Protein Phosphorylation Is Induced in Tobacco Cells by the Elicitor Cryptogein

Changes in plasmalemma ion fluxes were observed when tobacco (Nicotiana tabacum) cells were treated with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea. A strong alkalization of the culture medium, accompanied by a leakage of potassium, was induced within a few minutes of treatment. These effects reached a maximum after 30 to 40 min and lasted for several hours. This treatment also resulted in a rapid, but transient, production of activated oxygen species. All these physiological responses were fully sensitive to staurosporine, a known protein kinase inhibitor. Furthermore, a study of protein phosphorylation showed that cryptogein induced a staurosporine-sensitive phosphorylation of several polypeptides. These data suggest that phosphorylated proteins may be essential for the transduction of elicitor signals.     

隐地蛋白(cryptogein)基因定点突变及其广谱抗病烟草转化植株的获得

用PCR法从隐地疫霉 (Phytophthoracryptogea)基因组DNA中克隆了cryptogein(Cry)基因。将Cry基因的 13位赖氨酸 (K)突变成缬氨酸 (V) ,获突变基因CryK13V ,并将其构建于CaMV35S启动子控制的植物表达载体上。通过农杆菌介导的叶盘转化法转入烟草 ,经卡那霉素抗性筛选获 33株再生植株 ,PCR检测和Southern杂交分析表明CryK13V基因已整合到烟草基因组中。接种试验结果表明 ,转基因烟草植株对黑胫病菌、赤星病菌和野火病菌等的抗性均有提高。Northern杂交分析表明 ,微弱的CryK13V基因在转化植株中的表达就足以激活PR1和OPBP1等防卫反应相关基因的表达 ,而且表达丰度与转基因植株的抗病性有着一定的正相关性。研究结果还表明 ,隐地蛋白13位上的赖氨酸在诱导细胞死亡中起着关键的作用。     

Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein

Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the “membrane raft” hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment.The adaptive capacity of biological membranes is a primary determinant of cell survival in fluctuating conditions. In particular, membrane physical properties are adjusted in the perception of and response to environmental modifications (including temperature, mechanical, and osmotic stresses) in various organisms (Los and Murata, 2004; Vígh et al., 2007; Verstraeten et al., 2010), including plants (Vaultier et al., 2006; Königshofer et al., 2008). Moreover, it has been shown that modifications of plasma membrane (PM) physical properties induced by pharmacological treatments can trigger signaling events in tobacco (Nicotiana tabacum) suspension cells (Bonneau et al., 2010). This reinforces the need to analyze the relationships between membrane organization and signaling in greater detail.Fluidity, a physical property of the PM, is a measure of the rotational and translational motions of molecules within the membrane, and consequently this reflects the level of lipid order in the bilayer. Lipid order is comprised of structure, microviscosity, and membrane phase; the latter feature includes lipid shape, packing, and curvature (Rilfors et al., 1984; van der Meer et al., 1984; Bloom et al., 1991). Lipid self-association induces a physical segregation into lipid bilayers, wherein a liquid-ordered (Lo) phase coexists with a liquid-disordered (Ld) phase (Veatch and Keller, 2005; Gaus et al., 2006; Klymchenko et al., 2009; Heberle et al., 2010). The Lo phase couples a high rotational mobility with a high conformational order in the lipid acyl chain, two physical properties that could be spatially resolved by fluorescence microscopy (Kubiak et al., 2011). Moreover, some observations indicate that Lo size or proportion could be controlled by temperature or cholesterol content (Roche et al., 2008; Orth et al., 2011).This preferential association of some lipids in complex mixtures has resulted in the “membrane raft” hypothesis within the cell biology field. This theory postulates the existence of small (20–200 nm), short-lived, sterol-, and sphingolipid-enriched Lo assemblies within the membrane. An important feature is that these aggregations are believed to coalesce, upon a biological stimulus, into larger structures whose dynamics can regulate many cellular processes (Simons and Ikonen, 1997; Pike, 2006; Lingwood and Simons, 2010; Simons and Gerl, 2010). An increased resistance to solubilization by detergents of Lo versus Ld phases has led researchers to consider that membrane fractions insoluble to nonionic detergents at low temperatures could contain the putative “raft” fractions. One caveat of this theory is that recovered detergent-insoluble membrane fractions (DIMs) only exist after detergent treatment and do not correspond to the native membrane structure (Lichtenberg et al., 2005). Nevertheless, their significant enrichment in sterols, sphingolipids, and specific subsets of proteins, some of which displaying a clustered distribution within the PM (Simons and Gerl, 2010), has encouraged their use as a biochemical counterpart of Lo microdomains existing in biological membranes.Plant DIMs with a lipid content similar to animal DIMs have been isolated from several species, including tobacco cells, and are enriched in proteins involved in signaling and stress responses (Mongrand et al., 2004; Borner et al., 2005; Morel et al., 2006; Lefebvre et al., 2007; Kierszniowska et al., 2009). Moreover, immunoelectron microscopy experiments have revealed that lateral segregation of lipids and proteins occurs at the nanoscale level at the tobacco PM, thus correlating detergent insolubility with membrane domain localization of presumptive raft proteins (Raffaele et al., 2009; Furt et al., 2010; Demir et al., 2013). Together, these data point to the existence of specialized lipid domains in plants. Concomitantly, the presence of sterol-rich Lo membrane domains was observed in vivo at the tip of the growing pollen tube in Picea meyeri, using both filipin and the fluorescent probe 1-[2-hydroxy-3-(N,N-dimethyl-N-hydroxyethyl)ammoniopropyl]-4-[β-[2-(di-n-butylamino)-6-napthyl]vinyl] pyridinium dibromide (di-4-ANEPPDHQ; Liu et al., 2009). This observation argues in favor of a sterol-dependent organization of ordered domains at the plant PM surface. In addition, the combined use of fluorescent lipid analogs and the environmental dye laurdan has revealed different lipid phases that emerge in the PM of Arabidopsis (Arabidopsis thaliana) protoplasts during restoration of the cell wall (Blachutzik et al., 2012). Despite these details, necessary data concerning the presence and in vivo characterization of Lo domains at a micrometer to nanometer scale are still lacking.The importance of a more refined resolution for observing Lo domains was proposed in several recent reviews (Bagatolli, 2006; Duggan et al., 2008; García-Sáez and Schwille, 2010; Owen et al., 2010a; Stöckl and Herrmann, 2010; Klenerman et al., 2011). Although the physical properties of biological membranes have been studied in situ by various techniques, including two-channel ratiometric microscopy (Owen et al., 2010c) and microscopy imaging of partitioning of fluorescent lipids and proteins (Rosetti et al., 2010) or environmentally sensitive probes (Parasassi et al., 1990; Jin et al., 2006), membrane segregation into microscopic Lo- and Ld-like phases has been difficult to observe in living cells. Furthermore, only a few studies have demonstrated that a microscopic phase separation involving an ordered phase similar to the Lo domain of model membranes could occur in biomembranes using PM giant vesicles (Baumgart et al., 2007; Lingwood et al., 2008; Sengupta et al., 2008). A potentially powerful approach for imaging small ordered membrane domains relies on environment-sensitive probes coupled with fluorescence spectroscopy (Gaus et al., 2003, 2006; Oncul et al., 2010). In particular, analysis of the fluorescence of the di-4-ANEPPDHQ probe, which exhibits an emission shift independent of local chemical composition under different lipid packing conditions (Jin et al., 2005; Demchenko et al., 2009; Dinic et al., 2011), recently enabled the imaging of plant membrane domains at the micrometer scale (Liu et al., 2009). The relevance of this approach has been confirmed by mapping membrane domains using generalized anisotropy-based images of di-4-ANEPPDHQ-stained T cell immunological synapses (Owen et al., 2010c), together with the characterization of membrane organization of nonadherent cells (such as living zebrafish embryo tissues) labeled with this dye (Owen et al., 2012a).The function of dynamic PM compartmentalization in the detection and transduction of environmental signals in plant cells has only recently begun to emerge, along with a crucial role for sterols in this organization (for review, see Zappel and Panstruga, 2008; Mongrand et al., 2010; Simon-Plas et al., 2011). These observations make it indispensable to align how the surface membrane of living cells might reorganize during signaling with the membrane raft hypothesis. To investigate possible modifications of membrane organization during the initial steps of plant defense signaling, tobacco cells were treated with two well-described elicitors of defense reaction, cryptogein, a small protein able to trigger an hypersensitive reaction (HR) and an acquired resistance in tobacco plants (Ponchet et al., 1999; Garcia Brugger et al., 2006) together with a widely described signaling cascade in tobacco suspension cells, and flg22 (a 22-amino acid peptide corresponding to a conserved domain of bacterial flagellin). The latter peptide is also a potent elicitor in plants, yet it does not induce an HR type of necrosis (Gomez-Gomez and Boller, 2002; Chinchilla et al., 2007). The study of cryptogein response reveals that the earliest steps of the signal transduction pathway mainly involve PM activities (Ponchet et al., 1999; Garcia-Brugger et al., 2006). How the PM is laterally organized and possibly reorganized in response to this stress so it can efficiently trigger a signaling cascade remains unknown.Here, we have developed a confocal multispectral microscopy approach to generate in vivo ratiometric pictures of large areas of the tobacco cell PM labeled with di-4-ANEPPDHQ, allowing the in vivo characterization of the global level of order of this membrane. Although an increase in the proportion of ordered phase within the membrane transiently occurred in the early steps of the cryptogein and flg22 signaling cascades, the fluorescence recovery after photobleaching (FRAP) technique revealed an increase in PM fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of Lo phases on the membrane of living plant cells and monitored the variations induced by cryptogein elicitation. The results are discussed within the framework of the “membrane raft” hypothesis, in which we propose a new mechanism of signaling platform formation in the context of plant defense.     

Shear-Induced Migration of a Transmembrane Protein within a Vesicle

Biomembranes feature phospholipid bilayers and serve as the interface between cells or organelles and the extracellular and/or cellular environment. Lipids can move freely throughout the membrane; the lipid bilayer behaves like a fluid. Such fluidity is important in terms of the actions of membrane transport proteins, which often mediate biological functions; membrane protein motion has attracted a great deal of attention. Because the proteins are small, diffusion phenomena are often in play, but flow-induced transport has rarely been addressed. Here, we used a dissipative particle dynamics approach to investigate flow-induced membrane protein transport. We analyzed the drift of a membrane protein located within a vesicle. Under the influence of shear flow, the protein gradually migrated toward the vorticity axis via a random walk, and the probability of retention around the axis was high. To understand the mechanism of protein migration, we varied both shear strength and protein size. Protein migration was induced by the balance between the drag and thermodynamic diffusion forces and could be represented by the Péclet number. These results improve our understanding of flow-induced membrane protein transport.     

TMV介导的外源蛋白在植物中表达

烟草花叶病毒(Tobaccomosaicvirus,TMV)为Tobamovirus代表成员,以此病毒介导的外源蛋白在植物中表达,经过了十几年的研究和不断完善,已被证实为一种有效的表达外源蛋白的途径.这项技术已经在医用活性多肽以及疫苗的研制、功能基因的鉴定、植物体内生物合成途径的研究等方面发挥越来越重要的作用.重点阐述了TMV基因组RNA的结构和分子生物学特征,并着重对重组载体的构建及其利用加以了论述.     

Influence of Environmental Stress on Biomass Partitioning in Transgenic Tobacco Plants Expressing the Movement Protein of Tobacco Mosaic Virus

The influence of various environmental factors on biomass partitioning between shoots and roots in transgenic tobacco (Nicotiana tabacum) plants expressing the movement protein (MP) of tobacco mosaic virus (TMV) was investigated. TMV-MP-expressing transgenic plants exhibited a root-to-shoot ratio that was approximately 40% below that of transgenic vector control plants. When transgenic plants expressing the TMV-MP were subjected to water-stress conditions, the root-to-shoot ratio was increased to a value comparable to that of control plants subjected to the same water-stress treatment. Although the root-to-shoot ratio was increased by N or P deficiencies, the TMV-MP-induced alteration in biomass partitioning was not overcome. Surprisingly, under K+-deficient growth conditions, both TMV-MP-expressing and control plants exhibited reduced root-to-shoot ratios when compared with plants grown in the presence of sufficient K+. Furthermore, plant growth under K+-deficient conditions did not alleviate the influence of the TMV-MP over resource allocation to the roots. These results are discussed in terms of possible mechanisms by which stress signals could cause an alteration in biomass partitioning between shoots and roots in control and transgenic tobacco plants expressing the TMV-MP.     

Biosynthesis and Secretion of Cryptogein, a Protein Elicitor Secreted by Phytophthora cryptogea

The phytopathogenic fungi Phytophthora subspecies elicit hypersensitive-like necroses on their nonhost tobacco (Nicotiana tabacum), with the exception of the tobacco pathogen Phytophthora nicotianæ. In culture, these fungi—except P. nicotianæ—secrete proteins, called elicitins, that cause these remote leaf necroses and are responsible for the incompatible reaction. These proteins protect tobacco against invasion by the agent of the tobacco black shank, P. nicotianæ, which is unable to produce such an elicitor. Cryptogein, secreted by Phytophthora cryptogea, has been purified, sequenced, and characterized as an elicitin, a novel family of 10 kilodalton holoproteins. In the present paper, we examined the secretion and biosynthesis of this protein elicitor from P. cryptogea culture. Results showed that the secretion of cryptogein began later than its synthesis and stopped earlier, simultaneously with mycelium growth, when the nitrogen source in the culture medium was nearly exhausted. Electrophoretic patterns of total protein from mycelium extracts and N-terminal sequence analysis showed that cryptogein accumulated in the mycelium in its mature form. The comparison of the immunoselected in vitro translation products with ³⁵S in vivo-labeled cryptogein showed that cryptogein was synthesized as a preprotein with a signal peptide removed cotranslationally before the secretion into the culture medium. Immunoselected in vitro-synthesized products were subjected to radiosequencing to clearly determine the N-terminal position and the size (20 amino acids) of the signal peptide. Cryptogein did not undergo any other posttranslational modification.     

Involvement of Free Calcium in Action of Cryptogein,a Proteinaceous Elicitor of Hypersensitive Reaction in Tobacco Cells

Treatment of suspension-cultured tobacco (Nicotiana tabacum var Xanthi) cells with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea, induced a great stimulation of Ca2+ influx within the first minutes. Ca2+ influx is essential for the initiation of cryptogein-induced responses, since ethyleneglycol-bis([beta]-amino-ethyl ether)-N,N[prime]-tetraacetic acid or La3+, which block Ca2+ entrance, suppress cryptogein-induced responses such as extracellular alkalinization, active oxygen species, and phytoalexin production. Moreover, once initiated, these responses require sustained Ca2+ influx within the 1st h. A Ca2+ ionophore (A23187) was able to trigger an extracellular alkalinization but not the formation of active oxygen species and phytoalexins, even in the presence of cryptogein. Staurosporine, a protein kinase inhibitor that was recently reported to suppress cryptogein-induced responses (M.-P. Viard, F. Martin, A. Pugin, P. Ricci, J.-P. Blein [1994] Plant Physiol 104: 1245-1249), inhibited Ca2+ influx induced by cryptogein in a dose-dependent manner. These results suggest that protein phosphorylation followed by Ca2+ influx might be involved in the initial steps of cryptogein signal transduction.     

巴西固氮螺菌Yu62由水稻和烟草根部向茎、叶的迁移运动

巴西固氮螺菌(Azospirillrm brasilence)是重要的植物促生内生菌之一.用gfp基因标记固氮螺菌后接种无菌的水稻和烟草幼苗的根部,限制培养一定时间后,用共聚焦激光显微镜观察,结果表明:除了根部有发荧光的螺菌定殖外,螺菌还分布在茎、叶的表皮细胞,皮层细胞和维管系统组织的细胞间隙.从根、茎、叶器官分离固氮螺菌,都存在有较高的螺菌群体密度.这一结果证明螺菌在植物内存在着从根部向茎、叶顶端的迁移现象.这一发现为研究巴西固氮螺菌在窠主植物体内的迁移运动的机制、与植物细胞间的分子相互作用及其对植物的促生作用奠定了生态学和细胞形态学的基础,也为实际应用提示了进一步的科学依据,具有重要的科学和实践意义.     

巴西固氮螺菌Yu62由水稻和烟草根部向茎、叶的迁移运动(英文)

巴西固氮螺菌(Azospirillum brasilence)是重要的植物促生内生菌之一。用gfp基因标记固氮螺菌后接种无菌的水稻和烟草幼苗的根部,限菌培养一定时间后,用共聚焦激光显微镜观察,结果表明:除了根内部有发荧光的螺菌定殖外,螺菌还分布在茎、叶的表皮细胞,皮层细胞和维管系统组织的细胞和细胞间隙。从根、茎、叶器官分离固氮螺菌,都存在有较高的螺菌群体密度。这一结果证明螺菌在植物内存在着从根部向茎、叶顶端的迁移现象。这一发现为研究巴西固氮螺菌在宿主植物体内的迁移运动的机制、与植物细胞间的分子相互作用及其对植物的促生作用奠定了生态学和细胞形态学的基础,也为实际应用提供了进一步的科学依据,具有重要的科学和实践意义。     

转望江南核糖体失活蛋白基因cassin烟草及其对烟草花叶病毒的抗性

通过构建植物表达载体,由农杆菌介导,将望江南核糖体失活蛋白基因cassin转入烟草。PCR和Southern blot杂交结果证明：外源基因已经以单拷贝整合到烟草基因组内,并且在后代发生遗传分离。RT—PCR和Northern blot杂交结果显示：外源基因可以正常转录。用不同浓度的TMV机械摩擦接种转基因T1、T2代各3个自交株系,以非转基因烟草为阴性对照,实验结果表明转基因烟草对TMV表现出不同程度的抗性。     

Responses of Cultured Tobacco Cells to Cryptogein, a Proteinaceous Elicitor from Phytophthora cryptogea: Possible Plasmalemma Involvement

In culture, the phytopathogenic fungus Phytophthora cryptogea secretes a protein which elicits hypersensitive-like necroses and protects tobacco plants against invasion by the pathogen Phytophthora parasitica var. nicotianae. This protein, named cryptogein, has been purified and its amino acid sequence determined. In this work, we studied the effect of cryptogein on tobacco cell suspension cultures. Cryptogein was lethal at about 0.10 micromolar. When added at sublethal doses, it elicited the production of ethylene and phytoalexins. It also induced a rapid increase in pH and conductivity of the extracellular medium without affecting the integrity of the plasma membrane. Cryptogein reduced the fusicoccin-induced acidification of the extracellular medium. The concentration which inhibited the fusicoccin response by 50% was 0.8 nanomolar, while 1 micromolar erythrosine B, an ATPase inhibitor, was needed to produce the same inhibition. However, cryptogein did not inhibit the activity of a purified plasma membrane ATPase. Results of binding studies with whole cells suggested the presence of elicitor-binding sites with a high affinity for cryptogein. The involvement of the plasma membrane during the initial interaction between elicitor and cells is discussed.     

Direct Interaction between the Tobacco Mosaic Virus Helicase Domain and the ATP-bound Resistance Protein,N Factor during the Hypersensitive Response in Tobacco Plants

Plants cope with pathogens with distinct mechanisms. One example is a gene-for-gene system, in which plants recognize the pathogen molecule by specified protein(s), this being called the R factor. However, mechanisms of interaction between proteins from the host and the pathogen are not completely understood. Here, we analyzed the mode of interaction between the N factor, a tobacco R factor, and the helicase domain (p50) of tobacco mosaic virus (TMV). To this end, domain dissected proteins were prepared and subjected to Agroinfiltration into intact leaves, followed by yeast two hybrid and pull-down assays. The results pointed to three novel features. First, the N factor was found to directly bind to the p50 of TMV, second, ATP was pre-requisite for this interaction, with formation of an ATP/N factor complex, and third, the N factor was shown to possess ATPase activity, which is enhanced by the p50. Moreover, we found that intra- and/or inter-molecular interactions take place in the N factor molecule. This interaction required ATP, and was disrupted by the p50. Based on these results, we propose a following model for the TMV recognition mechanism in tobacco plants. The N factor forms a complex with ATP, to which the helicase domain interacts, and enhances ATP hydrolysis. The resulting ADP/N factor complex then changes its conformation, thereby facilitating further interaction with the down-stream signaling factor(s). This model is consistent with the idea of ‘protein machine’.     

Resistance and Susceptibility of Plants to Fungal Pathogens

Plants are under continuous threat of infection by pathogens endowed with diverse strategies to colonize their host. Comprehensive biochemical and genetic approaches are now starting to reveal the complex signaling pathways that mediate plant disease resistance. Initiation of defense signaling often involves specific recognition of invading pathogens by the products of specialized host resistance (R) genes. Potential resistance signaling components have been identified by mutational analyses to be required for specific resistance in the model Arabidopsis and some crop species. Strikingly, many of the components share similarity to that of innate immune systems in animals. Evidence is also accumulating that plant pathogens have a number of ways to evade host defenses during the early stages of infection, similar to animal pathogens. These strategies are becoming much better understood in a number of plant–pathogen interactions. In this review, we focus on the current knowledge of host factors that control plant resistance and susceptibility to fungal pathogens. The knowledge accumulated in these studies will serve a fundamental basis for combating diseases in strategic molecular agriculture.     

Tobacco Mosaic Virus Infection Results in an Increase in Recombination Frequency and Resistance to Viral,Bacterial, and Fungal Pathogens in the Progeny of Infected Tobacco Plants

Our previous experiments showed that infection of tobacco (Nicotiana tabacum) plants with Tobacco mosaic virus (TMV) leads to an increase in homologous recombination frequency (HRF). The progeny of infected plants also had an increased rate of rearrangements in resistance gene-like loci. Here, we report that tobacco plants infected with TMV exhibited an increase in HRF in two consecutive generations. Analysis of global genome methylation showed the hypermethylated genome in both generations of plants, whereas analysis of methylation via 5-methyl cytosine antibodies demonstrated both hypomethylation and hypermethylation. Analysis of the response of the progeny of infected plants to TMV, Pseudomonas syringae, or Phytophthora nicotianae revealed a significant delay in symptom development. Infection of these plants with TMV or P. syringae showed higher levels of induction of PATHOGENESIS-RELATED GENE1 gene expression and higher levels of callose deposition. Our experiments suggest that viral infection triggers specific changes in progeny that promote higher levels of HRF at the transgene and higher resistance to stress as compared with the progeny of unstressed plants. However, data reported in these studies do not establish evidence of a link between recombination frequency and stress resistance.Continuous exposure to stress leads to the evolutionary selection of adaptive traits beneficial in a particular environment. Such selection of the fittest of a population of plants grown under certain environmental conditions is believed to require a long time. However, it is known that plants also possess the ability to acclimate on much shorter time scales. A modification of homeostasis, also termed acclimatization, is a well-documented process that is used for adjusting metabolism to a new environment (Lichtenthaler, 1998; Mullineaux and Emlyn-Jones, 2005).Pathogens represent one of a variety of stresses that plants are constantly exposed to. In nature, the evolution of plant resistance to a particular pathogen, virus, bacterium, or fungus has been the result of constant interactions with said pathogen (McHale et al., 2006; Friedman and Baker, 2007). These interactions lead to a constant plant-pathogen arms race (Ingle et al., 2006).Plants are able to tolerate or resist pathogens in a variety of ways, which could be broadly attributed to mechanisms of innate immunity and actual gene-for-gene-based resistance. The latter one depends on direct or indirect recognition of pathogen avirulence gene products by plant resistance gene products (Whitham et al., 1994; Durrant and Dong, 2004). Pathogen recognition during this incompatible interaction triggers complex events, including a local hypersensitive response that manifests itself as a booster of radical production and activation of the salicylic acid-dependent pathway and necrotic lesions, which working together restrict pathogen spread. It also results in a plant-wide systemic acquired resistance response that provides protection and tolerance to future pathogen attacks (Durrant and Dong, 2004; Park et al., 2007; Vlot et al., 2008).If a functional pathogen resistance gene is absent (compatible interaction), then the interaction between a plant and a pathogen is more ambiguous. How do plants that lack a resistance gene respond to infection? We have previously reported that the compatible interaction between Tobacco mosaic virus (TMV) and tobacco (Nicotiana tabacum ‘SR1’) plants lacking the TMV resistance N gene results in the production of a systemic signal. The signal leads to an increase in the frequency of somatic homologous recombination (HRF; Kovalchuk et al., 2003a). Based on these observations, we hypothesized that these genomic changes could be inherited. Indeed, we found that the progeny of infected SR1 tobacco plants exhibited a higher frequency of RFLPs at the loci that have similarity (more than 60%) to the Leu-rich repeat region of the N gene (Boyko et al., 2007).Although several reports have shown an increase in genome instability in plants exposed to pathogens and pathogen elicitors (Lucht et al., 2002; Kovalchuk et al., 2003a; Molinier et al., 2006; Boyko et al., 2007), many questions still remained unanswered. What is the mechanism of occurrence of a pathogen-induced systemic increase in HRF? What is the mechanism of inheritance of high-frequency homologous recombination? Are elevated levels of HRF maintained throughout generations? What other changes occur in progeny of infected plants?Here, we attempted to answer the above questions by analyzing two consecutive progenies of TMV-infected tobacco cv SR1 plants. Both progenies of infected plants showed higher levels of somatic HRF, higher resistance to TMV infection and tolerance to methyl methane sulfonate (MMS), an increase in callose deposition, as well as a higher steady-state PATHOGENESIS-RELATED GENE1 (PR1) RNA level compared with the progeny of uninfected plants. Analysis of methylation patterns has revealed global genome hypermethylation in both progenies paralleled by hypomethylation in euchromatic areas.     

Phloem-Specific Expression of the Tobacco Mosaic Virus Movement Protein Alters Carbon Metabolism and Partitioning in Transgenic Potato Plants

The tobacco mosaic virus movement protein (TMV-MP) has pleiotropic effects when expressed in transgenic tobacco (Nicotiana tabacum) plants. In addition to its ability to increase the plasmodesmal size-exclusion limit, the TMV-MP alters carbohydrate metabolism in source leaves and dry matter partitioning between the various plant organs. In the present study the TMV-MP was expressed under the control of a phloem-specific promoter (rolC), and this system was employed to further explore the potential sites at which the TMV-MP exerts its influence over carbon metabolism and transport in transgenic potato (Solanum tuberosum) plants. Immunohistochemical analyses indicated that the TMV-MP was localized mainly to phloem parenchyma and companion cells. Starch and sucrose accumulated in source leaves of these plants to significantly higher levels compared with control potato lines. In addition, the rate of sucrose efflux from excised petioles was lower compared with control plants. Furthermore, under short-day conditions, carbon partitioning was lower to the roots and higher to tubers in rolC plants compared with controls. These results are discussed in terms of the mode(s) by which the TMV-MP exerts its influence over carbon metabolism and photoassimilate translocation.     

Resistance to Potato Virus X Infection in Transgenic Tobacco Plants with Coat Protein Gene of Virus

A major commercial cultivar of tobacco was transformed via Agrobacterium mediated procedure. Tobacco leaves started to form shoots on shoot inducing medium containing kanamycin after infected by Agrobacterium containing the plasmid with PVX CP gene. Regenerated plants were obtained in two weeks on hormone-free MS medium containing kanamycin. The transgenic tobacco plants were identified with nopaline detection,enzyme-linked immunosorbent assay and western blot analysis, symptom appearance was significantly delayed and virus accumulation was either absent or reduced in PVX CP gene transformed plants. Progenies of transgenic tobacco plants also gained resistance to PVX infection to a certain degree. These experiments demonstrate that CP protection is effective against PVX.