摘要
本实验室前期分别从大豆霜霉病抗病品种、感病品种中克隆得到丝氨酸/丙氨酸乙醛酸转氨酶蛋白的编码基因GmeR、GmeR2、假基因GmeR3及其启动子，原位杂交结果表明来自于抗病品种吉林小粒一号的GmeR基因编码产物定位于大豆叶片中的过氧化物体中，在植物光呼吸中起重要作用，是控制植物活性氧激增的关键酶，并且不属于R基因，不具有种属特异性，而是一种新的酶学抗病基因，因此，研究GmeR与大豆霜霉病抗性的相关性，对深入了解酶编码基因在植物抗病性中的作用、阐释植物对病原菌的抗性机制具有重要意义，本论文主要开展了以下工作：
1.大豆材料的霜霉病抗性和遗传背景的鉴定分析
为研究该基因与大豆霜霉病抗性的相关性，对从大豆种子资源库中收集得到的吉林小粒1号，灌水铁荚青，豫豆22，东农49，Amsoy，铁丰18，铁丰31，荆山扑，五星1号，滨海13-1，科丰1号，齐黄25号，中品95-5383，文丰7号，科丰14，彩豆2号16种具有不同大豆霜霉病抗性及其遗传背景的大豆材料进行了鉴定和分析，结果表明高抗品种4个，分别为吉林小粒一号，豫豆22，东农49，灌水铁荚青；抗病品种1个，为Amsoy；中抗品种2个，为铁丰18，铁丰31；中感品种1个，为荆山扑。由于这些材料具有不同的霜霉病抗性和遗传背景，在材料上具有代表性。
2.半定量RT-PCR检测GmeR基因在不同大豆材料中的表达
为了确定GmeR基因的表达量与大豆霜霉病抗性的相关性，首先通过半定量RT-PCR比较了9个霜霉病不同抗性种大豆材料中GmeR基因转录水平上相对表达量的差异。半定量RT-PCR结果显示，不同大豆品种中GmeR基因表达水平存在差异，GmeR基因与大豆霜霉病抗性具有相关性。以霜霉病中等感品种荆山扑为对照材料，霜霉病高抗品种吉林小粒一号、豫豆22号、东农49号GmeR基因的相对表量达分别是荆山扑GmeR基因表达量的3.1倍、2.7倍和2.4倍，方差分析差异显著。抗病材料Amsoy中GmeR基因相对表量是荆山扑GmeR基因表达量的2.2，方差分析差异显著。而其他材料中，如彩豆2号中GmeR基因相对表达量是荆山扑的1.9倍。
3.荧光定量PCR检测GmeR基因在不同大豆材料、不同生育期中的表达
荧光定量PCR检测显示15个大豆品种中GmeR基因相对表达量存在差异，霜霉病高抗品种吉林小粒1号，豫豆22，东农49中GmeR基因的相对表达量比较接近，都在27.5左右（以中品95-5383为对照），是该基因在荆山扑中相对表达量的3.7倍，方差分析差异显著，并且该结果与半定量PCR结果具有一致性。在抗病材料Amsoy中GmeR基因的相对表达量为21.34，中抗品种铁丰31中GmeR基因的相对表达量为13.15，都低于GmeR基因在上述3个中高抗霜霉病品种中的相对表达量。根据半定量RT-PCR和荧光定量PCR检测结果，可以得出结论：GmeR基因在不同大豆品种中表达量不同，并且在霜霉病抗性品种中相对表达量高于其在感病品种中的表达量。
实时荧光定量PCR对不同生育期大豆第二三片展开叶中GmeR基因表达水平的差异进行检测，结果显示：对于各个材料而言，在大豆不同生育期GmeR的表达量不同，均都随着植株的生长而增加。该结果与大豆光呼吸的强弱变化的规律具有一致性，这进一步证明GmeR参与植物的光呼吸过程中，铁丰31为例，GmeR基因在4月苗龄的叶片中的相对表达量是其在3月苗龄片中表达量的1.834倍；是其在1月苗龄叶片中表达量的3.6倍。
4.大豆材料中GmeR表达差异的Northern blot检测
为进一步验证半定量RT-PCR、实时荧光定量PCR的检测结果，进一步进行了Northern blot检测，检测结果显示，GmeR基因在吉林小粒一号中转录水平的表达是其在荆山扑中表达量的4.549倍，半定量RT-PCR和荧光定量PCR结果中吉林小粒中GmeR的相对表达量分别是荆山扑中的3.10倍，3.7倍，因此Northern blot检测结果与半定量RT-PCR、实时荧光定量PCR的检测结果具有一致性，说明GmeR基因与霜霉病抗性相关，在抗病材料中的表达量显著高于感病材料。
5. GmeR基因对转基因大豆霜霉病抗性的影响
为研究GmeR基因是在基因水平还是转录水平参与大豆霜霉病抗性，首先研究建立了大豆的组培体系，利用实验室克隆的GmeR基因，构建了GmeR正义植物表达载体pCGt2301和反义植物表达载体pCGr2301，分别转化大豆霜霉病抗病品种吉林小粒一号、感病品种黑农10号，目前已获得吉林小粒一号的pCGt2301转基因植株5株，pCGr2301转基因植株4株，并获得T0代转基因大豆种子，下一步将对T1代、T2代中目的基因的表达与大豆霜霉病抗性的相关性进行研究。
6. GmeR作为酶学抗病基因对转基因烟草抗病性的影响
由于GmeR基因的表达与植物体内活性氧的激增具有密切关系，因此从理论上讲，该基因的表达能够提高受体作物对病原菌的广谱抗性，为研究GmeR基因与植物对其它病原菌抗性的关系，通过农杆菌介导法将pCGt2301/pCGr2301分别导入烟草品种红花烟草中，分别得到7株，4株转基因烟草植株。GUS染色和PCR检测结果显示GmeR基因已经整合到烟草基因组中，下一步将对这些转基因植株进行青枯病、赤星病等鉴定，以及转基因烟草后代SGT/AGT活性、过氧化氢含量的改变明确酶学抗病基因在植物广谱抗病性中的作用。
大豆霜霉病抗病、感病品种中克隆得到的GmeR基因同源性为96.38%，本研究进行的荧光定量PCR检测显示15个大豆品种中GmeR基因的相对表达量差异显著，霜霉病高抗品种吉林小粒1号，豫豆22，东农49中GmeR基因的相对表达量是中感品种荆山扑的3.70倍，说明GmeR转录水平的差异与大豆的霜霉病抗性呈正相关性，本研究为植物抗病基因工程提供了一个新的策略和思路。
One of my research fellows in our lab has sucessfully cloned in soybean the GmeR gene and itspromoter, which encodes a protein with bioactivities of ser/ala glyoxylate apotransaminase., The GmeRgene is mainly expressed in leaves of soybean. The GMER protein is shown to be located in peroxisomeaccording to the in-situ hybridization, and palys an important role in photorespiratory process,as thekey enzyme that controls the actvition of outburst of reactive oxygen species in plant.Since we knowthat GmeR gene is not a R gene,but a gene encoding protein with bioactivities of ser/ala glyoxylateapotransaminase..The research of the correlation between GmeR gene and downy mildew resistance canlead us to have a better understanding of how the gene that encodes enzym functions in the immunresponses of a plant, as well as the mechanism of how plant responses to pathogens attacks. In myresearch, what I have conducted include the following parts:
1.The identification and analysis of the genetic background of different soybean varieties and theirresistance to downy mildew.
To identify the correlation between GmeR gene and the resistance of soybean to downy mildew, Ianalyzed and identified genetic background and the resistance to downy mildew of16different soybeanvarieties I have chosen from the repository of Soybean seed,including Wuxing1,Amsoy, Binhai13-1,Kefeng1,Qihuang25, Zhongpin95-5383, Jinshanpu,Wenfeng7, Guanshuitieyingqing,Yudou22,Tiefeng18, Dongnong49, Tiefeng31, kefeng14,Jilinxiaoli1,Caidou2.It turns out that four of them arehighly resistance varieties, including: Jilinxiaolii1,Yudou22,Dongnong49,Guanshuitiejiaqin; oneresistant breed, Amsoy;two medium resistant varieties, Tiefeng18,Tiefeng31and one mediumlysusceptible one, Jingshanpu.The varieties I have chosn are respectve since their genetic background andresistance to downy mildew are different from one another.
2.Semi-Quantitative RT-PCR analysis of the different expression level of GmeR in differentsoybean varieties.
The result of Semi-Quantitative RT-PCR indicates that the transcriptional expression of GmeR indifferent varieties differs apparently from one another, and GmeR gene is related to the host’s resistanceto downy mildew. Taking the mediumly susceptible soybean variety-Jingshanpu, as the control, we cansee highly resistant soybean varieties, Jinlixiaoli1, Yudou22and Dongnong49, is respectively3.1,2.7and2.4times that in Jingshanpu. Analysis of variance showed significant differences. In resistantvariety Amsoy2.2times that in Jingshanpu. Analysis of variance showed significant differences.Differences of in other varieties are also apparent in other varieties.
3.Real-time Quantitative PCR analysis of the different expression level of GmeR in differentsoybean varieties as well as in different growth stage of soybean
The result of RT-QPCR shows that the transcriptional expression of GmeR in highly resistantsoybean varieties, Jinlixiaoli1, Yudou22and Dongnong49are all around27.5,3.1times that inJingshanpu. Analysis of variance showed significant differences. The result of RT-QPCR is consistent with that of Semi-Quantitative RT-PCR. The the transcriptional expression of GmeR in resistant varietyAmsoy and is23.14，and it is13.15in mediumly resistant breed Tiefeng31, both of which are lowerthan that in the three highly resistant varieties mentioned above. It can be concluded that the expressionof GmeR differs from one another, which is be positively correlated with host’s resistance to downymildew. The RT-QPCR analysis of in the second/third leaves developed at different growth stage ofsoybean shows that that the expression of GmeR increases as the plant grows, which shares the the samechange trend with the photorespiratory process in soybean. It is a futher evidence for the involvement ofGMER protein in the photorespiratory process of soybean.
4.Northern blot analysis of the different expression level of GmeR in different soybean varieties
Northern Blot has been conducted to varify this conclution. It suggests that the expression of GmeRin Jilinxiaoli1is4.549times that in Jingshanpu, which is consistant with the results ofSemi-Quantitative RT-PCR and Real-time QPCR.
5.The influence of GmeR gene on transgenetic tobacco and soybean.
In order to test the influence of GmeR gene on the resistance of soybean to downy mildew, I firstlyestablished sense and antisense plant expression vector of GmeR, pCGt2301and pCGr2301,andtumefacted the vector into soybean variety Jinlinxiaoli1through Agrobacterium, obtaining5and4transgenetic breeds respectively. Meanwhile, through my study of the proper concentration ofkanamycin uesed in the resistance screening of transgenetic soybean in the tissure culture, I believe thesuit concentration shall be50mg/l.
To test whether function of GmeR gene remains the same in other plant species, I tumefacted thepCGt2301/pCGr2301vector into the tabacco variety, Honghua, through Agrobacterium, obtaining7and4transgenetic breeds respectively.The target sequences has been successfully inergrated into thegenome of tabacco, evidenced by gus staining and PCR analysis.
Generally speaking, according to my research, GmeR gene is further proved to be related with thesoybean’s resistance to downy mildew，and its expression level in the different soybean varities ispositively correlated with their resistance to downy mildew. It is of importance for the breeding ofsoybean with high resistance to downy mildew through genetic engineering.
引文
1.陈华民拟南芥中EIN3EIL1通过抑制SA信号途径负调控植物的先天免疫反应[D].上海交通大学,2008,8(5):26-31.
2.程曦,田彩娟,李爱宁等植物与病原微生物互作分子基础的研究进展[J]遗传,2012,34(2):134-144.
3.郭明NPR1基因对烟草的遗传转化及抗病性测定[D]华南农业大学,2008.19(14):35-39.
4.刘士旺,吴学龙,郭泽建等.拟南芥的抗病信号传导途径[J].植物病理学报,2003,33(2):104-111.
5.刘新,孟繁霞,张蜀秋等.Ca2+参与水杨酸诱导蚕豆气孔运动时的信号转导[J].植物生理与分子生物学学报,2003,29(1):59-64.
6.吕正光植物多糖免疫调节的希望靶点:甘露糖受体[D].第四军医大学,2006.17(12):54-56,60.
7.王炳楠,王双超,檀贝贝等.大丽轮枝菌蛋白激发子PevD1诱导的烟草对烟草花叶病毒(TMV)系统获得性抗性及其分子机制[J].农业生物技术学报,2012,20(2):188-195.
8.王钧.植物广州谱抗性及其信号转导[C].中国植物生理学会植物环境生理学术讨论会论文汇编.1999:34～44
9.钟申丽,孙玉诚,戈峰等.茉莉酮酸酯诱导植物抗性的分子机制[J].江西农业学报,2008,20(12):74-76,80.
10. Alfano J.R., and Collmer A., Type III secretion system effector proteins: double agents in bacterialdisease and plant defense. Annu Rev Phytopathol (2004)42,385-414.
11. Angelov D., and Krasteva L., Dominant inheritance of downy mildew resistance in melons. InPROCEEDINGS OF CUCURBITACEAE(2000),.273-275.
12. Asai T., Tena G., Plotnikova J., Willmann M.R., Chiu W.L., Gomez-Gomez L., Boller T., AusubelF.M., and Sheen J., MAP kinase signalling cascade in Arabidopsis innate immunity. Nature (2002)415,977-983.
13. Ausubel F.M., Are innate immune signaling pathways in plants and animals conserved? NatImmunol (2005)6,973-979.
14. Balass M., Cohen Y., and Barjoseph M., Identification of a constitutive45kda soluble-proteinassociated with resistance to downy mildew in muskmelon (cucumis-melo l), line pl124111f.Physiol Mol Plant P (1992)41,387-396.
15. Beach L.R., and Palmiter R.D., Amplification of the metallothionein-I gene in cadmium-resistantmouse cells. Proc Natl Acad Sci USA(1981)78,2110-2114.
16. Bent A.F., and Mackey D., Elicitors, effectors, and R genes: the new paradigm and a lifetimesupply of questions. Annu Rev Phytopathol (2007)45,399-436.
17. Chisholm S.T., Coaker G., Day B., and Staskawicz B.J. Host-microbe interactions: shaping theevolution of the plant immune response. Cell (2006)124,803-814.
18. Criswell A.D., Screening cucumber (Cucumis sativus) for resistance to downy mildew(Pseudoperonospora cubensis). MSc thesis North Carolina State University, Raleigh, NC, USA(2008)14,83-87
19. Cohen Y., Eyal H., Hanania J., and Malik Z., Ultrastructure of pseudoperonospora-cubensis inmuskmelon genotypes susceptible and resistant to downy mildew. Physiol Mol Plant P (1989)34,27-40.
20. Collinge D.B., Lund O.S. and Thordal-Christensen H., What are the prospects for geneticallyengineered, disease resistant plants? Eur J Plant Pathol (2008)121,217-231.
21. Dangl J.L., and Jones J.D., Plant pathogens and integrated defence responses to infection. Nature(2001)411,826-833.
22. Daxberger A,, Nemak A,, Mithofer A,, Fliegmann J,, Ligterink W,, Hirt H,, and Ebel J., Activationof members of a MAPK module in beta-glucan elicitor-mediated non-host resistance of soybean.Planta (2007)225,1559-1571.
23. Doruchowski R.W., kowska-Ryk, Inheritance of resistance to downy mildew(Pseudoperonospora cubensis Berk&Curt) in Cucumis sativus. In: Doruchowski RW (ed)Proceedings of the V Eucarpia Cucurbitaceae symposium. Skierniewice-Warszawa (1992)132–138
24. Ellis, J. Insights into nonhost disease resistance: can they assist disease control in agriculture? PlantCell (2006)18,523-528.
25. Epinat C., Pitrat M., Inheritance of resistance of three lines of muskmelon (Cucumis melo) todowny mildew (Pseudoperonospora cubensis). In: Thomas CE (ed) Evaluation and enhancement ofcucurbit germplasm. Cucurbitaceae Proceedings, Charleston, SC (1989),133–135
26. Fanourakis N.E., and Simon P.W. Analysis of genetic-linkage in the cucumber. J Hered (1987)78,238-242.
27. Favaloro J., Treisman R., and Kamen R., Transcription maps of polyoma virus-specific RNA:analysis by two-dimensional nuclease S1gel mapping. Methods Enzymol (1980)65,718-749.
28. Ferreira R.B., Monteiro S., Freitas R., Santos C.N., Chen Z., Batista L.M., Duarte J., Borges A.,and Teixeira A.R., The role of plant defence proteins in fungal pathogenesis.MOLECULARPLANT PATHOLOGY (2007)8,677-700.
29. Friedrich L., Lawton K., Dietrich R., Willits M., Cade R., and Ryals J., NIM1overexpression inArabidopsis potentiates plant disease resistance and results in enhanced effectiveness of fungicides.Mol Plant Microbe Interact (2001)14,1114-1124.
30. Glazebrook J., Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens.Annu Rev Phytopathol (2005)43,205-227.
31. Goker M., Voglmayr H., Riethmuller A., and Oberwinkler F., How do obligate parasites evolve? Amulti-gene phylogenetic analysis of downy mildews. Fungal Genet Biol (2007)44,105-122.
32. Gurr S.J., and Rushton P.J., Engineering plants with increased disease resistance: how are wegoing to express it? Trends Biotechnol (2005)23,283-290.
33. Habdas H., Staniaszek M., Doruchowski R.W., and Lqkowska-Ryk E., Anatomical and cytologicalmechanism of cucumber resistance and susceptibility to downy mildew (Pseudoperonosporacubensis Berk, Curt). In CUCURBITS TOWARDS (1996)2000,.263-268.
34. Hahlbrock K., Scheel D., Logemann E., Nurnberger T., Parniske M., Reinold S., Sacks W.R., andSchmelzer E., Oligopeptide elicitor-mediated defense gene activation in cultured parsley cells.Proc Natl Acad Sci USA (1995)92,4150-4157.
35. Heath M.C., Non-host resistance to plant pathogens: Nonspecific defense or the result of specificrecognition events? Physiol Mol Plant P(2001)58,53-54.
36. Heath M.C., A Personal Perspective of the Last40Years of Plant Pathology: Emerging Themes,Paradigm Shifts and Future Promise (2008),1-15.
37. Higuchi R., Fockler C., Dollinger G., and Watson R., Kinetic PCR analysis: real-time monitoringof DNA amplification reactions. Biotechnology (N Y)(1993)11,1026-1030.
38. Huang S., Li R., Zhang Z., Li L., Gu X., Fan Wet al.,The genome of the cucumber, Cucumissativus L. Nat Genet (2009)41:1275–1281
39. Inohara,Chamaillard, McDonald C., and Nunez G., NOD-LRR proteins: role in host-microbialinteractions and inflammatory disease. Annu Rev Biochem(2005)74,355-383.
40. Jackson A.O., and Taylor C.B. Plant-Microbe Interactions: Life and Death at the Interface. PlantCell (1996)8,1651-1668.
41. Janni M., Sella L., Favaron F., Blechl A.E., De Lorenzo G. and D'Ovidio R., The expression of abean PGIP in transgenic wheat confers increased resistance to the fungal pathogen Bipolarissorokiniana. Mol Plant Microbe Interact (2008)21,171-177.
42. Jha S, Tank H.G., Prasad B.D., and Chattoo B.B., Expression of Dm-AMP1in rice confersresistance to Magnaporthe oryzae and Rhizoctonia solani. Transgenic Res(2009)18,59-69.
43. Jonak C., Okresz L., Bogre L., and Hirt H., Complexity, cross talk and integration of plant MAPkinase signalling. Curr Opin Plant Biol (2002)5,415-424.
44. Jones J.D.G., and Dangl J.L., The plant immune system. Nature (2006)444,323-329.
45. Joubert D.A., Kars I., Wagemakers L., Bergmann C., Kemp G., Vivier M.A., and van Kan J.A.,A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of theendopolygalacturonase BcPG2from Botrytis cinerea in Nicotiana benthamiana leaves without anyevidence for in vitro interaction. Mol Plant Microbe Interact (2007)20,392-402.
46. Kafatos F.C., Jones C.W. and Efstratiadis A., Determination of nucleic acid sequence homologiesand relative concentrations by a dot hybridization procedure. Nucleic Acids Res (1979)7,1541-1552.
47. Kamoun, S., Nonhost resistance to Phytophthora: novel prospects for a classical problem. CurrOpin Plant Biol (2001)4,295-300.
48. Király L., Barna B., and Király Z., Plant Resistance to Pathogen Infection: Forms and Mechanismsof Innate and Acquired Resistance. J. Phytopathol.(2007)155,385-396.
49. Kostov K., Christova P., Slavov S. and Batchvarova R., Constitutive expression of a radishdefensin gene rs-afp2in tomato increases the resisstance to fungal pathogens. Biotechnol.Biotechnol. Equip (2009)23,1121-1125.
50. Kuc J., What's old and what's new in concepts of induced systemic resistance in plants, and itsapplication. In Multigenic and Induced Systemic Resistance in Plants,(2006).9-20.
51. Lange L., Eden U. and Olson L.W., Zoosporogenesis in pseudoperonospora-cubensis, cubensis, thecausal agent of cucurbit downy mildew. Nord J Bot (1989)8,497-504.
52. Li J., Brader G. and Palva E.T., The WRKY70transcription factor: a node of convergence forjasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell(2004)16,319-331.
53. Li J., Brader G., Kariola T. and Palva E.T., WRKY70modulates the selection of signalingpathways in plant defense. Plant J (2006)46,477-491.
54. Li J.W., Liu J., Zhang H., and Xie C.H.., Identification and transcriptional profiling ofdifferentially expressed genes associated with resistance to Pseudoperonospora cubensis incucumber. Plant Cell Rep (2011)30,345-357.
55. Lin W.C., Lu C.F., Wu J.W., Cheng M.L., Lin Y.M., Yang N.S., Black L., Green S.K., Wang J.F.,and Cheng C.P., Transgenic tomato plants expressing the Arabidopsis NPR1gene displayenhanced resistance to a spectrum of fungal and bacterial diseases. Transgenic Res (2004)13,567-581.
56. Makandar R., Essig J.S., Schapaugh M.A., Trick H.N., and Shah J., Genetically engineeredresistance to Fusarium head blight in wheat by expression of Arabidopsis NPR1. Mol PlantMicrobe Interact (2006)19,123-129.
57. Malnoy M., Jin Q., Borejsza-Wysocka E.E., He S.Y., and Aldwinckle H.S., Overexpression of theapple MpNPR1gene confers increased disease resistance in Malus x domestica Mol Plant MicrobeInteract (2007)20,1568-1580.
58. Mellersh D.G., and Heath M.C. An investigation into the involvement of defense signalingpathways in components of the nonhost resistance of Arabidopsis thaliana to rust fungi also revealsa model system for studying rust fungal compatibility.Mol Plant Microbe Interact (2003)16,398-404.
59. Meyers B.C., Dickerman A.W., Michelmore R.W., Sivaramakrishnan S., Sobral B.W., and YoungN.D. Plant disease resistance genes encode members of an ancient and diverse protein familywithin the nucleotide-binding superfamily. Plant J (1999)20,317-332.
60. Nakagami H., Pitzschke A., and Hirt, H. Emerging MAP kinase pathways in plant stress signalling.Trends Plant Sci (2005)10,339-346.
61. Neykov S., Dobrev D. Introduced cucumber cultivars relatively resistant to Pseudoperonosporacubensis in Bulgaria. Acta Hortic (1987)220:115–119
62. Nurnberger T., and Scheel D. Signal transmission in the plant immune response. Trends Plant Sci(2001)6,372-379.
63. Nurnberger T., Brunner F., Kemmerling B., and Piater L. Innate immunity in plants and animals:striking similarities and obvious differences. Immunol Rev(2004)198,249-266.
64. Pan Q., Wendel J., and Fluhr R. Divergent evolution of plant NBS-LRR resistance genehomologues in dicot and cereal genomes. J Mol Evol (2000)50,203-213.
65. Petrov L., Boogert K., Sheck L., Baider A., Rubin E., and Cohen Y. Resistance to downy mildew,Pseudoperonospora cubensis, in cucumbers. In PROCEEDINGS OF CUCURBITACEAE(2000)2000,.203-209.
66. Pieterse C.M., and Van Loon L.C. NPR1: the spider in the web of induced resistance signalingpathways. Curr Opin Plant Biol (2004)7,456-464.
67. Pitrat M., Risser G., Bertrand F., Blancard D., and Lecoq H. Evaluation of a melon collection fordisease resistances. In CUCURBITS TOWARDS (1996)2000,.49-58.
68. Shimizu S., Kanazawa K., Kato A., Studies on the breeding of cucumber for resistance to downymildew. Part2. Difference of resistance to downy mildew among the cucumber varieties and theutility of the cucumber variety resistance to downy mildew. Bul Hort Res Sta Jpn (1963)2:80–81
69. Song D., Chen J., Song F., and Zheng Z. A novel rice MAPK gene, OsBIMK2, is involved indisease-resistance responses. Plant Biol (Stuttg)(2006)8,587-596.
70. Staskawicz B.J., Ausubel F.M., Baker B.J., Ellis J.G., and Jones J.D., Molecular genetics of plantdisease resistance. Science (1995)268,661-667.
71. Taler D., Galperin M., Benjamin I., Cohen Y., and Kenigsbuch D., Plant eR genes that encodephotorespiratory enzymes confer resistance against disease. Plant Cell (2004)16,172-184.
72. Thomas C.E., Inaba T., and Cohen Y., Physiological specialization in pseudoperonospora-cubensis.Phytopathology (1987)77,1621-1624.
73. Thomas C.E., Cohen Y., Mccreight J.D., Jourdain E.L., and Cohen S., Inheritance of resistance todowny mildew in cucumis-melo.Plant Dis (1988)72,33-35.
74. Thomas P.S., Hybridization of denatured RNA and small DNA fragments transferred tonitrocellulose. Proc Natl Acad Sci USA (1980)77,5201-5205.
75. Thomma B.P., Eggermont K., Penninckx I.A., Mauch-Mani B., Vogelsang R., Cammue B.P., andBroekaert W.F., Separate jasmonate-dependent and salicylate-dependent defense-responsepathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc NatlAcad Sci USA (1998)95,15107-15111.
76. Tian D., Traw M.B., Chen J.Q., Kreitman M., and Bergelson J., Fitness costs of R-gene-mediatedresistance in Arabidopsis thaliana.Nature (2003)423,74-77.
77. Ton J., Pieterse C.M.J., and Van Loon L.C., The relationship between basal and induced resistancein Arabidopsis. In Multigenic and Induced Systemic Resistance in Plants,(2006).197-224.
78. Tuzun S.,Terminology related to induced systemic resistance: Incorrect use of synonyms may leadto a scientific dilemma by misleading interpretation of results. In Multigenic and Induced SystemicResistance in Plants,(2006).1-8.
79. van der Vossen E.A., Gros J., Sikkema A., Muskens M., Wouters D., Wolters P., Pereira A., andAllefs S. The Rpi-blb2gene from Solanum bulbocastanum is an Mi-1gene homolog conferringbroad-spectrum late blight resistance in potato. Plant J (2005)44,208-222.
80. Wally O., Jayaraj J., and Punja Z.K. Broad-spectrum disease resistance to necrotrophic andbiotrophic pathogens in transgenic carrots (Daucus carota L.) expressing an Arabidopsis NPR1gene. Planta (2009)231,131-141.
81. Williams D.L., Newman T.C., Shelness G.S., and Gordon D.A. Measurement of apolipoproteinmRNA by DNA-excess solution hybridization with single-stranded probes. Methods Enzymol(1986)128,671-689.
82. Williamson B., Tudzynski B., Tudzynski P., and van Kan J.A. Botrytis cinerea: the cause of greymould disease. Mol Plant Pathol (2007)8,561-580.
83. Yamaguchi T., Yamada A., Hong N., Ogawa T., Ishii T., and Shibuya N. Differences in therecognition of glucan elicitor signals between rice and soybean: beta-glucan fragments from therice blast disease fungus Pyricularia oryzae that elicit phytoalexin biosynthesis insuspension-cultured rice cells. Plant Cell (2000)12,817-826.
84. Yang S., Gao M., Xu C., Gao J., Deshpande S., Lin S., Roe B.A., and Zhu H., Alfalfa benefitsfrom Medicago truncatula: the RCT1gene from M. truncatula confers broad-spectrum resistance toanthracnose in alfalfa. Proc Natl Acad Sci USA (2008)105,12164-12169.
85. Yoshikawa M., Keen N.T., and Wang M.C. A receptor on soybean membranes for a fungal elicitorof phytoalexin accumulation. Plant Physiol (1983)73,497-506.
86. Zhang S., and Liu Y., Activation of salicylic acid-induced protein kinase, a mitogen-activatedprotein kinase, induces multiple defense responses in tobacco. Plant Cell (2001)13,1877-1889.
87. Zhang Z., Feechan A., Pedersen C., Newman M.A., Qiu J.L., Olesen K.L., andThordal-Christensen H., A SNARE-protein has opposing functions in penetration resistance anddefence signalling pathways. Plant J (2007)49,302-312.
88. Zhao B, Lin X, Poland J, Trick H, Leach J, and Hulbert S., A maize resistance gene functionsagainst bacterial streak disease in rice. Proc Natl Acad Sci USA(2005)102,15383-15388.
89. Zhou Y, Xu J, Zhou S, Yu J, Xie X, Xu M, Sun Y, Zhu L, Fu B, Gao Y, and Li Z.,Pyramiding Xa23and Rxo1for resistance to two bacterial diseases into an elite indica rice varietyusing molecular approaches. Mol Breeding (2009)23,279-287.