Alpha-picolinic acid (PA),a metabolite of tryptophan and an inducer of apoptosis in the animal cell,has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistan...Alpha-picolinic acid (PA),a metabolite of tryptophan and an inducer of apoptosis in the animal cell,has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here,we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS),rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride,indicating that the generation of reactive oxygen species was NADPHoxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence,it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice,which also provides a simple eliciting tool for studying apoptosis in the plant cell.展开更多
Plant diseases heavily affct plant growth and crop yield even in modern agriculture. Control its difficult because pathogens mutate frequently, and this leads in frequent breaking of disease resistance in commercial c...Plant diseases heavily affct plant growth and crop yield even in modern agriculture. Control its difficult because pathogens mutate frequently, and this leads in frequent breaking of disease resistance in commercial cultivars. The excessive application of chemical pesticides is not only producing pesticide-resistant pathogens, but it is harming the environment threatening the health of human beings. Therefore, the use of biological control agents (BCA) may provide an environmental friendly alternative to chemicals for plant disease control. Hypersensitive response (HR) and systemic acquired resistance (SAR) are the typical expressions of plant defense reactions. Once SAR is established,, the plants exhibits a broad-spectrum of disease resistance against pathogen attack. Researchers have identified elicitor proteins, such as elicitins and harpins, which activate plant defense reactions. It would be useful to explore the possibility of using biological control agents to induce a status of SAR in crop plants. Trichoderma viride is an ubiquitous soil saprophyte and a biological control agent acting by competition for nutrients, antibiosis, and mycoparasitism. If T. viride could be used as a producer and carrier of an elicitor protein, it may be used as a novel BCA specifically active on some plants. To test this possibility, we used cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea, to bio-engineering T. viride . The plasmid containing the Crypt gene or its mutated form, was introduced into T. viride genome by using the restriction enzyme mediated integration (REMI) method. The transformed T. viride was able to produce the Crypt protein and to improve disease resistance when the mutants were applied on tobacco plants. In summary our study included: 1. Construction of pCSNTCC and pCSNTCCm plasmids: Crypt gene was mutated by changing the K at position 13 of Crypt into a V (the mutated form was named CryK13V) as described elsewhere. In order allow secretion of the transgenic protein in T. viride cells, a signal sequence of a chitinase gene from Trichoderma (ThChi) was fused to the 5’ end of Crypt and CryK13V. The chimeric genes were placed under the control of trpC promoter in the vector pCSN43. A hygromycin resistant gene was introduced into the vectors, thus obtaining the plasmids pCSNTCC (for Crypt gene) and pCSNTCCm (CrypK13V) . 2. Establishment of a T. viride transformation system:The optimum conditions for T. viride protoplasts isolation and regeneration from were determined. For protoplast isolation, 24 hours-old hyphae of T. viride were digested with 4 mg/mL of Glucanex in phosphate buffer (pH 6.98) for 4 hours at 30 ℃, with a protoplast yield of 4.7×107 colony forming unit/mL. The maximum regeneration rate (14.5%) was obtained in the CM medium containing 0.3 mol/L KCl and 0.3 mol/L inositol. Plasmids pCSNTCC and pCSNTCCm were transformed into the protoplasts of T. viride by a Xho I restriction enzyme-mediated integration, with an efficiency of 1-2 transformants per microgram of DNA. Thirty transformants were obtained, TV-1 to TV-20 for Crypt gene and TV-21 to TV-30 for CrypK13V gene. The presence of the hygromycin resistance gene in the transformants was determined by polymerase chain reactions. The elicitor protein was detected in the culture media by western blot analysis but not inside the cells. The result indicated that the exogenous gene was expressed in T. viride , but the transgenic protein was entirely secreted into the culture media. 3. Expression of Crypt gene in T. viride enhanced plant disease resistance:Tobacco plants (4-6 week-old) were treated with spores of the transgenic or the wild-type T. viride applied to the soil. After ten days the plants or detached leaves were inoculated with Phytophthora parasitica var nicotianae, Alternaria alternata, Pseudomonas syringae pv. tabaci (Pst), or Tobacco mosaic virus (TMV). The lesions caused by TMV were suppressed by the treatment with the transgenic T. viride as compared with the wild-type展开更多
WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cD...WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of Os- WRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.展开更多
Cryptogein (Crypt), an elicitin secreted from Phytophthora cryptogea, was used for genetic engineering of biotic and abiotic resistance plants. We generated trans-genic tobacco plants harboring a rice phenylalanine am...Cryptogein (Crypt), an elicitin secreted from Phytophthora cryptogea, was used for genetic engineering of biotic and abiotic resistance plants. We generated trans-genic tobacco plants harboring a rice phenylalanine ammo-nia-lyase (PAL) promoter and Crypt fusion gene (PAL::Crypt) or the mutated Crypt (mutation of the lysine at the position 13 to valine) under the control CaMV35S promoter (CaMV35S::CryK13V). T2 progeny of the transgenic plants showed significantly enhanced disease resistance to patho-gens of fungal Phytophthora parasitica var nicotiana (Ppn) and Alternaria alternata, and bacterial Pseudomonas syringae pv tabaci. The amount of mRNA accumulation of Crypt and CryK13V was quite low in the transgenic lines analyzed by Northern blot, and was detected by a reverse transcription PCR method. Plants harboring PAL::Crypt construct showed faster and stronger induction of PR-1a gene after Ppn inoculation than that in the wild-type plants. The re-sults suggested that the inducible PAL promoter could rap-idly respond to pathogen attack and efficiently suppress the pathogen infection. Furthermore, the enhanced tolerance to salt stress in both of the Crypt and CryK13V expressing tobacco plants was also observed compared with that in the control plants. The constitutive expression of PR and tran-scription factor genes in the transformants was probably associated with the salt tolerance. The above observations suggested that a cross-talk between biotic and abiotic stresses existed in tobacco plants.展开更多
基金supported by the National Natural Science Foudation of China(30125030)the Chinese Academy of Sciences project(KSCX2-SW-301-02)Z.He is a fellow of the CAS“One-Hundred Talent”program.
文摘Alpha-picolinic acid (PA),a metabolite of tryptophan and an inducer of apoptosis in the animal cell,has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here,we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS),rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride,indicating that the generation of reactive oxygen species was NADPHoxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence,it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice,which also provides a simple eliciting tool for studying apoptosis in the plant cell.
文摘Plant diseases heavily affct plant growth and crop yield even in modern agriculture. Control its difficult because pathogens mutate frequently, and this leads in frequent breaking of disease resistance in commercial cultivars. The excessive application of chemical pesticides is not only producing pesticide-resistant pathogens, but it is harming the environment threatening the health of human beings. Therefore, the use of biological control agents (BCA) may provide an environmental friendly alternative to chemicals for plant disease control. Hypersensitive response (HR) and systemic acquired resistance (SAR) are the typical expressions of plant defense reactions. Once SAR is established,, the plants exhibits a broad-spectrum of disease resistance against pathogen attack. Researchers have identified elicitor proteins, such as elicitins and harpins, which activate plant defense reactions. It would be useful to explore the possibility of using biological control agents to induce a status of SAR in crop plants. Trichoderma viride is an ubiquitous soil saprophyte and a biological control agent acting by competition for nutrients, antibiosis, and mycoparasitism. If T. viride could be used as a producer and carrier of an elicitor protein, it may be used as a novel BCA specifically active on some plants. To test this possibility, we used cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea, to bio-engineering T. viride . The plasmid containing the Crypt gene or its mutated form, was introduced into T. viride genome by using the restriction enzyme mediated integration (REMI) method. The transformed T. viride was able to produce the Crypt protein and to improve disease resistance when the mutants were applied on tobacco plants. In summary our study included: 1. Construction of pCSNTCC and pCSNTCCm plasmids: Crypt gene was mutated by changing the K at position 13 of Crypt into a V (the mutated form was named CryK13V) as described elsewhere. In order allow secretion of the transgenic protein in T. viride cells, a signal sequence of a chitinase gene from Trichoderma (ThChi) was fused to the 5’ end of Crypt and CryK13V. The chimeric genes were placed under the control of trpC promoter in the vector pCSN43. A hygromycin resistant gene was introduced into the vectors, thus obtaining the plasmids pCSNTCC (for Crypt gene) and pCSNTCCm (CrypK13V) . 2. Establishment of a T. viride transformation system:The optimum conditions for T. viride protoplasts isolation and regeneration from were determined. For protoplast isolation, 24 hours-old hyphae of T. viride were digested with 4 mg/mL of Glucanex in phosphate buffer (pH 6.98) for 4 hours at 30 ℃, with a protoplast yield of 4.7×107 colony forming unit/mL. The maximum regeneration rate (14.5%) was obtained in the CM medium containing 0.3 mol/L KCl and 0.3 mol/L inositol. Plasmids pCSNTCC and pCSNTCCm were transformed into the protoplasts of T. viride by a Xho I restriction enzyme-mediated integration, with an efficiency of 1-2 transformants per microgram of DNA. Thirty transformants were obtained, TV-1 to TV-20 for Crypt gene and TV-21 to TV-30 for CrypK13V gene. The presence of the hygromycin resistance gene in the transformants was determined by polymerase chain reactions. The elicitor protein was detected in the culture media by western blot analysis but not inside the cells. The result indicated that the exogenous gene was expressed in T. viride , but the transgenic protein was entirely secreted into the culture media. 3. Expression of Crypt gene in T. viride enhanced plant disease resistance:Tobacco plants (4-6 week-old) were treated with spores of the transgenic or the wild-type T. viride applied to the soil. After ten days the plants or detached leaves were inoculated with Phytophthora parasitica var nicotianae, Alternaria alternata, Pseudomonas syringae pv. tabaci (Pst), or Tobacco mosaic virus (TMV). The lesions caused by TMV were suppressed by the treatment with the transgenic T. viride as compared with the wild-type
基金This work was supported by the State Basic Research and Development Plan(G200001 6203)the National Natural Science Foundation of China(Grant Nos.30370139&30471122).
文摘WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of Os- WRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.
文摘Cryptogein (Crypt), an elicitin secreted from Phytophthora cryptogea, was used for genetic engineering of biotic and abiotic resistance plants. We generated trans-genic tobacco plants harboring a rice phenylalanine ammo-nia-lyase (PAL) promoter and Crypt fusion gene (PAL::Crypt) or the mutated Crypt (mutation of the lysine at the position 13 to valine) under the control CaMV35S promoter (CaMV35S::CryK13V). T2 progeny of the transgenic plants showed significantly enhanced disease resistance to patho-gens of fungal Phytophthora parasitica var nicotiana (Ppn) and Alternaria alternata, and bacterial Pseudomonas syringae pv tabaci. The amount of mRNA accumulation of Crypt and CryK13V was quite low in the transgenic lines analyzed by Northern blot, and was detected by a reverse transcription PCR method. Plants harboring PAL::Crypt construct showed faster and stronger induction of PR-1a gene after Ppn inoculation than that in the wild-type plants. The re-sults suggested that the inducible PAL promoter could rap-idly respond to pathogen attack and efficiently suppress the pathogen infection. Furthermore, the enhanced tolerance to salt stress in both of the Crypt and CryK13V expressing tobacco plants was also observed compared with that in the control plants. The constitutive expression of PR and tran-scription factor genes in the transformants was probably associated with the salt tolerance. The above observations suggested that a cross-talk between biotic and abiotic stresses existed in tobacco plants.
