Construction of cryptogein mutants, a proteinaceous elicitor from Phytophthora, with altered abilities to induce a defense reaction in tobacco cells

We prepared a series of cryptogein mutants, an elicitor from Phytophthora cryptogea, with altered abilities to bind sterols and fatty acids. The induction of the early events, i.e., synthesis of active oxygen species and pH changes, in suspension tobacco cells by these mutated proteins was proportional to their ability to bind sterols but not fatty acids. Although the cryptogein-sterol complex was suggested to be a form triggering a defense reaction in tobacco, some proteins unable to bind sterols induced the synthesis of active oxygen species and pH changes. The modeling experiments showed that conformational changes after the introduction of bulky residues into the omega loop of cryptogein resemble those induced by sterol binding. These changes may be necessary for the ability to trigger the early events by elicitins. However, the ability to stimulate necrosis in suspension tobacco cells and the expression of defense proteins in tobacco plants were linked neither to the lipid binding capacity nor to the capacity to provoke the early events. On the basis of these experiments and previous results, we propose that elicitins could stimulate two signal pathways. The first one induces necroses and the expression of pathogen-related proteins, includes tyrosine protein kinases and mitogen-activated protein kinases, and depends on the overall structure and charge distribution. The second type of interaction is mediated by phospholipase C and protein kinase C. It triggers the synthesis of active oxygen species and pH changes. This interaction depends on the ability of elicitins to bind sterols.     

Mediation of elicitin activity on tobacco is assumed by elicitin-sterol complexes

Elicitins secreted by phytopathogenic Phytophthora spp. are proteinaceous elicitors of plant defense mechanisms and were demonstrated to load, carry, and transfer sterols between membranes. The link between elicitor and sterol-loading properties was assessed with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues, postulated to be involved in sterol binding. Mutated cryptogeins were tested for their ability to load sterols, bind to plasma membrane putative receptors, and trigger biological responses. For each mutated elicitin, the chemical characterization of the corresponding complexes with stigmasterol (1:1 stoichiometry) demonstrated their full functionality. However, these proteins were strongly altered in their sterol-loading efficiency, specific binding to high-affinity sites, and activities on tobacco cells. Ligand replacement experiments strongly suggest that the formation of a sterol-elicitin complex is a requisite step before elicitins fasten to specific binding sites. This was confirmed with the use of two sterol-preloaded elicitins. Both more rapidly displaced labeled cryptogein from its specific binding sites than the unloaded proteins. Moreover, the binding kinetics of elicitins are related to their biological effects, which constitutes the first evidence that binding sites could be the biological receptors. The first event involved in elicitin-mediated cell responses is proposed to be the protein loading with a sterol molecule.     

The plant defense elicitor cryptogein stimulates clathrin-mediated endocytosis correlated with reactive oxygen species production in bright yellow-2 tobacco cells

The plant defense elicitor cryptogein triggers well-known biochemical events of early signal transduction at the plasma membrane of tobacco (Nicotiana tabacum) cells, but microscopic observations of cell responses related to these early events were lacking. We determined that internalization of the lipophilic dye FM4-64, which is a marker of endocytosis, is stimulated a few minutes after addition of cryptogein to tobacco Bright Yellow-2 (BY-2) cells. This stimulation is specific to the signal transduction pathway elicited by cryptogein because a lipid transfer protein, which binds to the same receptor as cryptogein but without triggering signaling, does not increase endocytosis. To define the nature of the stimulated endocytosis, we quantified clathrin-coated pits (CCPs) forming on the plasma membrane of BY-2 cells. A transitory stimulation of this morphological event by cryptogein occurs within the first 15 min. In the presence of cryptogein, increases in both FM4-64 internalization and clathrin-mediated endocytosis are specifically blocked upon treatment with 5 microm tyrphostin A23, a receptor-mediated endocytosis inhibitor. The kinetics of the transient increase in CCPs at the plasma membrane coincides with that of transitory reactive oxygen species (ROS) production occurring within the first 15 min after elicitation. Moreover, in BY-2 cells expressing NtrbohD antisense cDNA, which are unable to produce ROS when treated with cryptogein, the CCP stimulation is inhibited. These results indicate that the very early endocytic process induced by cryptogein in tobacco is due, at least partly, to clathrin-mediated endocytosis and is dependent on ROS production by the NADPH oxidase NtrbohD.     

Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance.

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr 203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea-induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.     

Characterization of the cryptogein binding sites on plant plasma membranes

Cryptogein is a 98-amino acid proteinaceous elicitor of tobacco defense reactions. Specific binding of cryptogein to high affinity binding sites on tobacco plasma membranes has been previously reported (K(d) = 2 nM; number of binding sites: 220 fmol/mg of protein). In this study, biochemical characterization of cryptogein binding sites reveals that they correspond to a plasma membrane glycoprotein(s) with an N-linked carbohydrate moiety, which is involved in cryptogein binding. Radiation inactivation experiments performed on tobacco plasma membrane preparations indicated that cryptogein bound specifically to a plasma membrane component with an apparent functional molecular mass of 193 kDa. Moreover, using the homobifunctional cross-linking reagent disuccinimidyl suberate and tobacco plasma membranes incubated with (125)I-cryptogein, we identified, after SDS-polyacrylamide gel electrophoresis and autoradiography, two (125)I-cryptogein linked N-glycoproteins of about 162 and 50 kDa. Similar results were obtained using Arabidopsis thaliana and Acer pseudoplatanus plasma membrane preparations, whereas cryptogein did not induce any effects on the corresponding cell suspensions. These results suggest that either cryptogein binds to nonfunctional binding sites, homologues to those present in tobacco plasma membranes, or that a protein involved in signal transduction after cryptogein recognition is absent or inactive in both A. pseudoplatanus and A. thaliana.     

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。     

Disruption of microtubular cytoskeleton induced by cryptogein, an elicitor of hypersensitive response in tobacco cells

The dynamics of microtubular cytoskeleton were studied in tobacco (Nicotiana tabacum cv Xanthi) cells in response to two different plant defense elicitors: cryptogein, a protein secreted by Phytophthora cryptogea and oligogalacturonides (OGs), derived from the plant cell wall. In tobacco plants cryptogein triggers a hypersensitive-like response and induces systemic resistance against a broad spectrum of pathogens, whereas OGs induce defense responses, but fail to trigger cell death. The comparison of the microtubule (MT) dynamics in response to cryptogein and OGs in tobacco cells indicates that MTs appear unaffected in OG-treated cells, whereas cryptogein treatment caused a rapid and severe disruption of microtubular network. When hyperstabilized by the MT depolymerization inhibitor, taxol, the MT network was still disrupted by cryptogein treatment. On the other hand, the MT-depolymerizing agent oryzalin and cryptogein had different and complementary effects. In addition to MT destabilization, cryptogein induced the death of tobacco cells, whereas OG-treated cells did not die. We demonstrated that MT destabilization and cell death induced by cryptogein depend on calcium influx and that MT destabilization occurs independently of active oxygen species production. The molecular basis of cryptogein-induced MT disruption and its potential significance with respect to cell death are discussed.     

The structure of "defective in induced resistance" protein of Arabidopsis thaliana, DIR1, reveals a new type of lipid transfer protein

Screening of transfer DNA (tDNA) tagged lines of Arabidopsis thaliana for mutants defective in systemic acquired resistance led to the characterization of dir1-1 (defective in induced resistance [systemic acquired resistance, SAR]) mutant. It has been suggested that the protein encoded by the dir1 gene, i.e., DIR1, is involved in the long distance signaling associated with SAR. DIR1 displays the cysteine signature of lipid transfer proteins, suggesting that the systemic signal could be lipid molecules. However, previous studies have shown that this signature is not sufficient to define a lipid transfer protein, i.e., a protein capable of binding lipids. In this context, the lipid binding properties and the structure of a DIR1-lipid complex were both determined by fluorescence and X-ray diffraction. DIR1 is able to bind with high affinity two monoacylated phospholipids (dissociation constant in the nanomolar range), mainly lysophosphatidyl cholines, side-by-side in a large internal tunnel. Although DIR1 shares some structural and lipid binding properties with plant LTP2, it displays some specific features that define DIR1 as a new type of plant lipid transfer protein. The signaling function associated with DIR1 may be related to a specific lipid transport that needs to be characterized and to an additional mechanism of recognition by a putative receptor, as the structure displays on the surface the characteristic PxxP structural motif reminiscent of SH3 domain signaling pathways.     

The elicitor cryptogein blocks glucose transport in tobacco cells

Cryptogein is a 10-kD protein secreted by the oomycete Phytophthora cryptogea that induces a hypersensitive response on tobacco (Nicotiana tabacum var. Xanthi) plants and a systemic acquired resistance against various pathogens. The mode of action of this elicitor has been studied using tobacco cell suspensions. Our previous data indicated that within minutes, cryptogein signaling involves various events including changes in ion fluxes, protein phosphorylation, sugar metabolism, and, eventually, cell death. These results suggested that transport of sugars could be affected and, thus, involved in the complex relationships between plant and microorganisms via elicitors. This led us to investigate the effects of cryptogein on glucose (Glc) uptake and mitochondrial activity in tobacco cells. Cryptogein induces an immediate inhibition of Glc uptake, which is not attributable to plasma membrane (PM) depolarization. Conversely, cryptogein-induced valine uptake is because of PM depolarization. Inhibition of the PM Glc transporter(s) was shown to be mediated by a calcium-dependent phosphorylation process, and is independent of active oxygen species production. This inhibition was associated with a strong decrease in O(2) uptake rate by cells and a large mitochondrial membrane depolarization. Thus, inhibition of Glc uptake accompanied by inhibition of phosphorylative oxidation may participate in hypersensitive cell death. These results are discussed in the context of competition between plants and microorganisms for apoplastic sugars.     

Questioning the role of salicylic acid and cytosolic acidification in mitogen-activated protein kinase activation induced by cryptogein in tobacco cells

Elicitors of plant defence reactions, oligogalacturonides and cryptogein, an elicitin produced by Phytophthora cryptogea, were previously shown to induce a rapid and transient activation of two mitogen-activated protein kinases (MAPKs) in cells of tobacco [ Nicotiana tabacum L. cv. Xanthi; A. Lebrun-Garcia et al. (1998) Plant J 15:773-781]. We verified that these two MAPKs correspond to the salicylic acid-induced protein kinase (SIPK) and the wound-induced protein kinase (WIPK). The involvement of salicylic acid (SA) in cryptogein-induced MAPK activation was investigated using transgenic NahG tobacco cells expressing the salicylate hydroxylase gene and thus unable to accumulate SA. The large and sustained activation of both MAPKs by cryptogein was maintained in transgenic cells compared with non-transgenic tobacco cells. Moreover, weak acids, namely SA, 4-hydroxybenzoic acid, an ineffective analogue of SA in plant resistance, and butyric acid acidified the cytosol, a physiological event also induced by cryptogein, but activated both MAPKs only slightly and transiently in tobacco cells. These results indicate that MAPK activation by cryptogein is not mediated by SA, that cytosolic acidification can be transduced by MAPKs, and that in cryptogein-treated cells, cytosolic acidification should contribute poorly to MAPK activation.     

Synergistic biosynthesis of biphasic ethylene and reactive oxygen species in response to hemibiotrophic Phytophthora parasitica in tobacco plants

We observed the biphasic production of ethylene and reactive oxygen species (ROS) in susceptible tobacco (Nicotiana tabacum 'Wisconsin 38') plants after shoot inoculation with Phytophthora parasitica var nicotianae. The initial transient increase in ROS and ethylene at 1 and 3 h (phase I), respectively, was followed by a second massive increase at 48 and 72 h (phase II), respectively, after pathogen inoculation. This biphasic pattern of ROS production significantly differed from the hypersensitive response exhibited by cryptogein-treated wild-type tobacco plants. The biphasic increase in ROS production was mediated by both NADPH oxidase isoforms, respiratory burst oxidase homolog (Rboh) D and RbohF. Conversely, different 1-aminocyclopropane-1-carboxylic acid synthase members were involved in specific phases of ethylene production: NtACS4 in the first phase and NtACS1 in the second phase. Biphasic production of ROS was inhibited in transgenic antisense plant lines expressing 1-aminocyclopropane-1-carboxylic acid synthase/oxidase or ethylene-insensitive3 as well as in transgenic plants impaired in ROS production. All tested transgenic plants were more tolerant against P. parasitica var nicotianae infection as determined based on trypan blue staining and pathogen proliferation. Further, silencing of NtACS4 blocked the second massive increase in ROS production as well as pathogen progression. Pathogen tolerance was due to the inhibition of ROS and ethylene production, which further resulted in lower activation of ROS-detoxifying enzymes. Accordingly, the synergistic inhibition of the second phase of ROS and ethylene production had protective effects against pathogen-induced cell damage. We conclude that the levels of ethylene and ROS correlate with compatible P. parasitica proliferation in susceptible plants.     

Elicitin genes expressed in vitro by certain tobacco isolates of Phytophthora parasitica are down regulated during compatible interactions

Phytophthora spp. secrete proteins called elicitins in vitro that can specifically induce hypersensitive response and systemic acquired resistance in tobacco. In Phytophthora parasitica, the causal agent of black shank, most isolates virulent on tobacco are unable to produce elicitins in vitro. Recently, however, a few elicitin-producing P. parasitica strains virulent on tobacco have been isolated. We investigated the potential diversity of elicitin genes in P. parasitica isolates belonging to different genotypes and with various virulence levels toward tobacco as well as elicitin expression pattern in vitro and in planta. Although elicitins are encoded by a multigene family, parAl is the main elicitin gene expressed. This gene is highly conserved among isolates, regardless of the elicitin production and virulence levels toward tobacco. Moreover, we show that elicitin-producing P. parasitica isolates virulent on tobacco down regulate parAl expression during compatible interactions, whichever host plant is tested. Conversely, one elicitin-producing P. parasitica isolate that is pathogenic on tomato and avirulent on tobacco still expresses parAl in the compatible interaction. Therefore, some P. parasitica isolates may evade tobacco recognition by down regulating parA1 in planta. The in planta down regulation of parA1 may constitute a suitable mechanism for P. parasitica to infect tobacco without deleterious consequences for the pathogen.     

Microsomal Membrane Changes during the Ripening of Apple Fruit

The changes in leakage and viscosity of microsomal membranes from apples (Malus sylvestris cv Calville de San Sauveur) at different stages of ripening were examined. These changes were correlated with those in the lipid composition of the membranes, sterols, phospholipids, and fatty acids of the phospholipids. The greatest changes in membrane properties occurred as the fruit reached its climacteric and this corresponded with a change in the sterol:phospholipid ratio in the membranes. Changes were also found in fatty acid unsaturation level, but primarily in the postclimacteric stage of ripening.     

Diverse subcellular locations of cryptogein-induced reactive oxygen species production in tobacco Bright Yellow-2 cells

Reactive oxygen species (ROS) play a crucial role in many cellular responses and signaling pathways, including the oxidative burst defense response to pathogens. We have examined very early events in cryptogein-induced ROS production in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells. Using Amplex Red and Amplex Ultra Red reagents, which report real-time H2O2 accumulation in cell populations, we show that the internal signal for H2O2 develops more rapidly than the external apoplastic signal. Subcellular accumulation of H2O2 was also followed in individual cells using the 2',7'-dichlorofluorescein diacetate fluorescent probe. Major accumulation was detected in endomembrane, cytoplasmic, and nuclear compartments. When cryptogein was added, the signal developed first in the nuclear region and, after a short delay, in the cell periphery. Interestingly, isolated nuclei were capable of producing H2O2 in a calcium-dependent manner, implying that nuclei can serve as a potential active source of ROS production. These results show complex spatial compartmentalization for ROS accumulation and an unexpected temporal sequence of events that occurs after cryptogein application, suggesting novel intricacy in ROS-signaling cascades.     

Solution structure of a tobacco lipid transfer protein exhibiting new biophysical and biological features

Plant lipid transfer proteins are small soluble extracellular proteins that are able to bind and transfer a variety of lipids in vitro. Recently, it has been proposed that lipid transfer proteins may play a key role in plant defence mechanisms, especially during the induction of systemic acquired resistance. However, very little is known about the proteins expressed in developing plants and tissues, since almost all the biophysical and structural data available to date on lipid transfer proteins originate from proteins present in storage tissues of monocot cereal seeds. In this paper, we report the structural and functional characteristics of a lipid transfer protein (named LTP1_1) constitutively expressed in young aerial organs of Nicotiana tabacum (common tobacco). The unlabelled and uniformly labelled proteins were produced in the yeast Pichia pastoris, and we determined the three-dimensional (3D) structure of LTP1_1 using nuclear magnetic resonance (NMR) spectroscopy and molecular modeling techniques. The global fold of LTP1_1 is very close to the previously published structures of LTP1 extracted from cereal seeds, including an internal cavity. However, the chemical shift variations of several NMR signals upon lipid binding show that tobacco LTP1_1 is able to bind only one LysoMyristoylPhosphatidylCholine (LMPC), while wheat and maize LTPs can bind either one or two. Titration experiments using intrinsic tyrosine fluorescence confirm this result not only with LMPC but also with two fatty acids. These differences can be explained by the presence in tobacco LTP1_1 of a hydrophobic cluster closing the second possible access to the protein cavity. This result suggests that LTP1 lipid binding properties could be modulated by subtle changes in a conserved global structure. The biological significance of this finding is discussed in the light of the signalling properties of the tobacco LTP1_1-jasmonate complex described elsewhere.