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K⁺ channels of Cf-9 transgenic tobacco guard cells as targets for Cladosporium fulvum Avr9 elicitor-dependent signal transduction

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PLANT JOURNAL 1999年第4期19卷 453-462页

作者： Blatt, MR Grabov, A Brearley, J Hammond-Kosack, K Jones, JDG Univ London Wye Coll Lab Plant Physiol & Biophys Wye TN25 5AH Kent England John Innes Ctr Plant Sci Res Sainsbury Lab Norwich NR4 7UH Norfolk England

The Cf-9 gene encodes an extracytosolic leucine-rich repeat (LRR) protein that is membrane anchored near its C-terminus. The protein confers resistance in tomato to races of the fungus Cladosporium fulvum expressing the corresponding avirulence gene Avr9. In Nicotiana tabacum the Cf-9 transgene confers sensitivity to the Avr9 elicitor, and leads on elicitation to a subset of defence responses qualitatively similar to those normally seen in the tomato host. One of the earliest responses, both in the native and transgenic hosts, results in K+ salt loss from the infected tissues. However, the mechanism(s) underlying this solute flux and its control is poorly understood. We have explored the actions of Avr9 on Cf-9 transgenic Nicotiana using guard cells as a model. Much detail of guard cell ion channels and their regulation is already known. Measurements were carried out on intact guard cells in epidermal peels, and the currents carried by inward- (I-K,I-in) and outward-rectifying (I-K,I-out) K+ channels were characterized under voltage clamp. Exposures to Avr9-containing extracts resulted in a 2.5- to 3-fold stimulation of I-K,I-out and almost complete suppression of I-K,I-in within 3-5min. The K+ channel responses were irreversible. They were specific for the Avr9 elicitor, were not observed in guard cells of Nicotiana lacking the Cf-9 transgene and, from kinetic analyses, could be ascribed to changes in channel gating. Both K+ channel responses were found to be saturable functions of Avr9 concentration and were completely blocked in the presence of 0.5 mu M staurosporine and 100 mu M H7, both broad-range protein kinase antagonists. These results demonstrate the ability of the Cf-9 transgene to couple Avr9 elicitation specifically to a concerted action on two discrete K+ channels and they indicate a role for protein phosphorylation in Avr9/Cf-9 signal transduction leading to transport control.

关键词： 1-(5-异喹啉磺酰基)-2-甲基哌嗪/药理学分枝孢子菌属/致病力分枝孢子菌属/生理学导电性真菌蛋白质类/拮抗剂和抑制剂真菌蛋白质类/药理学离子通道闸门/药物作用动力学膜糖蛋白类/遗传学膜糖蛋白类/生理学膜电位/药物作用膜片钳术磷酰化/药物作用植物疾病/微生物学植物蛋白质类/遗传学植物蛋白质类/生理学植物基因修饰植物有毒钾/代谢钾通道/代谢钾通道内向整流钾通道双孔蛋白质丝氨酸苏氨酸激酶/拮抗剂和抑制剂蛋白质丝氨酸苏氨酸激酶/代谢信号传导/药物作用星状孢子素/药理学烟草/细胞学烟草/药物作用烟草/代谢烟草/微生物学动物

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Effect of killer toxin K1 on yeast membrane potential reported by the diS-C₃(3) probe reflects strain- and physiological state-dependent variations

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FOLIA MICROBIOLOGICA 1999年第3期44卷 283-288页

作者： Eminger, M Gásková, D Brodská, B Holoubek, A Stadler, N Sigler, K Charles Univ Inst Phys CR-12116 Prague Czech Republic Acad Sci Czech Republ Inst Microbiol CR-14220 Prague Czech Republic Univ Bonn Inst Bot D-53115 Bonn Germany

The rate and extent of uptake of the fluorescent probe diS-C-3(3) reporting on membrane potential in S. cerevisiae is affected by the strain under study, cell-growth phase, starvation and by the concentration of glucose both in the growth medium and in the monitored cell suspension under non-growth conditions. Killer toxin K1 brings about changes in membrane potential. In all types of cells tested, viz. in glucose-supplied stationary or exponential cells of the killer-sensitive strain S6/1 or a conventional strain RXII, or in glucose-free exponential cells of both strains, both active and heat-inactivated toxin slow down the potential-dependent uptake of diS-C-3(3) into the cells. This may reflect "clogging" of pores in the cell wall that hinders, but does not prevent, probe passage to the plasma membrane and its equilibration. The clogging effect of heat-inactivated toxin is stronger than that exerted by active toxin. In susceptible cells, ie. in exponential-phase glucose-supplied cells of the sensitive strain S6/1, this phase of probe uptake retardation is followed by an irreversible red shift in probe fluorescence maximum lambda(max) indicating damage to membrane integrity and cell permeabilization. A similar fast red shift in IZ,, signifying lethal cell damage was found in heat-killed or nystatin-treated cells.

关键词：羰花青/代谢荧光染料/代谢真菌蛋白质类/药理学杀伤因子酵母膜电位/药物作用真菌毒素类/药理学制霉菌素/药理学酿酒酵母菌/生理学

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Enhanced detection of chemiluminescence intensity during an oxidative burst reaction in suspension cultured Nicotiana tabacum cells through the use of ficoll

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ANALYTICAL BIOCHEMISTRY 2002年第1期306卷 152-154页

作者： Gerber, IB Dubery, IA RAU Univ Dept Biochem ZA-20060 Auckland Pk South Africa

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Rad54 protein is targeted to pairing loci by the Rad51 nucleoprotein filament

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MOLECULAR CELL 2000年第3期6卷 583-592页

作者： Mazin, AV Bornarth, CJ Solinger, JA Heyer, WD Kowalczykowski, SC Univ Calif Davis Div Biol Sci Microbiol Sect Davis CA 95616 USA Univ Calif Davis Sect Mol & Cellular Biol Davis CA 95616 USA

Rad51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. Rad51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA;Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature Rad51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to Rad51 within the filament. Reciprocally, the Rad51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the Rad51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.

关键词：腺苷三磷酸酶类/代谢细胞核/遗传学细胞核/代谢 DNA解旋酶类 DNA修复/药物作用 DNA修复/生理学 DNA修复酶类 DNA 单链/代谢 DNA 超螺旋/代谢 DNA结合蛋白质类/代谢 DNA结合蛋白质类/药理学大肠杆菌真菌蛋白质类/代谢真菌蛋白质类/药理学 Rad51重组酶酿酒酵母菌酿酒酵母蛋白质类种特异性人类

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Purification, characterization, and amino acid sequence of cerato-platanin, a new phytotoxic protein from Ceratocystis fimbriata f. sp platani

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第35期274卷 24959-24964页

作者： Pazzagli, L Cappugi, G Manao, G Camici, G Santini, A Scala, A Univ Florence Ist Patol & Zool Forestale & Agr I-50144 Florence Italy Univ Florence Dipartimento Sci Biochim I-50134 Florence Italy

A new phytotoxic protein (cerato-platanin) of about 12.4 kDa has been identified in culture filtrates of the Ascomycete Ceratocystis fimbriata f. sp, platani, the causal agent of canker stain disease. The toxicity of the pure protein was bioassayed by detecting the inducing necrosis in tobacco leaves. The pure protein also elicited host synthesis of fluorescent substances in tobacco and plane (Platanus acerifolia) leaves. We purified the protein from culture medium to homogeneity, Its complete amino acid sequence was determined;this protein consists of 120 amino acid residues, contains 4 cysteines (S-S-bridged), and has a high percentage of hydrophobic residues. The molecular weight calculated from the amino acid sequence agrees with that determined by mass spectrometry, suggesting that no post-transnational modification occurs. Searches performed by the BLAST program in data banks (Swiss-Prot, EBI, and GenBank(TM)) revealed that this protein is highly homologous with two proteins produced by other Ascomycete fungi, One, produced during infection of wheat leaves, is codified by the snodprot1 gene of Phaeosphaeria nodorum (the causal agent of glume blotch of wheat), whereas the other is the rAsp f13 allergen from Aspergillus fumigatus. Furthermore, the N terminus of cerato-platanin is homologous with that of cerato-ulmin, a phytotoxic protein belonging to the hydrophobin family and produced by Ophiostoma (Ceratocystis) ulmi, a fungus responsible for Dutch elm disease.

关键词：氨基酸序列子囊菌纲/化学子囊菌纲/致病力数据库事实型真菌蛋白质类/化学真菌蛋白质类/药理学分子序列数据真菌毒素类植物疾病/微生物学植物有毒序列比对序列分析烟草/药物作用烟草/微生物学

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Role of Suc1 in the activation of the cyclosome by protein kinase Cdk1 cyclin B

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第1期257卷 12-18页

作者： Shteinberg, M Hershko, A Technion Israel Inst Technol Biochem Unit B Rappaport Fac Med IL-31096 Haifa Israel Technion Israel Inst Technol Rappaport Inst Res Med Sci IL-31096 Haifa Israel

A large complex, called the cyclosome or anaphase-promoting complex, has specific and regulated protein-ubiquitin ligase activity that targets mitotic regulators (such as cyclin B) for degradation at the end of mitosis. In early embryonic cell cycles the cyclosome is inactive in the interphase, but is subsequently converted by protein kinase Cdk1/cyclin B to an active, phosphorylated form, in a process that includes an initial lag period. This time lag may be important to prevent premature self-inactivation of Cdk1/cyclin B before the end of mitosis. We have previously observed that the phosphorylated form of the cyclosome binds to Suc1, a protein that associates with Cdk1 and with phosphate-containing compounds. We now report that low, physiological concentrations of Suc1 stimulate the activation of the interphase form of the cyclosome by the protein kinase. When Suc1 was present from the beginning of the incubation together with protein kinase Cdk1/cyclin B, activation of the cyclosome took place with the normal lag kinetics. However, when interphase cyclosome was first incubated with protein kinase Cdk1/cyclin B without Suc1, the subsequent addition of Suc1 caused a rapid burst of cyclosome activation and the lag was completely abolished. These findings are consistent with the interpretation that following initial slow phosphorylations of the cyclosome by the protein kinase, Suc1 accelerates multiple phosphorylations that culminate in the full activation of the cyclosome. In support of this interpretation, we find that Suc1 stimulates the phosphorylation of several proteins in the preparation of interphase cyclosome and that the effect of Suc1 on phosphorylation was augmented by prior incubation of interphase cyclosome with protein kinase Cdk1/cyclinB. (C) 1999 Academic Press.

关键词：腺苷三磷酸/代谢 CDC2蛋白激酶/代谢催化作用/药物作用细胞周期蛋白质类细胞周期蛋白B/代谢剂量效应关系药物酶激活/药物作用真菌蛋白质类/代谢真菌蛋白质类/药理学组蛋白类/代谢分裂间期动力学连接酶类/代谢分子量磷酰化/药物作用蛋白质类/代谢酵母菌酿酒/酶学非洲粟酒人体酵母蛋白质类星状孢子素/药理学时间因素泛素蛋白连接酶复合物泛素蛋白连接酶类

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Interaction of calicheamicin γ₁^I and its related carbohydrates with DNA-protein complexes

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1999年第19期96卷 10643-10648页

作者： Sissi, C Aiyar, J Boyer, S Depew, K Danishefsky, S Crothers, DM Yale Univ Dept Chem New Haven CT 06511 USA Univ Padua Dept Pharmaceut Sci I-35131 Padua Italy Columbia Univ Dept Chem New York NY 10027 USA Sloan Kettering Inst Canc Res New York NY 10021 USA

We report studies of the contribution of DNA structure, holding the sequence constant, to the affinity of calicheamicin gamma(1)(I) and its aryltetrasaccharide moiety for DNA. We used polynucleotide chains as models of known protein-binding sequences [the catabolite activator protein (CAP) consensus sequence, AP-1 and cAMP response element (CRE) sites] in their free and protein-bound forms. The proteins were selected to provide examples in which the minor-groove binding site for the carbohydrate is (CAP) or is not (GCN4) covered by the protein. Additionally, peptides related to the GCN4 and CREB families, which have different bending effects on their DNA-binding sites, were used. We observe that proteins of the CREB class, which induce a tendency to bend toward the minor groove at the center of the site, inhibit drug-cleavage sites located at the center of the free AP-1 or CRE DNA sites. In the case of GCN4, which does not induce DNA bending, there is no effect on calicheamicin cleavage of the CRE site, but we observe a GCN4-induced rearrangement of the cutting pattern in the AP-1 site. This effect may arise from either a subtle local conformational rearrangement not accompanied by bending or a localized reduction in DNA flexibility. Whereas GCN4 binding is not inhibited by the calicheamicin aryltetrasaccharide, binding of CAP to its DNA target is significantly inhibited, and calicheamicin cutting of DNA at the center of the CAP-DNA complex site is strongly reduced by protein binding. This result probably reflects steric inhibition of drug binding by the protein.

关键词：氨基糖苷类抗菌药/化学抗菌药/代谢碱基序列结合竞争性 DNA/药物作用 DNA结合蛋白质类/代谢剂量效应关系药物电泳聚丙烯酰氨凝胶烯二炔类大肠杆菌/遗传学真菌蛋白质类/药理学分子序列数据核酸构象寡糖类/化学寡糖类/代谢多糖类/化学多糖类/遗传学多糖类/药理学蛋白激酶类/药理学酿酒酵母蛋白质类三糖类/化学三糖类/代谢

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Isolation and characterization of a novel antifungal peptide from Aspergillus niger

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1999年第3期263卷 646-651页

作者： Lee, DG Shin, SY Maeng, CY Jin, ZZ Kim, KL Hahm, KS Korea Res Inst Biosci & Biotechnol Peptide Engn Res Unit Taejon South Korea Yanbian Univ Coll Med Yanji 133000 Peoples R China

A novel antifungal peptide (termed as Anafp) was isolated from the culture supernatant of the filamentous fungi, Aspergillus niger, The whole amino acid sequence of Anafp was determined and the peptide was found to be composed of a single polypeptide chain with 58 amino acids including six cysteine residues. The peptide shows some degree of sequence homology to a cysteine-rich antifungal peptides reported from the seeds of Sinapis alba and Arabidopsis thaliana or the extracellular media of Aspergillus giganteus and Penicillium chrysogenumsome, Cysteine-spacing pattern of Anafp was similar to that of the antifungal peptide from Penicillium chrysogenum, The Anafp exhibited potent growth inhibitory activities against yeast strains as well as filamentous fungi at a range from 4 to 15 mu M. In contrast, Anafp did not show antibacterial activity against Echerichia coli and Bacillus subtilis even at 50 mu M. (C) 1999 Academic Press.

关键词：氨基酸序列抗生素类抗真菌/化学抗生素类抗真菌/分离和提纯抗生素类抗真菌/药理学拟南芥属/化学曲霉菌黑/化学细菌/药物作用半胱氨酸真菌蛋白质类/化学真菌蛋白质类/分离和提纯真菌蛋白质类/药理学真菌/药物作用微生物敏感性试验分子序列数据芥子植物/化学青霉产黄/化学植物药用种子/化学序列比对序列同源性氨基酸

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Oligandrin.: A proteinaceous molecule produced by the mycoparasite Pythium oligandrum induces resistance to Phytophthora parasitica infection in tomato plants

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PLANT PHYSIOLOGY 2000年第1期124卷 379-395页

作者： Picard, K Ponchet, M Blein, JP Rey, P Tirilly, Y Benhamou, N Univ Brest Lab Microbiol & Secur Alimentaire F-29200 Plouzane France INRA Unite Rech Sante Vegetale & Environm F-06606 Antibes France INRA UMR 692 Lab Phytopharm & Biochim Interact Cellulaires F-21034 Dijon France Univ Laval Ste Foy PQ G1K 7P4 Canada

A low-molecular weight protein, termed oligandrin, was purified to homogeneity from the culture filtrate of the mycoparasitic fungus Pythium oligandrum. When applied to decapitated tomato (Lycopersicon esculentum Mill. var. Prisca) plants, this protein displayed the ability to induce plant defense reactions that contributed to restrict stem cell invasion by the pathogenic fungus Phytophthora parasitica. According to its N-terminal sequence, low-molecular weight, acidic isoelectric Feint, ultraviolet spectrum, and migration profile, the P. oligandrum-produced oligandrin was found to share some similarities with several elicitins from other Phytophthora spp. and Pythium spp. However, oligandrin did not induce hypersensitive reactions. A significant decrease in disease incidence was monitored in oligandrin-treated plants as compared with water-treated plants. Ultrastructural investigations of the infected tomato stem tissues from non-treated plants showed a rapid colonization of all tissues associated with a marked host cell disorganization. In stems from oligandrin-treated plants, restriction of fungal growth to the outermost tissues and decrease in pathogen viability were the main features of the host-pathogen interaction. Invading fungal cells were markedly damaged at a time when the cellulose component of their cell walls was quite well preserved. Host reactions included the plugging of intercellular spaces as well as the occasional formation of wall appositions at sites of potential pathogen entry. In addition, pathogen ingress in the epidermis was associated with the deposition of an electron-opaque material in most invaded intercellular spaces. This material, lining the primary walls, usually extended toward the inside to form deposits that frequently interacted with the wall of invading hyphae. In the absence of fungal challenge, host reactions were not detected.

关键词：藻蛋白质类/化学藻蛋白质类/分离和提纯藻蛋白质类/药理学氨基酸序列载体蛋白质类色谱法高压液相真菌蛋白质类/化学真菌蛋白质类/分离和提纯真菌蛋白质类/药理学胶体金番茄/微生物学番茄/超微结构显微镜检查电子分子序列数据疫霉属/致病力植物疾病/微生物学腐霉菌属/化学序列比对序列分析蛋白质

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Synthesis of a Trichoderma chitinase which affects the Sclerotium rolfsii and Rhizoctonia solani cell walls

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FOLIA MICROBIOLOGICA 1999年第1期44卷 45-49页

作者： Lima, LHC De Marco, JL Ulhoa, CJ Felix, CR Empresa Brasileira Pesquisa Agropecuaria Cent Nacl Pesquisa Recursos Genet & Biotecnol BR-70770900 Brasilia DF Brazil Univ Brasilia Dept Biol Celular BR-70910900 Brasilia DF Brazil Univ Fed Goias Dept Bioquim BR-74001970 Goiania Go Brazil

A Trichoderma sp. isolate, hereafter called Tg, produces a 46-kDa endochitinase (CHIT 46) which had been shown to drastically affect in vitro the cell walls of the phytopathogens Sclerotium rolfsii and Rhizoctonia solani. We attempted to gain insight into its properties. The CHIT 46 N-terminal amino acid sequence shares a very high homology with other fungal chitinases. Western blot analysis using polyclonal antibodies anti-CHIT 46 revealed that this enzyme is immunologically distinct from other proteins produced by the same Trichoderma isolate T-6, but is immunologically identical with proteins having equivalent molar mass, probably chitinases, produced by other Trichoderma spp. isolates. In addition, the antibodies revealed also that a substantial amount of this enzyme is secreted into the culture medium 2 d after the Trichoderma isolate T-6 comes into contact with chitin.

关键词：印迹法蛋白质细胞壁/药物作用电泳聚丙烯酰氨凝胶真菌蛋白质类/化学真菌蛋白质类/分类真菌蛋白质类/遗传学真菌蛋白质类/免疫学真菌蛋白质类/药理学丝核菌属/药物作用序列同源性木霉属/酶学木霉属/生长和发育动物小鼠

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