利用启动子陷阱技术（Promoter trapping）构建稻瘟病菌（Magnaporthe grisea）突变体库
摘要
稻瘟病是水稻上重要的病害之一，其病原为子囊菌Magnaporthe grisea。了解M.grisea的致病机理不仅有利于稻瘟病的防治，而且作为研究植物病原真菌与寄主互作的理想模式系统，对于了解其它真菌的致病机理也有重要意义。致病相关基因的克隆和分析是达到这一目标的有效途径。突变体的获得及表型分析是鉴定致病相关基因的有效策略。
获得突变体的方法很多，如限制性内切酶介导的转化，农杆菌介导的转化，转座子标签技术等。然而，这些方法不能研究以下基因：在实验室条件下功能上多余的基因；在多个水平上都有作用的基因；低水平表达的重要基因。启动子陷阱的方法，并不需要产生可见的功能缺陷，只需根据报告基因的表达就可将转化子筛选出来，然后通过启动子陷阱载体上的已知序列克隆目的基因。本研究，将启动子陷阱的技术应用于稻瘟病菌，通过构建启动子陷阱载体，利用限制性内切酶介导转化，建立的突变体库。研究结果如下：
1．构建了启动子陷阱载体pEGFP-HPH和pCB1003-EGFP，均具有具有潮霉素抗性基因和无启动子的eGFP基因；
2．建立了稻瘟病菌遗传转化体系，转化效率达到40-60个突变体／微克DNA；
3．获得突变体1077株，通过荧光显微镜检测，489株突变体荧光表达，eGFP基因表达率45.4％；生长速度明显比野生型减慢的突变体3株；菌落白色的突变体9株；产孢减少的有35株；致病性有所降低的菌株18株；随机挑取8株突变体进行Southern杂交分析，可以杂交到强弱不同的信号。
实验数据表明，启动子陷阱技术具有良好的前景，通过这种方法，我们可以得到许多在形态、代谢、致病性等各个方面有变化的突变体。
Magnaporthe grisea is a causal agent of rice blast and diseases of many other different grasses. Understanding of the molecular basis of this disease is not only benificial for rice blast control, but also can serve as a model for revealing of other fungal pathogen-plant interactions. Obviously, identification of genes required for pathogenicity is a key step toward achieving this goal. The parastic interaction between the pathogenic fungus and its plant host is a complex biological process. Mutation analysis of pathogen is a powerful strategy in study of this process. To elucidate the mechanisms of the pathogenesis, we clone genes by using of insertional mutagenesis. As we know, strategies, such as REMI, ATMT and trasnposon tagging have cloned many interesting genes in many plant pathogenic fungi so far. However, those methods will not permit isolation of genes without clear mutant phenotypes during development either because of gene redundancy or due to the gene product not being essential for development
under the conditions in the laboratory. One way to gain access to such genes is to use a method called promoter trapping, in which expression of a reporter gene can be initiated only from an endogenous promoter because the reporter gene lacks its own promoter. In this method, it is not necessary to obtain the obvious phenotype mutants. Here we discribe this promoter trapping method for M. grisea that depends upon restriction enzyme-mediated insertion of a vector carrying a promoterless eGFP (green fluorescent protein) gene. New promoter trapping vectors pEGFP-HPH and pCB 1003-EGFP have been developed to trap genes in M. grisea. The redults are shown as follows:
1. New promoter trapping vectors pEGFP-HPH and pCB1003-EGFP have been constructed which possess the marker hygromycin gene and a promoterless eGFP gene;
2. Transformation system is stabilized. The frequency of the transformation is 40-60mutants/ug DNA;
3. We constructed a library in Magnaporthe grisea which consisted of 1077
mutants. eGFP expression was detected by fluoroscopy. eGFP expression in 489 mutants. The frequency of the eGFP expression is 45.4%. By analysing of the 489 mutants, 3 mutants grow slowly, 9 mutants'clony are white, the spore production of 35 mutants are decreased and 18 mutants deduced in pathogenecity. We use Southern blot analysis to check the library and get the different signals. These data indicated that the promoter trapping technique has a good future. By
analyzing the mutants, we can get the useful mutants which have defects in
pathogenicity, metabolism, and alterations in morphology.
获得突变体的方法很多，如限制性内切酶介导的转化，农杆菌介导的转化，转座子标签技术等。然而，这些方法不能研究以下基因：在实验室条件下功能上多余的基因；在多个水平上都有作用的基因；低水平表达的重要基因。启动子陷阱的方法，并不需要产生可见的功能缺陷，只需根据报告基因的表达就可将转化子筛选出来，然后通过启动子陷阱载体上的已知序列克隆目的基因。本研究，将启动子陷阱的技术应用于稻瘟病菌，通过构建启动子陷阱载体，利用限制性内切酶介导转化，建立的突变体库。研究结果如下：
1．构建了启动子陷阱载体pEGFP-HPH和pCB1003-EGFP，均具有具有潮霉素抗性基因和无启动子的eGFP基因；
2．建立了稻瘟病菌遗传转化体系，转化效率达到40-60个突变体／微克DNA；
3．获得突变体1077株，通过荧光显微镜检测，489株突变体荧光表达，eGFP基因表达率45.4％；生长速度明显比野生型减慢的突变体3株；菌落白色的突变体9株；产孢减少的有35株；致病性有所降低的菌株18株；随机挑取8株突变体进行Southern杂交分析，可以杂交到强弱不同的信号。
实验数据表明，启动子陷阱技术具有良好的前景，通过这种方法，我们可以得到许多在形态、代谢、致病性等各个方面有变化的突变体。
Magnaporthe grisea is a causal agent of rice blast and diseases of many other different grasses. Understanding of the molecular basis of this disease is not only benificial for rice blast control, but also can serve as a model for revealing of other fungal pathogen-plant interactions. Obviously, identification of genes required for pathogenicity is a key step toward achieving this goal. The parastic interaction between the pathogenic fungus and its plant host is a complex biological process. Mutation analysis of pathogen is a powerful strategy in study of this process. To elucidate the mechanisms of the pathogenesis, we clone genes by using of insertional mutagenesis. As we know, strategies, such as REMI, ATMT and trasnposon tagging have cloned many interesting genes in many plant pathogenic fungi so far. However, those methods will not permit isolation of genes without clear mutant phenotypes during development either because of gene redundancy or due to the gene product not being essential for development
under the conditions in the laboratory. One way to gain access to such genes is to use a method called promoter trapping, in which expression of a reporter gene can be initiated only from an endogenous promoter because the reporter gene lacks its own promoter. In this method, it is not necessary to obtain the obvious phenotype mutants. Here we discribe this promoter trapping method for M. grisea that depends upon restriction enzyme-mediated insertion of a vector carrying a promoterless eGFP (green fluorescent protein) gene. New promoter trapping vectors pEGFP-HPH and pCB 1003-EGFP have been developed to trap genes in M. grisea. The redults are shown as follows:
1. New promoter trapping vectors pEGFP-HPH and pCB1003-EGFP have been constructed which possess the marker hygromycin gene and a promoterless eGFP gene;
2. Transformation system is stabilized. The frequency of the transformation is 40-60mutants/ug DNA;
3. We constructed a library in Magnaporthe grisea which consisted of 1077
mutants. eGFP expression was detected by fluoroscopy. eGFP expression in 489 mutants. The frequency of the eGFP expression is 45.4%. By analysing of the 489 mutants, 3 mutants grow slowly, 9 mutants'clony are white, the spore production of 35 mutants are decreased and 18 mutants deduced in pathogenecity. We use Southern blot analysis to check the library and get the different signals. These data indicated that the promoter trapping technique has a good future. By
analyzing the mutants, we can get the useful mutants which have defects in
pathogenicity, metabolism, and alterations in morphology.
引文
1. Adachi K, Hamer JE. Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea, Plant Cell, 1998,10(8):1361-74.
2. Adams MD, Kelley J-M, Gocayne JD, et al. Complementary DNA sequencing: Expressed sequence tags and human genome project. Science, 1991, 252:1651-1656
3. AhnN, Kim S, Choi W, Im KH, Lee YH, Extracellular matrix protein gene, EMP1, is required for appressorium form ationand pathogenicity of the rice blast fungus, Magmaporthe grisea Mol Cells. 2004, 17(1):166-73.
4. Asiegbu FO, Choi W, Jeong JS, Dean RA. Cloning, sequencing and functional analysis of Magnaporthe grisea MVP1 gene, a hex-1 homolog encoding a putative 'woronin body' protein. FEMS Microbiol Lett. 2004, 230(1):85-90.
5. Baker B, Zambryski P, Staskawicz B, Dinesh-gumar SP, Signaling in plant-microbe interactions, Science, 1997, 276:726-733
6. Balhadère PV, Foster AJ, Talbot NJ, Identification of pathogenicity mutants of the rice blast gungus Nagnaporthe grisea by insertional mutagnesis, MPMI, 1999, 12:129-142
7. Balhadère PV, Talbot NJ, Fungal infection of plants: using Magnaporthe grisea as a model for understanding fungal pathogenesis. Annual Plant Reviews,4: (Eds. J. Roberts, M. Dickinson, Sheffield Academic Press). (2000)pp 1-25
8. Balhadère PV, Talbot NJ, Fungal pathogenicity-establishing infection. In: Dickson M(ed.) Molecular Plant Pathology(Annual plant Review, 2000, 4:1-25
9. Balhadère PV, Talbot NJ, PDE1 encodes a P-typeATPase involved in appressorium mediated plant infection by Magnaporthe grisea. The Plant Cell, 2001, 13:1-19
10. Beckerman JL, Ebbole DJ, MPG1, a gene encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition. MPMI. 1996, 9:450-456.
11. Bellen HJ, O'Kane CJ, Wilson C, Grossniklaus U, Pearson RK, and Gehring WJ, 1989, P-element -mediated enhancer detection: a versatile method to study
development in Drosophila, Genes Dev, 1989, 3(9):1288-300
12. Bellen HJ, Ten years of enhancer detection: Lessons from the fly, Plant Cell, 1999, 11:2271-2281.
13. Blker M, Bhnert HU, Braun KH, Grl J and Kahmann R, Tagging pathogenicity genes in Ustilagomaydisby restriction enzyme-mediated integration (PEMI). Mol. Gen. Genet. 1995, 248: 547-552
14. Bourett T, Howard R, In vitro development ofpenetration structures in the rice blast fungus Magnaporthe grisea. Can. J. Bot. 1990, 69:329-342.
15. Brown PO, Bostein D, Exploring the new world of genomic with DNA microarrays [J], Nature Genetics(Supplement), 1999, 21:33.
16. Casadaban MJ, and Cohen SN, Lactose genes fused to exogenous promoters in one step using a Mu-lac acteriophage: In vivo probe for transcriptional control sequences. Proc. Natl. Acad. Sci. USA, 1979, 76:4530-4533.
17. Choi W, DeannA, The adenylate cyclase gene MAClof magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 1997, 9:1973-1983.
18. Clergeot PH, Gourgues M, Cots J, Laurans F, Latorse MP, Pepin R, Tharreau D, Notteghem JL, and Lebrun MH, PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea PNAS, 2001, 98:6963-6968.
19. Covert SF, Kapoor P, Lee Mei-ho, et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinnatum, Mycological Research, 2001, 105(3):259-264
20. De Jong JC, Mccormack J, Sminoff N , Talbot NJ, Glycerol generates turgor in rice blast. Nature, 1997, 389, 244-255.
21. DeRisi J, Penland L, Brown PO, et al. Use of a cDNA microarray to analysis gene expression patterns in human cancer [J] , Nat Genet, 1996, 14:457.
22. DeZwaan TM, Carroll AM, Valent B, Magnaporthe grisea pthllp is a novel plasma membrane protein that mediates appressorium differenciation in response to inductive substrate cues. Plant cell, 1999, 11(10):2013-2030
23. Dixon KP, XuJR, Smirnoff N, Talbot NJ. Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea, Plant Cell, 1999, 11(10):2045-58.
24. Epstein L, Lusnak K. and Kaur S. Transformation-mediated developmental mutants of Glomerella graminicola (Colletotrichum graminicola). Fungal Genet. Biol. 1998, 23:189-203
25. Fang EG, Dean RA, Site-directed mutagnesis of the magB gene affects growth and development in Magnaporthe grisea, MPMI, 2000, 13(11),1214-1227
26. Faria-Campos AC, Cerqueira GC, Anacleto C, de Carvalho CM, Ortega JM. Mining microorganism EST databases in the quest for new proteins. Genet Mol Res. 2003, 31;2(1):169-77.
27. ProeligerEH, Carpenter BE, NUTI a major nitrogen regulatory gene in Magnaporthe grisea, is dispensable for pathogenicity. Molec. Gen Genet, 1996,251:647-656
28. Goffeau A, Barrell BG, Bussey H, et al. Life with 6000 genes. Science. 1996, 274:546,563-567
29. Gold SE, Carcia-Pedrajas MD, Martinez-Epinoza AD, New (and used) approaches to the study of fungal pathogenicity. Annu. Rev. Phytopathol. 2001, 39:337-365
30. Hamer JE, Horword RJ, Chumley FG, et al, A mechanism for surface attachment in spores of plant pathogenic fungus, Science, 1988,239,84:509-512
31. Hua-Van A, Hericourt F, Capy F, Daboussi M.-J. and Langin T, Three highly divergent subfamilies of the impala transposable element coexist in the genome of the fungus Fusarium oxysporum. Mol. Gen Genet. 1998, 259:354-362
32. Jelinsky S and Samson L. Global response of Saccharomyces cerevisiae to a al kylating agent,proc [J] .Natl Acad Sci USA, 1999, 96: 1486.
33. Jin-rong Xu, Chaoyang Xue, Time for a blast:genomics of Magnaporthe grisea, Molecular plant pathology, 2002, 3(3),173-176
34. Kahmann R. and Basse C. REMI (Restriction Enzyme Mediated Integration) and its impact on the isolation of pathogenicity genes in fungi attacking plants. Eur. J. Plant Pathol. 1999, 105:221-229
35. Kuspa A, Loomis WF, Tagging development genes in bictyostelium by restriction
enzyme-mediated integration of plasmid DNA. Proc Nail Acad Sci USA, 1992, 89:8803-8807
36. Linnemannstns P, Vob T, Hedden P, Gaskin P and Tudzynski B, Deletions in the gibberellin biosynthesis gene cluster of Gibberella fujikuroi by restriction enzyme-mediated integration and conventional transformation-mediated mutagenesis. Appl. Env. Microbiol. 1999, 65: 2558-2564
37. Liu S, Dean RA, G protein a subunit genes control growth, development, and pathogenicity of Magnaporthe grisea. MPMI, 1997, 10:1075-1086
38. Liu-S-j, Wei R-x, Arie T and Yamaguchi I, Obtaining insertion mutants in Magnaporthe grisea by restriction enzyme-mediated integration of plasmid. Chinese Journal of Genetics, 1998, 25:137-142
39. Liu-S-j, Wei R-x, Arie T and Yamaguchi I, REMI mutagenesis and identification of pathogenic mutants in Magnaporthe grisea (in Chinese). Chinese Journal of Biotechnology, 1998, 14:254-259
40. Lockhart DJ, Dong H, Byrne MC, et al, Expression monitoring by hybridization to high-density oligonucleotide arrays [J], Nature Biotechnology, 1997, 14: 1675.
41. Lu S, Lyngholm L, Yang G, Bronson C, Yoder OC and Turgen BG, Tagged mutations at the Toxl locus of CochlJobolus heterostrophus by restriction enzyme-mediated integration. Proc. Natl. Acad. Sci. USA ,1994, 91: 12649- 12653
42. Maier FJ, Schafer W, Mutagenesis via insertionalor restriction enzyme-mediated-integration (REMI) as a tool to tag pathogenicity related genes in plant pathogenic fungi. Biol. Chem. 1999, 380: 855-864
43. Marshall A, Hodgson J, DNA chips: an array of possibilities [J], Nature biotechnology, 1998, 16:731
44. Mishra NC, Tatum EL. Non-Mendelian inheritance of DNA-induced inositol independence in Neurospora. Proc Natl Acad Sci USA. 1973, 70(12):3875-9.
45. Mitchell TK, Dean RA, The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea. Plant Cell, 1995, 7:1869-1878.
46. Motoyama T, Imanishi K, Yamaguchi I, cDNA cloning, expression and mutagnesis of scytalone dehydratase needed for pathogenicity of the rice blast fungus, Pyricularia grisea. Biosci Biotechnol Biochem, 1998, 62:564-566
47. Mullins ED, Kang S, Transformation: a tool for studying fungal pathogens of plants, Cellular and Molecular Life Sciences, 2001, 58 (14), 2043-2052
48. Mullins ED, Chen PX, Romaine P et al, Agrobacterium tumefaciens-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 2001, 91:173-180
49. Mullins ED, Kang S, Transformation: a tool for studying fungal pathogens of plants, CMLS, 2001, 58:2043-2052
50. Nishimura M, Park G, Xu JR. The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Mafnaporthe grisea. Mol Microbiol. 2003, 50(1):231-43.
51. Niwa H, Araki K, Kimura S, Taniguchi S, Wakasugi S, Yamamura K, An efficient gene-trap method using poly A trap vectors and characterization of gene-trap events, J Biochem (Tokyo), 1993, 113(3):343-9
52. OUSH. Rice disease. 2nd ed. (M). Surrey, Commonwealth Mycology Institute, 1985
53. Park C H, Kim S, Park J Y, Ahn IP, Jwa NS, Im KH, Lee YH. Molecular characterization of a pathogenesis-related protein 8 gene encoding a class Ⅲ chitinase in rice. Mol Cells. 2004, 17(1):144-50.
54. Park SM, Choi ES, Kim MJ, Cha BJ, Yang MS, Kim DH. Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress. Mol Microbiol. 2004, 51(5):1267-77
55. Park, G., Xue, C., Zheng, L., Lam, S. and Xu, J.R.. (2002). MST12 regulates infectious growth but not appressorium formation in the rice blast fungus Magnaporthe grisea. MPMI. 15, 183-92.
56. Patricia S. Springer, Gene Traps: Tools for Plant Development and Genomics, The Plant Cell, 2000, 12:1007-1020
57. Paul B. Rainey, Adaptation of Pseudomonas fluorescens to the plant rhizosphere,
Environmental Microbiology ,1999, 1(3):243-257
58. Ramsay R. DNA chips: State-of-the-art [J].Nature Biotechnology, 1998, 16:40.
59. Rho HS, Kang S, Lee YH. Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea. Mol Cells. 2001, 12(3):407-11.
60. Rossman AY, Howard RJ, Valent B, Pricularia grisea the correct name for the rice blast disease fungus. Mycologia 1990, 82:509-512
61. Saitoh K, Arie T, Teraoka T, Yamaguchi I, Kamakura T. Targeted gene disruption of the neuronal calcium sensor 1 homologue in rice blast fungus, Magnaporthe grisea. Biosci Biotechnol Biochem. 2003, 67(3):651-3.
62. Saitoh K, Arie T, Teraoka T, Yamaguchi I, Kamakura T. Targeted gene disruption of the neuronal calcium sensor 1 homologue in rice blast fungus, Magnaporthe grisea. Biosci Biotechnol Biochem. 2003, 67(3):651-3.
63. Sanchez 0., Navarro R. E. and Aguirre J. Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (RFMI). Mol. Gen. Genet. 1998, 258:89-94
64. Schiestl R. H. and Petes T. D. Integration of DNA fragmentsby illegitimate recombination in Saccharomyces cerevisiae Proc. Natl. Acad. Sci. 1991, USA 88: 7585-7589
65. Shi Z, Christian D, Leung H, Enchanced transformation in Magnaporthe grisea by chemical and insertional mutagenesis. Phytopathol, 1995a, 85:329-333
66. Sweigard JA, Carroll AM, Kang S, Farrall L, Chumley FG, Yalent B, Identification cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus. Plant cell, 1995,7:1221-1233
67. Sweigard JA, Carroll AM, Kang S, Farrall L, Chumley FG, Valent B, Magnaporthe grisea pathogenecity genes obtained through insertional mutagenesis, MPMI, 1998, 11(5):404-412
68. Sweigard JA, Carroll AM, Valent B, Cloning and analysis of CUTI, a cutinase gene from Magnaporthe grisea. Mol. Gen. Genet. 1992a, 232:174-182
69. T Irie, H Matsumura, R Terauchi, H Saitoh, Serial analysis of gene expression
(SAGE) of Magnaporthe grisea: genes involved in appressorium formation, Mol Gen Genomics, 2003, 270:181-189
70. Takano Y, Choi W, Mitchell TK, Okuno T, and Dean RA. Large scale parallel analysis of gene expression during infection-related morphogenesis of Magnaporthe grisea. Molecular Plant Pathology. 2003, 4, 337
71. Talbot NJ, Having a blast: exploring the pathogenicity of Magnaporthe grisea. Trends Microbiol. 1995, 3:9-16
72. Talbot NJ, Kershaw, M J, Wakley GE, de Vries, O. M. H., Wessels, J. G. H., and Hamer, J. E. MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related of Nagnaporthe grisea. Plant Cell, 1996, 8, 985-999.
73. Tharreau D, Lebrun M-H, Talbot NJ, Notteghem JL, Advances in Rice Blast Research, 2000, pp. 376, Kluwer, Dordrecht
74. Thines E, Weber RW, Talbot NJ. MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea, Plant Cell, 2000, 12(9):1703-18.
75. Thomas SW, Glaring MA, Rasmussen SW et al, Transcript profiling in the barley mildrew pathogen Blumeria graminis by serial analysis of gene expression(SAGE), MPMI, 2002, 15:847-856
76. Thon MR, Nuckles EM, Vaillancourt LJ, Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrlchum graminicola. MPMI, 2000, 13: 1356-1365
77. Urban M, Bhargava T, Hamer JE. An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease. EMBO J. 1999, 18(3):512-21.
78. Velent B, Chumley FG, Molecular genetic analysis of the rice blast fungus Magnaporthe grisea Anun. Rev. Phytopathol, 1991, 29:443-467
79. Vidal-Cros A, Viviani F, Labesse G, Boccara M, Gaudry M., Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea. Purification, cDNA cloning and sequencing, Eur J Biochem, 1994, 1:219(3):985-92
80. Villaba F, Lebrun M-H, Hua-Van A, Daboussi M-J and Grosjean-Cournoyer M-C, Transposon impala, a novel tool for gene tagging in the rice blast fungus
Magnaporthe grisea. MPMI. 2001, 14(3): 308-315
81. Wang R, Hong G, Han B. Transcript abundance of rmll encoding a putative GT1-like factor in rice, is up-regulated by Magnaporthe grisea and down-regulated by light. Gene. 2004 Jan 7;324:105-15.
82. Weber RWS, Thines E, Wakley GE and Talbot NJ, The vacuole acts as a sink for lipid droplets during appressorium turgor generation by Magnaporthe grisea. Protoplasma, 2001, 216:101-112
83. Wen-Tsan Chang, Julian D. Gross, Peter C. Newell, Trapping developmental promoters in Dictyostelium, Plasmid, 1995, 34:175-183
84. Wodicka L, Dong H, Mittmann M, et al, Genome-wide expression monitoring in Saccharomyces cerevisiae [J] , Nature biotecnology, 1997,15:1359
85. Xu JR, Staiger CJ, Hamer JE. Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses. Proc Natl Acad Sci U S A. 1998, 95(21):12713-8.
86. Yang G, Rose MS, Turgeon BG et al, A polyketide synthase is required for the wA gene of Aspergillus nidulans. Genetics, 1996,126:81-90
87. Yang GP, Ross DT, Kuang WW, et al. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes [J], Nucl Res, 1999, 27: 1517-1523
88. Yun SH, TurgeonBG, Yoder OC, REMI-induced mutants of Mycosphaerella zeae-maydis lacking the polyketide PM-toxin are deficient in pathogenesis to corn. Physiol Mol Plant Pathol, 1998, 52:53-66
89. Zeigler RS, Recombination in Msgnaporthe grisea. Annu Rev Phytopathol, 1998, 36:249-75.
90. Zwiers LH, De Waad MA, Efficient Agrobacterium tumefaciens-mediated gene disruption in the phytopathogen Mycosphserella graminicola. Current Genetic, 2001, 39:388-393
91．J．萨姆布鲁克，D．W．拉塞尔，分子克隆实验指南(第三版)，2002，科学出版社
92．方卫国，张永军，肖月华，根癌农杆菌介导球孢白僵菌的遗传转化及T-DNA插入突变体
的获得，中国科学C辑
93．方卫国，张永军，杨星永等，根癌农杆菌介导真菌遗传转化的研究现状，中国生物工程杂志，2002，22(5)：40-44
94．何月秋，唐文华，水稻稻瘟病菌研究进展(一) 水稻稻瘟病菌多样性及变异机制，云南农大学学报，2001，16(1)：60-64
95．李宏宇，潘初沂，陈涵等，稻瘟病菌T-DNA插入方法优化及其突变体分析，生物工程学报，2003，7，V19(4)，419-423
96．林福呈，稻瘟病菌附着胞形成的细胞生物学，植物病理学报，2001，31(2)：97-101
97．林福呈，稻瘟病菌附着胞形成机理的研究，博士学位论文，1999，
98．林福呈，李德葆等，稻瘟病菌侵入机理，微生物学通报，24(3)：167-1270
99．陆凡，郑小波等，江苏省稻瘟病菌的毒性多样性及水稻品种的抗病性，生物多样性，2001，9(3)：201-206
100．沈瑛，李成云等，稻瘟病菌的菌丝融合现象及其后代致病性变异，中国农业科学，1997，30(6)：16-22
101．沈瑛等，我国稻瘟病菌的遗传多样性及其地理分布(英)，浙江农业大学学报，1998，24(5)：493-501
102．王洪凯，林福呈，李德葆，稻瘟病菌致病相关基因研究进展，菌物系统，2002，21(3)：459-464
103．徐炳森、邵健忠几种新型生物芯片的研究进展，生物化学与生物物理进展，2000，27(3)：251-253．
104．张正光，郑小波，稻瘟病菌不同小种菌株间无性重组的研究，植物病理学报，2000，30(4)：312-318
105．张正光，郑小波，稻瘟病菌营养体亲和和配对筛选及其亲和能力遗传，植物病理学报，1999．29(4)：304-308
http://cogeme.ex.ac.uk
http://fungi.fgl.ncsu.edu/tdsblastscript.html
http://www.broad.mit.edu/annotation/fungi/magnaporthe/index.html
http://www.ddbj.nig.ac.jp
http://www.ebi.ac.uk
http://www.ncbi.nlm.nih.gov
2. Adams MD, Kelley J-M, Gocayne JD, et al. Complementary DNA sequencing: Expressed sequence tags and human genome project. Science, 1991, 252:1651-1656
3. AhnN, Kim S, Choi W, Im KH, Lee YH, Extracellular matrix protein gene, EMP1, is required for appressorium form ationand pathogenicity of the rice blast fungus, Magmaporthe grisea Mol Cells. 2004, 17(1):166-73.
4. Asiegbu FO, Choi W, Jeong JS, Dean RA. Cloning, sequencing and functional analysis of Magnaporthe grisea MVP1 gene, a hex-1 homolog encoding a putative 'woronin body' protein. FEMS Microbiol Lett. 2004, 230(1):85-90.
5. Baker B, Zambryski P, Staskawicz B, Dinesh-gumar SP, Signaling in plant-microbe interactions, Science, 1997, 276:726-733
6. Balhadère PV, Foster AJ, Talbot NJ, Identification of pathogenicity mutants of the rice blast gungus Nagnaporthe grisea by insertional mutagnesis, MPMI, 1999, 12:129-142
7. Balhadère PV, Talbot NJ, Fungal infection of plants: using Magnaporthe grisea as a model for understanding fungal pathogenesis. Annual Plant Reviews,4: (Eds. J. Roberts, M. Dickinson, Sheffield Academic Press). (2000)pp 1-25
8. Balhadère PV, Talbot NJ, Fungal pathogenicity-establishing infection. In: Dickson M(ed.) Molecular Plant Pathology(Annual plant Review, 2000, 4:1-25
9. Balhadère PV, Talbot NJ, PDE1 encodes a P-typeATPase involved in appressorium mediated plant infection by Magnaporthe grisea. The Plant Cell, 2001, 13:1-19
10. Beckerman JL, Ebbole DJ, MPG1, a gene encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition. MPMI. 1996, 9:450-456.
11. Bellen HJ, O'Kane CJ, Wilson C, Grossniklaus U, Pearson RK, and Gehring WJ, 1989, P-element -mediated enhancer detection: a versatile method to study
development in Drosophila, Genes Dev, 1989, 3(9):1288-300
12. Bellen HJ, Ten years of enhancer detection: Lessons from the fly, Plant Cell, 1999, 11:2271-2281.
13. Blker M, Bhnert HU, Braun KH, Grl J and Kahmann R, Tagging pathogenicity genes in Ustilagomaydisby restriction enzyme-mediated integration (PEMI). Mol. Gen. Genet. 1995, 248: 547-552
14. Bourett T, Howard R, In vitro development ofpenetration structures in the rice blast fungus Magnaporthe grisea. Can. J. Bot. 1990, 69:329-342.
15. Brown PO, Bostein D, Exploring the new world of genomic with DNA microarrays [J], Nature Genetics(Supplement), 1999, 21:33.
16. Casadaban MJ, and Cohen SN, Lactose genes fused to exogenous promoters in one step using a Mu-lac acteriophage: In vivo probe for transcriptional control sequences. Proc. Natl. Acad. Sci. USA, 1979, 76:4530-4533.
17. Choi W, DeannA, The adenylate cyclase gene MAClof magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell 1997, 9:1973-1983.
18. Clergeot PH, Gourgues M, Cots J, Laurans F, Latorse MP, Pepin R, Tharreau D, Notteghem JL, and Lebrun MH, PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea PNAS, 2001, 98:6963-6968.
19. Covert SF, Kapoor P, Lee Mei-ho, et al. Agrobacterium tumefaciens-mediated transformation of Fusarium circinnatum, Mycological Research, 2001, 105(3):259-264
20. De Jong JC, Mccormack J, Sminoff N , Talbot NJ, Glycerol generates turgor in rice blast. Nature, 1997, 389, 244-255.
21. DeRisi J, Penland L, Brown PO, et al. Use of a cDNA microarray to analysis gene expression patterns in human cancer [J] , Nat Genet, 1996, 14:457.
22. DeZwaan TM, Carroll AM, Valent B, Magnaporthe grisea pthllp is a novel plasma membrane protein that mediates appressorium differenciation in response to inductive substrate cues. Plant cell, 1999, 11(10):2013-2030
23. Dixon KP, XuJR, Smirnoff N, Talbot NJ. Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea, Plant Cell, 1999, 11(10):2045-58.
24. Epstein L, Lusnak K. and Kaur S. Transformation-mediated developmental mutants of Glomerella graminicola (Colletotrichum graminicola). Fungal Genet. Biol. 1998, 23:189-203
25. Fang EG, Dean RA, Site-directed mutagnesis of the magB gene affects growth and development in Magnaporthe grisea, MPMI, 2000, 13(11),1214-1227
26. Faria-Campos AC, Cerqueira GC, Anacleto C, de Carvalho CM, Ortega JM. Mining microorganism EST databases in the quest for new proteins. Genet Mol Res. 2003, 31;2(1):169-77.
27. ProeligerEH, Carpenter BE, NUTI a major nitrogen regulatory gene in Magnaporthe grisea, is dispensable for pathogenicity. Molec. Gen Genet, 1996,251:647-656
28. Goffeau A, Barrell BG, Bussey H, et al. Life with 6000 genes. Science. 1996, 274:546,563-567
29. Gold SE, Carcia-Pedrajas MD, Martinez-Epinoza AD, New (and used) approaches to the study of fungal pathogenicity. Annu. Rev. Phytopathol. 2001, 39:337-365
30. Hamer JE, Horword RJ, Chumley FG, et al, A mechanism for surface attachment in spores of plant pathogenic fungus, Science, 1988,239,84:509-512
31. Hua-Van A, Hericourt F, Capy F, Daboussi M.-J. and Langin T, Three highly divergent subfamilies of the impala transposable element coexist in the genome of the fungus Fusarium oxysporum. Mol. Gen Genet. 1998, 259:354-362
32. Jelinsky S and Samson L. Global response of Saccharomyces cerevisiae to a al kylating agent,proc [J] .Natl Acad Sci USA, 1999, 96: 1486.
33. Jin-rong Xu, Chaoyang Xue, Time for a blast:genomics of Magnaporthe grisea, Molecular plant pathology, 2002, 3(3),173-176
34. Kahmann R. and Basse C. REMI (Restriction Enzyme Mediated Integration) and its impact on the isolation of pathogenicity genes in fungi attacking plants. Eur. J. Plant Pathol. 1999, 105:221-229
35. Kuspa A, Loomis WF, Tagging development genes in bictyostelium by restriction
enzyme-mediated integration of plasmid DNA. Proc Nail Acad Sci USA, 1992, 89:8803-8807
36. Linnemannstns P, Vob T, Hedden P, Gaskin P and Tudzynski B, Deletions in the gibberellin biosynthesis gene cluster of Gibberella fujikuroi by restriction enzyme-mediated integration and conventional transformation-mediated mutagenesis. Appl. Env. Microbiol. 1999, 65: 2558-2564
37. Liu S, Dean RA, G protein a subunit genes control growth, development, and pathogenicity of Magnaporthe grisea. MPMI, 1997, 10:1075-1086
38. Liu-S-j, Wei R-x, Arie T and Yamaguchi I, Obtaining insertion mutants in Magnaporthe grisea by restriction enzyme-mediated integration of plasmid. Chinese Journal of Genetics, 1998, 25:137-142
39. Liu-S-j, Wei R-x, Arie T and Yamaguchi I, REMI mutagenesis and identification of pathogenic mutants in Magnaporthe grisea (in Chinese). Chinese Journal of Biotechnology, 1998, 14:254-259
40. Lockhart DJ, Dong H, Byrne MC, et al, Expression monitoring by hybridization to high-density oligonucleotide arrays [J], Nature Biotechnology, 1997, 14: 1675.
41. Lu S, Lyngholm L, Yang G, Bronson C, Yoder OC and Turgen BG, Tagged mutations at the Toxl locus of CochlJobolus heterostrophus by restriction enzyme-mediated integration. Proc. Natl. Acad. Sci. USA ,1994, 91: 12649- 12653
42. Maier FJ, Schafer W, Mutagenesis via insertionalor restriction enzyme-mediated-integration (REMI) as a tool to tag pathogenicity related genes in plant pathogenic fungi. Biol. Chem. 1999, 380: 855-864
43. Marshall A, Hodgson J, DNA chips: an array of possibilities [J], Nature biotechnology, 1998, 16:731
44. Mishra NC, Tatum EL. Non-Mendelian inheritance of DNA-induced inositol independence in Neurospora. Proc Natl Acad Sci USA. 1973, 70(12):3875-9.
45. Mitchell TK, Dean RA, The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea. Plant Cell, 1995, 7:1869-1878.
46. Motoyama T, Imanishi K, Yamaguchi I, cDNA cloning, expression and mutagnesis of scytalone dehydratase needed for pathogenicity of the rice blast fungus, Pyricularia grisea. Biosci Biotechnol Biochem, 1998, 62:564-566
47. Mullins ED, Kang S, Transformation: a tool for studying fungal pathogens of plants, Cellular and Molecular Life Sciences, 2001, 58 (14), 2043-2052
48. Mullins ED, Chen PX, Romaine P et al, Agrobacterium tumefaciens-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 2001, 91:173-180
49. Mullins ED, Kang S, Transformation: a tool for studying fungal pathogens of plants, CMLS, 2001, 58:2043-2052
50. Nishimura M, Park G, Xu JR. The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Mafnaporthe grisea. Mol Microbiol. 2003, 50(1):231-43.
51. Niwa H, Araki K, Kimura S, Taniguchi S, Wakasugi S, Yamamura K, An efficient gene-trap method using poly A trap vectors and characterization of gene-trap events, J Biochem (Tokyo), 1993, 113(3):343-9
52. OUSH. Rice disease. 2nd ed. (M). Surrey, Commonwealth Mycology Institute, 1985
53. Park C H, Kim S, Park J Y, Ahn IP, Jwa NS, Im KH, Lee YH. Molecular characterization of a pathogenesis-related protein 8 gene encoding a class Ⅲ chitinase in rice. Mol Cells. 2004, 17(1):144-50.
54. Park SM, Choi ES, Kim MJ, Cha BJ, Yang MS, Kim DH. Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress. Mol Microbiol. 2004, 51(5):1267-77
55. Park, G., Xue, C., Zheng, L., Lam, S. and Xu, J.R.. (2002). MST12 regulates infectious growth but not appressorium formation in the rice blast fungus Magnaporthe grisea. MPMI. 15, 183-92.
56. Patricia S. Springer, Gene Traps: Tools for Plant Development and Genomics, The Plant Cell, 2000, 12:1007-1020
57. Paul B. Rainey, Adaptation of Pseudomonas fluorescens to the plant rhizosphere,
Environmental Microbiology ,1999, 1(3):243-257
58. Ramsay R. DNA chips: State-of-the-art [J].Nature Biotechnology, 1998, 16:40.
59. Rho HS, Kang S, Lee YH. Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea. Mol Cells. 2001, 12(3):407-11.
60. Rossman AY, Howard RJ, Valent B, Pricularia grisea the correct name for the rice blast disease fungus. Mycologia 1990, 82:509-512
61. Saitoh K, Arie T, Teraoka T, Yamaguchi I, Kamakura T. Targeted gene disruption of the neuronal calcium sensor 1 homologue in rice blast fungus, Magnaporthe grisea. Biosci Biotechnol Biochem. 2003, 67(3):651-3.
62. Saitoh K, Arie T, Teraoka T, Yamaguchi I, Kamakura T. Targeted gene disruption of the neuronal calcium sensor 1 homologue in rice blast fungus, Magnaporthe grisea. Biosci Biotechnol Biochem. 2003, 67(3):651-3.
63. Sanchez 0., Navarro R. E. and Aguirre J. Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (RFMI). Mol. Gen. Genet. 1998, 258:89-94
64. Schiestl R. H. and Petes T. D. Integration of DNA fragmentsby illegitimate recombination in Saccharomyces cerevisiae Proc. Natl. Acad. Sci. 1991, USA 88: 7585-7589
65. Shi Z, Christian D, Leung H, Enchanced transformation in Magnaporthe grisea by chemical and insertional mutagenesis. Phytopathol, 1995a, 85:329-333
66. Sweigard JA, Carroll AM, Kang S, Farrall L, Chumley FG, Yalent B, Identification cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus. Plant cell, 1995,7:1221-1233
67. Sweigard JA, Carroll AM, Kang S, Farrall L, Chumley FG, Valent B, Magnaporthe grisea pathogenecity genes obtained through insertional mutagenesis, MPMI, 1998, 11(5):404-412
68. Sweigard JA, Carroll AM, Valent B, Cloning and analysis of CUTI, a cutinase gene from Magnaporthe grisea. Mol. Gen. Genet. 1992a, 232:174-182
69. T Irie, H Matsumura, R Terauchi, H Saitoh, Serial analysis of gene expression
(SAGE) of Magnaporthe grisea: genes involved in appressorium formation, Mol Gen Genomics, 2003, 270:181-189
70. Takano Y, Choi W, Mitchell TK, Okuno T, and Dean RA. Large scale parallel analysis of gene expression during infection-related morphogenesis of Magnaporthe grisea. Molecular Plant Pathology. 2003, 4, 337
71. Talbot NJ, Having a blast: exploring the pathogenicity of Magnaporthe grisea. Trends Microbiol. 1995, 3:9-16
72. Talbot NJ, Kershaw, M J, Wakley GE, de Vries, O. M. H., Wessels, J. G. H., and Hamer, J. E. MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related of Nagnaporthe grisea. Plant Cell, 1996, 8, 985-999.
73. Tharreau D, Lebrun M-H, Talbot NJ, Notteghem JL, Advances in Rice Blast Research, 2000, pp. 376, Kluwer, Dordrecht
74. Thines E, Weber RW, Talbot NJ. MAP kinase and protein kinase A-dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea, Plant Cell, 2000, 12(9):1703-18.
75. Thomas SW, Glaring MA, Rasmussen SW et al, Transcript profiling in the barley mildrew pathogen Blumeria graminis by serial analysis of gene expression(SAGE), MPMI, 2002, 15:847-856
76. Thon MR, Nuckles EM, Vaillancourt LJ, Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrlchum graminicola. MPMI, 2000, 13: 1356-1365
77. Urban M, Bhargava T, Hamer JE. An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease. EMBO J. 1999, 18(3):512-21.
78. Velent B, Chumley FG, Molecular genetic analysis of the rice blast fungus Magnaporthe grisea Anun. Rev. Phytopathol, 1991, 29:443-467
79. Vidal-Cros A, Viviani F, Labesse G, Boccara M, Gaudry M., Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea. Purification, cDNA cloning and sequencing, Eur J Biochem, 1994, 1:219(3):985-92
80. Villaba F, Lebrun M-H, Hua-Van A, Daboussi M-J and Grosjean-Cournoyer M-C, Transposon impala, a novel tool for gene tagging in the rice blast fungus
Magnaporthe grisea. MPMI. 2001, 14(3): 308-315
81. Wang R, Hong G, Han B. Transcript abundance of rmll encoding a putative GT1-like factor in rice, is up-regulated by Magnaporthe grisea and down-regulated by light. Gene. 2004 Jan 7;324:105-15.
82. Weber RWS, Thines E, Wakley GE and Talbot NJ, The vacuole acts as a sink for lipid droplets during appressorium turgor generation by Magnaporthe grisea. Protoplasma, 2001, 216:101-112
83. Wen-Tsan Chang, Julian D. Gross, Peter C. Newell, Trapping developmental promoters in Dictyostelium, Plasmid, 1995, 34:175-183
84. Wodicka L, Dong H, Mittmann M, et al, Genome-wide expression monitoring in Saccharomyces cerevisiae [J] , Nature biotecnology, 1997,15:1359
85. Xu JR, Staiger CJ, Hamer JE. Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses. Proc Natl Acad Sci U S A. 1998, 95(21):12713-8.
86. Yang G, Rose MS, Turgeon BG et al, A polyketide synthase is required for the wA gene of Aspergillus nidulans. Genetics, 1996,126:81-90
87. Yang GP, Ross DT, Kuang WW, et al. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes [J], Nucl Res, 1999, 27: 1517-1523
88. Yun SH, TurgeonBG, Yoder OC, REMI-induced mutants of Mycosphaerella zeae-maydis lacking the polyketide PM-toxin are deficient in pathogenesis to corn. Physiol Mol Plant Pathol, 1998, 52:53-66
89. Zeigler RS, Recombination in Msgnaporthe grisea. Annu Rev Phytopathol, 1998, 36:249-75.
90. Zwiers LH, De Waad MA, Efficient Agrobacterium tumefaciens-mediated gene disruption in the phytopathogen Mycosphserella graminicola. Current Genetic, 2001, 39:388-393
91．J．萨姆布鲁克，D．W．拉塞尔，分子克隆实验指南(第三版)，2002，科学出版社
92．方卫国，张永军，肖月华，根癌农杆菌介导球孢白僵菌的遗传转化及T-DNA插入突变体
的获得，中国科学C辑
93．方卫国，张永军，杨星永等，根癌农杆菌介导真菌遗传转化的研究现状，中国生物工程杂志，2002，22(5)：40-44
94．何月秋，唐文华，水稻稻瘟病菌研究进展(一) 水稻稻瘟病菌多样性及变异机制，云南农大学学报，2001，16(1)：60-64
95．李宏宇，潘初沂，陈涵等，稻瘟病菌T-DNA插入方法优化及其突变体分析，生物工程学报，2003，7，V19(4)，419-423
96．林福呈，稻瘟病菌附着胞形成的细胞生物学，植物病理学报，2001，31(2)：97-101
97．林福呈，稻瘟病菌附着胞形成机理的研究，博士学位论文，1999，
98．林福呈，李德葆等，稻瘟病菌侵入机理，微生物学通报，24(3)：167-1270
99．陆凡，郑小波等，江苏省稻瘟病菌的毒性多样性及水稻品种的抗病性，生物多样性，2001，9(3)：201-206
100．沈瑛，李成云等，稻瘟病菌的菌丝融合现象及其后代致病性变异，中国农业科学，1997，30(6)：16-22
101．沈瑛等，我国稻瘟病菌的遗传多样性及其地理分布(英)，浙江农业大学学报，1998，24(5)：493-501
102．王洪凯，林福呈，李德葆，稻瘟病菌致病相关基因研究进展，菌物系统，2002，21(3)：459-464
103．徐炳森、邵健忠几种新型生物芯片的研究进展，生物化学与生物物理进展，2000，27(3)：251-253．
104．张正光，郑小波，稻瘟病菌不同小种菌株间无性重组的研究，植物病理学报，2000，30(4)：312-318
105．张正光，郑小波，稻瘟病菌营养体亲和和配对筛选及其亲和能力遗传，植物病理学报，1999．29(4)：304-308
http://cogeme.ex.ac.uk
http://fungi.fgl.ncsu.edu/tdsblastscript.html
http://www.broad.mit.edu/annotation/fungi/magnaporthe/index.html
http://www.ddbj.nig.ac.jp
http://www.ebi.ac.uk
http://www.ncbi.nlm.nih.gov