pentapeptide TKLGE to VKGT inhibited the elicitation activity but not the beta-1-4-endoxylanase activity of the EIX protein further demonstrating that elicitation and enzyme activity are independent properties. Elucidation of a peptide sequence that is essential for elicitation of HR creates the opportunity to understand the control and signaling of plant defense.     

CRT1, an Arabidopsis ATPase that interacts with diverse resistance proteins and modulates disease resistance to turnip crinkle virus

Plant immunity frequently involves the recognition of pathogen-encoded avirulence (avr) factors by their corresponding plant resistance (R) proteins. This triggers the hypersensitive response (HR) where necrotic lesions formed at the site(s) of infection help restrict pathogen spread. HRT is an Arabidopsis R protein required for resistance to turnip crinkle virus (TCV). In a genetic screen for mutants compromised in the recognition of TCV's avr factor, we identified crt1 (compromised recognition of TCV), a mutant that prematurely terminates an ATPase protein. Following TCV infection, crt1 developed a spreading HR and failed to control viral replication and spread. crt1 also suppressed HR-like cell death induced by ssi4, a constitutively active R protein, and by Pseudomonas syringae carrying avrRpt2. Furthermore, CRT1 interacts with HRT, SSI4, and two other R proteins, RPS2 and Rx. These data identify CRT1 as an important mediator of defense signaling triggered by distinct classes of R proteins.     

Rice Pti1a negatively regulates RAR1-dependent defense responses

Tomato (Solanum lycopersicum) Pto encodes a protein kinase that confers resistance to bacterial speck disease. A second protein kinase, Pti1, physically interacts with Pto and is involved in Pto-mediated defense signaling. Pti1-related sequences are highly conserved among diverse plant species, including rice (Oryza sativa), but their functions are largely unknown. Here, we report the identification of a null mutant for the Pti1 homolog in rice and the functional characterization of Os Pti1a. The rice pti1a mutant was characterized by spontaneous necrotic lesions on leaves, which was accompanied by a series of defense responses and resistance against a compatible race of Magnaporthe grisea. Overexpression of Pti1a in rice reduced resistance against an incompatible race of the fungus recognized by a resistance (R) protein, Pish. Plants overexpressing Pti1a were also more susceptible to a compatible race of the bacterial pathogen Xanthomonas oryzae pv oryzae. These results suggest that Os Pti1a negatively regulates defense signaling for both R gene-mediated and basal resistance. We also demonstrated that repression of the rice RAR1 gene suppressed defense responses induced in the pti1a mutant, indicating that Pti1a negatively regulates RAR1-dependent defense responses. Expression of a tomato Pti1 cDNA in the rice pti1a mutant suppressed the mutant phenotypes. This contrasts strikingly with the previous finding that Sl Pti1 enhances Pto-mediated hypersensitive response (HR) induction when expressed in tobacco (Nicotiana tabacum), suggesting that the molecular switch controlling HR downstream of pathogen recognition has evolved differently in rice and tomato.     

The sesquiterpene botrydial produced by Botrytis cinerea induces the hypersensitive response on plant tissues and its action is modulated by salicylic acid and jasmonic acid signaling

Botrytis cinerea, as a necrotrophic fungus, kills host tissues and feeds on the remains. This fungus is able to induce the hypersensitive response (HR) on its hosts, thus taking advantage on the host's defense machinery for generating necrotic tissues. However, the identity of HR effectors produced by B. cinerea is not clear. The aim of this work was to determine whether botrydial, a phytotoxic sesquiterpene produced by B. cinerea, is able to induce the HR on plant hosts, using Arabidopsis thaliana as a model. Botrydial induced the expression of the HR marker HSR3, callose deposition, and the accumulation of reactive oxygen species and phenolic compounds. Botrydial also induced the expression of PR1 and PDF1.2, two pathogenesis-related proteins involved in defense responses regulated by salicylic acid (SA) and jasmonic acid (JA), respectively. A. thaliana and tobacco plants defective in SA signaling were more resistant to botrydial than wild-type plants, as opposed to A. thaliana plants defective in JA signaling, which were more sensitive. It can be concluded that botrydial induces the HR on its hosts and its effects are modulated by host signaling pathways mediated by SA and JA.     

The hypersensitive response and the induction of cell death in plants

The hypersensitive response, or HR, is a form of cell death often associated with plant resistance to pathogen infection. Reactive oxygen intermediates and ion fluxes are proximal responses probably required for the HR. Apoptosis as defined in animal systems is, thus far, not a strict paradigm for the HR. The diversity observed in plant cell death morphologies suggests that there may be multiple pathways through which the HR can be triggered. Signals from pathogens appear to interfere with these pathways. HR may play in plants the same role as certain programmed cell deaths in animals with respect to restricting pathogen growth. In addition, the HR could regulate the defense responses of the plant in both local and distant tissues.     

植物幼苗原发性超弱光子成像的研究

采用光子计数成像系统（PIAS）对植物幼苗萌发过程的超弱发光进行观察。结果表明,自发光子长时间积累可形成二维图象;光子计数和采集图象均可得到植物体的自发发光;通过实验探测到幼苗的根,叶在同一平面图象有不同的发光表现;光子成像系统可客观地比较生物自发超弱发光,为进一步研究超弱发光机理提供实验基础。     

Biophotons from stressed and dying organisms: toxicological aspects

Cells and organisms exposed to detrimental and toxic substances show different responses in photon emission dependent on amount, kind and exposure time of toxin as well as on the organism investigated. Radical reaction-generating substances and dehydrating, lipid dissolving and protein denaturating toxins which do not induce direct chemiluminescence resulting from reactive oxygen species were applied. Lethal doses of toxins and stress factors such as osmotics and temperature evoke increase in the intensity of photon emission resulting from a rapid and irreversible perturbation of homeostasis. Bacterial and fungal toxins that elicit hypersensitive death of plant cells or defense response correlated with photon emission are also briefly discussed. Collective molecular interactions contribute to the photon-generating degradative processes in stressed and dying organisms. The measurements of biophoton signals and analysis of their parameters are used to elucidate the possible mechanisms of the toxin-organism interaction and the resistance of organisms. Toxicological perspectives of the use of these sensitive and rapid measurements as a part of direct toxicity assessment are discussed.     

New insights into resistance protein-mediated signaling against turnip crinkle virus in Arabidopsis

The Arabidopsis-turnip crinkle virus (TCV) system is one of the few tractable plant-virus systems that allow simultaneous characterization of host components required for basal- and/or resistance (R) protein-mediated defenses. Another unique feature is that hypersensitive response (HR) and resistance can be studied as two distinct phenotypes in this pathosytem. The R protein HRT confers HR to TCV but requires a recessive locus rrt to confer resistance. The pathways leading to HR and resistance are mutually exclusive. HRT interacts with EDS1, which potentiates HR to TCV and is also required for resistance signaling. HRT-mediated signaling is also dependent on the EDS1-interacting proteins PAD4 and SAG101, which form binary and ternary complexes with EDS1. HRT-mediated resistance is also dependent on light and more specifically on the blue-light photoreceptors, cryptochromes (CRY) and phototropins (PHOT). Of these, CRY2 and PHOT2 are required for the stability of HRT. HRT is degraded in a proteasome-dependent manner, which correlates with its interaction with the E3 ubiquitin ligase, COP1. Together, these results suggest that components of light signaling modulate plant defense against TCV by regulating the stability of, and signaling mediated by, the R protein HRT.     

Proteomic analysis of S-nitrosylated proteins in Arabidopsis thaliana undergoing hypersensitive response

Nitric oxide (NO) has a fundamental role in the plant hypersensitive disease resistance response (HR), and S-nitrosylation is emerging as an important mechanism for the transduction of its bioactivity. A key step toward elucidating the mechanisms by which NO functions during the HR is the identification of the proteins that are subjected to this PTM. By using a proteomic approach involving 2-DE and MS we characterized, for the first time, changes in S-nitrosylated proteins in Arabidopsis thaliana undergoing HR. The 16 S-nitrosylated proteins identified are mostly enzymes serving intermediary metabolism, signaling and antioxidant defense. The study of the effects of S-nitrosylation on the activity of the identified proteins and its role during the execution of the disease resistance response will help to understand S-nitrosylation function and significance in plants.     

Comparative analysis of the capacity of tombusvirus P22 and P19 proteins to function as avirulence determinants in Nicotiana species

We have used an agroinfiltration assay for a comparative study of the roles of tombusvirus P22 and P19 proteins in elicitation of hypersensitive response (HR)-like necrosis and the role of P19 in silencing suppression in Nicotiana species. The advantage of agroinfiltration rather than expression in plant virus vectors is that putative viral avirulence proteins can be evaluated in isolation, eliminating the possibility of synergistic effects with other viral proteins. We found that tombusvirus P22 and P19 proteins elicited HR-like necrosis in certain Nicotiana species but, also, that Nicotiana species could recognize subtle differences in sequence between these proteins. Furthermore, Nicotiana species that responded with systemic necrosis to virion inoculations responded to agroinfiltration of tombusvirus P19 with a very weak and delayed necrosis, indicating that the rapid HR-like necrosis was associated with putative resistance genes and a plant defense response that limited the spread of the virus. Tombusvirus P19 proteins also appeared to differ in their effectiveness as silencing suppressors; in our assay, the P19 proteins of Cymbidium ringspot virus and Tomato bushy stunt virus were stronger silencing suppressors than Cucumber necrosis virus P20. Finally, we show that agroinfiltration can be used to track the presence of putative plant resistance genes in Nicotiana species that target either tombusvirus P19 or P22.     

植物抗病反应的信号传导网络

植物由抗病基因介导的防卫过程存在一系列生理生化和分子生物学反应,这些反应从病原菌侵染点开始的超敏反应(HR)并延伸到远处组织的系统抗性或获得性抗性(SAR),受制于一种信号传导网络的调控。这个信号系统由抗病蛋白和病原菌非毒性蛋白在一种配体-受体的互作模式下激发,并由信号分子H2O2,NO和系统信号分子SA,JA和乙烯和通过关键调控基因传递和放大,最终诱导一系列防卫反应基因的表达和代谢的变化而产生抗性。植物防卫信号的产生有类似于动物免疫系统因子的介导,并可由非寄主病原菌或诱导子诱发。这些信号途径所产生的广谱抗性为植物抗病基因工程的应用奠定了基础。     

盐胁迫下绿豆幼苗的超微弱发光

对不同NaCl浓度胁迫下绿豆种子早期萌发时的超微弱发光变化进行了初步研究。结果表明,随。NaCl浓度的增加,绿豆胚根的生长速度(根长)减慢,生长受到明显抑制,其超微弱发光的强度显著下降。萌发期间,SOD活性随着盐浓度的增加而降低,其活性与生物光子强度有极为密切的关系。这些结果表明生物超微弱发光探测技术有可能成为植物盐胁迫研究的有效工具,对于进一步理解盐胁迫机理有一定的意义。     

寄主植物与病原菌免疫反应的分子遗传基础

近年来,大量的植物抗病基因和病原菌无毒基因被克隆,抗病基因和无毒基因的结构、功能及其互作关系的研究也取得重大进展。在植物中,由病原菌模式分子(pathogen-associated molecular patterns, PAMPs)引发的免疫反应(PAMP-triggered immunity, PTI)和由效应因子引发的免疫反应(effector-triggered immunity, ETI)是植物在长期进化过程中形成的两类抵抗病原物的机制。PTI反应主要通过细胞表面受体(patternrecognition receptors, PRRs)识别并结合PAMPs从而激活下游免疫反应,而在ETI反应中,则通过植物R基因(resistance gene,R)与病原菌无毒基因(avirulence gene, Avr)产物间的直接或间接相互作用来完成免疫反应。本文对植物PTI反应和ETI反应分别进行了概述,重点探讨了植物R基因与病原菌Avr基因之间的互作遗传机理,并对目前植物抗性分子遗传机制研究和抗病育种中的问题进行了探讨和展望。     

Phospholipases in action during plant defense signaling

Eukaryotic organisms rely on intricate signaling networks to connect recognition of microbes with the activation of efficient defense reactions. Accumulating evidence indicates that phospholipids are more than mere structural components of biological membranes. Indeed, phospholipid-based signal transduction is widely used in plant cells to relay perception of extracellular signals. Upon perception of the invading microbe, several phospholipid hydrolyzing enzymes are activated that contribute to the establishment of an appropriate defense response. Activation of phospholipases is at the origin of the production of important defense signaling molecules, such as oxylipins and jasmonates, as well as the potent second messenger phosphatidic acid (PA), which has been shown to modulate the activity of a variety of proteins involved in defense signaling. Here, we provide an overview of recent reports describing the different plant phospholipase pathways that are activated during the establishment of plant defense reactions in response to pathogen attack.Key words: lipid signaling, PA, PLA, PLC, PLD, plant immunityIn plant cells, perception of pathogenic microbes largely relies on transmembrane pattern recognition receptors that specifically recognize highly conserved pathogen-derived molecules called PAMPs/MAMPs (pathogen-/microbial-associated molecular patterns), such as bacterial flagellin.¹ PAMP recognition by the plant leads to basal defense responses. A second layer of defense is based on the recognition of specific pathogen-derived molecules, called effectors, primarily by an additional class of plant cytoplasmic receptor proteins [nucleotide-binding leucine-rich repeat (NB-LRR) proteins] but also by protein receptors predicted to be located at the plasma membrane [receptor-like proteins (RLPs) and receptor-like kinases (RLKs)]. This recognition leads to the activation of plant immune responses that are frequently associated to the development of hypersensitive cell death (HR) at the inoculation site, which has been shown to contribute to plant resistance.²The activation of plant immunity involves a variety of early signaling events, including rapid accumulation of reactive oxygen species (ROS), changes in cellular ion fluxes, activation of protein kinase cascades, changes in gene expression and production of stress-related hormones.^3,4 During recent years, a substantial number of reports have also shown the importance of lipids and lipid-related molecules, including glycerolipids, sphingolipids, fatty acids, oxylipins, jasmonates and sterols, in the regulation of plant defense responses.⁵Phospholipids are more than structural components in biological membranes. Indeed, evidence that phospholipases and phospholipid-derived molecules are involved in plant signaling, and more particularly in plant immunity, is rapidly accumulating.^6,7 In plants, phosphatidic acid (PA) can be produced from phospholipids by phospholipase D (PLD) enzymes or from diacylyglycerol (DAG) by DAG kinases (DGKs) in the phospholipase C (PLC) pathway. PA is a potent secondary signal messenger molecule that modulates the activity of kinases, phosphatases, phospholipases and proteins involved in membrane-trafficking, Ca²⁺ signaling and the oxidative burst.^8,9 In addition, a growing body of evidence indicates that phospholipase A (PLA) [and related molecules such as lysophospholipids (LPLs) and free fatty acids (FFAs)] and phospholipase C (PLC) (and its related molecules DAG and DGK) play important roles in the control of the plant defense response to the attack by invading pathogens.⁷Here, we review the recent advances in understanding phospholipase-mediated signaling and its importance in the control of plant immune responses.     

Arsenal of elevated defense proteins fails to protect tomato against Verticillium dahliae

Although the hypersensitive reaction in foliar plant diseases has been extensively described, little is clear regarding plant defense strategies in vascular wilt diseases affecting numerous economically important crops and trees. We have examined global genetic responses to Verticillium wilt in tomato (Lycopersicon esculentum Mill.) plants differing in Ve1 resistance alleles. Unexpectedly, mRNA analyses in the susceptible plant (Ve1-) based on the microarrays revealed a very heroic but unsuccessful systemic response involving many known plant defense genes. In contrast, the response is surprisingly low in plants expressing the Ve1+ R-gene and successfully resisting the pathogen. Similarly, whole-cell protein analyses, based on 2D gel electrophoresis and mass spectrometry, demonstrate large systemic increases in a variety of known plant defense proteins in the stems of susceptible plants but only modest changes in the resistant plant. Taken together, the results indicate that the large systemic increases in plant defense proteins do not protect the susceptible plant. Indeed, since a number of the highly elevated proteins are known to participate in the plant hypersensitive response as well as natural senescence, the results suggest that some or all of the disease symptoms, including ultimate plant death, actually may be the result of this exaggerated plant response.     

An S-Type Anion Channel SLAC1 Is Involved in Cryptogein-Induced Ion Fluxes and Modulates Hypersensitive Responses in Tobacco BY-2 Cells

Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis SLAC1, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl⁻ and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of SLAC1 enhanced cryptogein-induced Cl⁻ efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl⁻ efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.