百脉根结瘤信号通路蛋白NSP1/NSP2相互作用的研究
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摘要
根瘤菌与豆科植物能特异地形成共生体-根瘤。这种共生关系的建立涉及到低等微生物与其宿主植物之间复杂的信号识别、基因表达调控、分子间相互作用等过程。百脉根是研究共生固氮体系的模式豆科植物之一,对其根瘤菌结瘤因子信号转导途径和作用机制的研究,将有助于揭示根瘤菌与豆科植物共生关系建立和共生体形成的奥秘。
百脉根结瘤信号通路NSP1和NSP2是根瘤菌共生信号转导途径中关键的信号蛋白,都属于GRAS家族转录因子,位于CCaMK (Ca2+/calmodulin-dependent protein kinase)下游,并受其调控。本研究的侧重点在于探究NSP1与NSP2间是否存在相互作用,相互作用区段以及检测NSP1基因是否具有结合启动子的能力以及行使此功能的区段,进而揭示NSP1、NSP2在结瘤因子信号传导途径的调控机制。其研究结果如下：
1.构建了一系列nsp1和nsp2缺失突变体,利用酵母双杂交系统研究了百脉根结瘤信号通路NSP1和NSP2蛋白间的相互作用。结果表明NSP1具有与NSP2相互作用的功能,而且NSP2单独的LHR1结构域(105-186氨基酸)则能与NSP1相互作用,说明NSP1与NSP2在行使功能时可能形成异源二聚体。
2.构建了一系列在大肠杆菌中表达的缺失突变体,并表达和纯化缺失不同结构域的截短蛋白,利用pull-down和western印迹转移技术进行了体外蛋白质相互作用。结果表明,NSP1缺失LHR1、VHIID两个结构域后仍具有与NSP2蛋白相互作用的功能,而缺失LHRII区段后则不能相互作用,说明NSP1的LHRII区段是两蛋白间相互作用所必需的。
3.利用凝胶阻滞(EMSA)实验,初步检测了NSP1是否结合启始结瘤基因NIN的启动子的功能,但没有得到理想的实验结果,其原因可能是所用的DNA片段太长(500bp),在EMSA凝胶上只见到一些弥散的DNA带,因此该结果需要进一步证实。
The leguminous plants can specifically establish a symbiosis "nodule" with rhizobia. The establishment of the symbiotic relationship is involved in the complex process incloding signal perception, gene expression and regulation, the interactions of intermolecules between microbes and host plants. Lotus japonicus is one of the model legumes plants for studying the system on the symbiotic nitrogen fixation. The research of the Nod factors signaling pathway and the mechanism of intermolecular interaction in Lotus japonicus will uncover the secret about the establishment of symbiotic relationship and the formation of the symbiosis.
In Lotus japonicus, the proteins of NSP1 (Nodulation Signaling Pathway 1) and NSP2 are belonged to the GRAS family transcription factors, which are the pivotal signal proteins in the symbiotic signaling pathway, and in the downstream of the CCaMK. In this study, we focus on the protein-protein interactions between NSP1 and NSP2, characterization of interaction domain, and identification of the role of the NSP1 binding to the promoter of taget genes. The results are as follows:
1. Constructing a series of deletion mutants of NSP1 and NSP2, we assessed the interactions between NSP1 and NSP2 using yeast two-hybrid approach. The results indicate that NSP1 can interact with NSP2 and the LHRI domain alone (105-186aa) of NSP2 is necessary for the interaction. This suggests that NSP1 and NSP2 may form heteropolymers for performing Nod factors signaling in L. japonicus.
2. A series of full and truncated proteins of NSP1 and NSP2 were expressed and purfitied from E. coli, which contains different domains. The protein-protein interactions in vitro were performed using pull-down and detected with western blotting. The results showed that the NSP1 in the absence of LHR1 and VHIID could be interacted with NSP2, and the interaction was abolished without the domain of the LHRII. This reveals that the LHRII of NSP1 is involved in the interaction between NSP1 and NSP2 in L. japonicus.
3. Using the electrophoretic mobility shift analysis (EMSA), we tried to test whether NSP1 could directly bind to the NIN gene promoter which is a initial nodulation gene. The preliminarily results just showed some DNA smear on the EMSA gel and need to be further demonstrated.
百脉根结瘤信号通路NSP1和NSP2是根瘤菌共生信号转导途径中关键的信号蛋白,都属于GRAS家族转录因子,位于CCaMK (Ca2+/calmodulin-dependent protein kinase)下游,并受其调控。本研究的侧重点在于探究NSP1与NSP2间是否存在相互作用,相互作用区段以及检测NSP1基因是否具有结合启动子的能力以及行使此功能的区段,进而揭示NSP1、NSP2在结瘤因子信号传导途径的调控机制。其研究结果如下：
1.构建了一系列nsp1和nsp2缺失突变体,利用酵母双杂交系统研究了百脉根结瘤信号通路NSP1和NSP2蛋白间的相互作用。结果表明NSP1具有与NSP2相互作用的功能,而且NSP2单独的LHR1结构域(105-186氨基酸)则能与NSP1相互作用,说明NSP1与NSP2在行使功能时可能形成异源二聚体。
2.构建了一系列在大肠杆菌中表达的缺失突变体,并表达和纯化缺失不同结构域的截短蛋白,利用pull-down和western印迹转移技术进行了体外蛋白质相互作用。结果表明,NSP1缺失LHR1、VHIID两个结构域后仍具有与NSP2蛋白相互作用的功能,而缺失LHRII区段后则不能相互作用,说明NSP1的LHRII区段是两蛋白间相互作用所必需的。
3.利用凝胶阻滞(EMSA)实验,初步检测了NSP1是否结合启始结瘤基因NIN的启动子的功能,但没有得到理想的实验结果,其原因可能是所用的DNA片段太长(500bp),在EMSA凝胶上只见到一些弥散的DNA带,因此该结果需要进一步证实。
The leguminous plants can specifically establish a symbiosis "nodule" with rhizobia. The establishment of the symbiotic relationship is involved in the complex process incloding signal perception, gene expression and regulation, the interactions of intermolecules between microbes and host plants. Lotus japonicus is one of the model legumes plants for studying the system on the symbiotic nitrogen fixation. The research of the Nod factors signaling pathway and the mechanism of intermolecular interaction in Lotus japonicus will uncover the secret about the establishment of symbiotic relationship and the formation of the symbiosis.
In Lotus japonicus, the proteins of NSP1 (Nodulation Signaling Pathway 1) and NSP2 are belonged to the GRAS family transcription factors, which are the pivotal signal proteins in the symbiotic signaling pathway, and in the downstream of the CCaMK. In this study, we focus on the protein-protein interactions between NSP1 and NSP2, characterization of interaction domain, and identification of the role of the NSP1 binding to the promoter of taget genes. The results are as follows:
1. Constructing a series of deletion mutants of NSP1 and NSP2, we assessed the interactions between NSP1 and NSP2 using yeast two-hybrid approach. The results indicate that NSP1 can interact with NSP2 and the LHRI domain alone (105-186aa) of NSP2 is necessary for the interaction. This suggests that NSP1 and NSP2 may form heteropolymers for performing Nod factors signaling in L. japonicus.
2. A series of full and truncated proteins of NSP1 and NSP2 were expressed and purfitied from E. coli, which contains different domains. The protein-protein interactions in vitro were performed using pull-down and detected with western blotting. The results showed that the NSP1 in the absence of LHR1 and VHIID could be interacted with NSP2, and the interaction was abolished without the domain of the LHRII. This reveals that the LHRII of NSP1 is involved in the interaction between NSP1 and NSP2 in L. japonicus.
3. Using the electrophoretic mobility shift analysis (EMSA), we tried to test whether NSP1 could directly bind to the NIN gene promoter which is a initial nodulation gene. The preliminarily results just showed some DNA smear on the EMSA gel and need to be further demonstrated.
引文
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2. Ane JM, Kiss GB, Riely BK, et al. Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes. Science,2004,303:1364-1367
3. Bolle C. The role of GRAS proteins in plant signal transduction and development. Planta,2004,218:683-692
4. Brundrett M. Coevolution of roots and mycorrhizas of land plants. New Phytol,2002, 154:275-304
5. Capoen W, Goormachtig S, De Rycke R, et al. A plant receptor essential for symbiosome formation. Proc Natl Acad Sci USA,2005,102:10369-10374
6. Catoira R, Galera C, de Billy F, et al. Four genes of Medicago truncatula controlling components of a nod factor transduction pathway. Plant Cell,2000,12:1647-1665
7. Charron D, Pingret J L, Chabaud M, et al. Pharmacological evidence that multiple phospholipids signaling pathways link rhizobium nodulation factor perception in Medicago truncatula root hairs to intracellular responses, including Ca2+ spiking and specific ENOD gene expression. Plant Physiol,2004,136:3582-3593
8. de Lucas M, Daviere J M, Rodriguez-Falcon, et al. A molecular framework for light and gibberellin control of cell elongation. Nature,2008,451:480-484
9. Denarie J, Debelle F, Prome JC. Rhizobium lipo-chitooligosaccharide nodulation factor:Signaling molecules mediating recognition and morphogenesis. Annu Rev Biochem,1996,65:503-535
10. Denztie J and Cullimore J. Lipooligosaccharide nodulation factors:A minnireview new class of signaling molecules mediating recognition and morphogenesis. Cell, 1993,74:951-954
11. Downie JA and Walker SA. Plant responses to nodulation factors. Curr Opin Plant Biol,1999,2:483-489
12. Edwards A, Heckmann AB, Yousafzai F, Due G, Downie JA. Structural implications of mutations in the pea SYM8 symbiosis gene, the DMI1 ortholog, encoding a predicted ion channel. Mol Plant-Microbe Interact,2007,20:1183-1191
13. Ehrhardt DW, Wais R, Long SR. Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Cell,1996,85:673-681
14. Endre G, Kereszt A, Kevei Z, et al. A receptor kinase gene regulating symbiotic nodule development. Nature,2002,417:962-966
15. Endre G, Kereszt A, Kevei Z, et al. A receptor kinase gene regulating symbiotic nodule development. Nature,2002,417:962-966
16. Feng, S., Martinez C, Gusmaroli G, et al. Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature,2008,451:475-479
17. Gherbi H, Markmann K, Svistoonoff S, et al. SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria. Proc Natl Acad Sci U S A,2008,105:4928-4932
18. Gleason C, Chaudhuri S, Yang T B, et al. Nodulation independent of rhizobia induced by a calcium-activated kinase lacking autoinhibition. Nature,2006, 441:1149-1152
19. Heckmann AB, Lombardo F, Miwa H, et al. Lotus japonicus nodulation requires two GRAS domain regulators, one of which is functionally conserved in a non-legume. Plant Physiol,2006,142:1739-1750
20. Heidstra R, Bisseling T. Nod factor-induced host resoponses and mechanisms of Nod factor perception. New Phytol,1996,133:25-43
21. Hirsch S, Kim J, Munoz A, et al. GRAS proteins form a DNA binding complex to induce gene expression during Nodulation signaling in Medicago truncatula. Plan cell,2009,21:545-557
22. Imaizumi-Anraku H, Takeda N, Charpentier M, et al. Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots. nature,2005,433:527-531
23. Kalo P, Gleason C,Edwards A, et al. Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science,2005, 308:1786-1789
24. Kanamori N, Madsen LH, Radutoiu S, et al. A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis. Proc Natl Acad Sci USA,2006,103:359-364
25. Kistner C, Winzer T, Pitzschke A, et al. Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis. Plant Cell,2005,17:2217-2229
26. Lerouge P, Roche P, Faucher C, et al. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature,1990,344:781-784
27. Levy J, Bres C, Geurts R, Chalhoub B, et al. A putative Ca2+ and almodulin dependent protein kinase required for bacterial and fungal symbioses. Science,2004, 303:1361-1364
28. Limpens E and Bisseling T. Signaling in symbiosis. Curr Opin Plant Biol,2003, 6:343-350
29. Limpens E, Mirabella R, Fedorova E, et al. Formation of organelle-like N2-fixing symbiosomes in legume root nodules is controlled by DMI2. PNAS,2005, 102:10375-10380
30. Madsen E B, Madsen L H, Radutoiu S, et al. A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature,2003,425:637-640
31. Messinese E, Mun J-H, Yeun L, et al. A novel nuclear protein interacts with the symbiotic DMI3 calcium and calmodulin dependent protein kinase of Medicago truncatula. Mo Plant-Microbe Interact,2007,20:912-921
32. Middleton PH, Jakab J, Penmetsa RV, et al. An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction. Plant Cell,2007, 19:1221-1234
33. Miller, J. H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1972
34. Mitra RM, Gleason CA, Edwards A, et al. A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development:Gene identification by transcript-based cloning. Proc Natl Acad Sci USA,2004,101:4701-4705
35. Miwa H, Sun J, Oldroyd GED, Downie JA. Analysis of nod-factorinduced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus. Mol Plant Microbe Interact,2006,19:914-923
36. Murakami Y, Miwa H, Imaizumi-Anraku H, et al. Positional cloning identifies Lotus japonicus NSP2, a putative transcription factor of the GRAS family, required for NIN and ENOD40 gene expression in nodule initiation. DNA Res,2006,13:255-265
37. Oldroyd G E D and Downie J A. Coordinating nodule morphogenesis with rhizobial infection in legumes. Annu Rev Plant Biol,2008,59:519-546
38. Oldroyd G E D and Long S R. Identification and characterization of NSP2, a gene of Medicago truncatula involved in Nod factor signaling. Plant Physiol,2003, 131:1027-1032
39. Oldroyd G E and Downie J A Nuclear calcium changes at the core of symbiosis signalling. Curr Opin Plant Biol,2006,9:351-357
40. Oldroyd, G E D and Downie, J A. Calcium, kinases and nodulation signalling in legumes. Nat Rev Mol Cell Biol,2004,5:566-576
41. Parniske M and Downie J A. Locks, keys and symbioses. Nature Publishing Group, 2003,425:569-570
42. Patil S, Takezawa D, Poovaiah BW. Chimeric plant calcium/calmodulin-dependent protein kinase gene with a neural visinin-like calcium-binding domain. Proc Natl Acad Sci USA 1995,92:4897-901
43. Peters N K, Vermat D P S. Penolic compounds as regulators of gene expression in plant-microbe interaction. Plant-Microbe Interact,1990,3:4-8
44. Pysh LD, Wysocka-Diller JW, Camilleri C, et al. The GRAS gene family in Arabidopsis:sequence characterization and basic expression analysis of the SCARECROW-LIKE genes. Plant J,1999,18:111-119
45. Radutoiu S, Madsen L H, Madsen E B, et al. Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature,2003,425:585-592
46. Radutoiu S, Madsen LH, Madsen EB, et al. LysM domains mediate lipochitin-oligosaccharide recognition and Nfr genes extend the symbiotic host range. EMBO J,2007,26:3923-3935
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53. Smit P, Raedts J, Portyanko V, et al. NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription. Science,2005,308:1789-1791
54. Stracke S, Kistner C, Yoshida S, et al. A plant receptor-like kinase required for both bacterial and fungal symbiosis. Nature,2002,417:959-962
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57. Wais R.J., Galera C., Oldroyd G., et al. Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula. Proc Natl Acad Sci USA,2000, 97:13407-13412
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2. Ane JM, Kiss GB, Riely BK, et al. Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes. Science,2004,303:1364-1367
3. Bolle C. The role of GRAS proteins in plant signal transduction and development. Planta,2004,218:683-692
4. Brundrett M. Coevolution of roots and mycorrhizas of land plants. New Phytol,2002, 154:275-304
5. Capoen W, Goormachtig S, De Rycke R, et al. A plant receptor essential for symbiosome formation. Proc Natl Acad Sci USA,2005,102:10369-10374
6. Catoira R, Galera C, de Billy F, et al. Four genes of Medicago truncatula controlling components of a nod factor transduction pathway. Plant Cell,2000,12:1647-1665
7. Charron D, Pingret J L, Chabaud M, et al. Pharmacological evidence that multiple phospholipids signaling pathways link rhizobium nodulation factor perception in Medicago truncatula root hairs to intracellular responses, including Ca2+ spiking and specific ENOD gene expression. Plant Physiol,2004,136:3582-3593
8. de Lucas M, Daviere J M, Rodriguez-Falcon, et al. A molecular framework for light and gibberellin control of cell elongation. Nature,2008,451:480-484
9. Denarie J, Debelle F, Prome JC. Rhizobium lipo-chitooligosaccharide nodulation factor:Signaling molecules mediating recognition and morphogenesis. Annu Rev Biochem,1996,65:503-535
10. Denztie J and Cullimore J. Lipooligosaccharide nodulation factors:A minnireview new class of signaling molecules mediating recognition and morphogenesis. Cell, 1993,74:951-954
11. Downie JA and Walker SA. Plant responses to nodulation factors. Curr Opin Plant Biol,1999,2:483-489
12. Edwards A, Heckmann AB, Yousafzai F, Due G, Downie JA. Structural implications of mutations in the pea SYM8 symbiosis gene, the DMI1 ortholog, encoding a predicted ion channel. Mol Plant-Microbe Interact,2007,20:1183-1191
13. Ehrhardt DW, Wais R, Long SR. Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Cell,1996,85:673-681
14. Endre G, Kereszt A, Kevei Z, et al. A receptor kinase gene regulating symbiotic nodule development. Nature,2002,417:962-966
15. Endre G, Kereszt A, Kevei Z, et al. A receptor kinase gene regulating symbiotic nodule development. Nature,2002,417:962-966
16. Feng, S., Martinez C, Gusmaroli G, et al. Coordinated regulation of Arabidopsis thaliana development by light and gibberellins. Nature,2008,451:475-479
17. Gherbi H, Markmann K, Svistoonoff S, et al. SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria. Proc Natl Acad Sci U S A,2008,105:4928-4932
18. Gleason C, Chaudhuri S, Yang T B, et al. Nodulation independent of rhizobia induced by a calcium-activated kinase lacking autoinhibition. Nature,2006, 441:1149-1152
19. Heckmann AB, Lombardo F, Miwa H, et al. Lotus japonicus nodulation requires two GRAS domain regulators, one of which is functionally conserved in a non-legume. Plant Physiol,2006,142:1739-1750
20. Heidstra R, Bisseling T. Nod factor-induced host resoponses and mechanisms of Nod factor perception. New Phytol,1996,133:25-43
21. Hirsch S, Kim J, Munoz A, et al. GRAS proteins form a DNA binding complex to induce gene expression during Nodulation signaling in Medicago truncatula. Plan cell,2009,21:545-557
22. Imaizumi-Anraku H, Takeda N, Charpentier M, et al. Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots. nature,2005,433:527-531
23. Kalo P, Gleason C,Edwards A, et al. Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science,2005, 308:1786-1789
24. Kanamori N, Madsen LH, Radutoiu S, et al. A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis. Proc Natl Acad Sci USA,2006,103:359-364
25. Kistner C, Winzer T, Pitzschke A, et al. Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis. Plant Cell,2005,17:2217-2229
26. Lerouge P, Roche P, Faucher C, et al. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature,1990,344:781-784
27. Levy J, Bres C, Geurts R, Chalhoub B, et al. A putative Ca2+ and almodulin dependent protein kinase required for bacterial and fungal symbioses. Science,2004, 303:1361-1364
28. Limpens E and Bisseling T. Signaling in symbiosis. Curr Opin Plant Biol,2003, 6:343-350
29. Limpens E, Mirabella R, Fedorova E, et al. Formation of organelle-like N2-fixing symbiosomes in legume root nodules is controlled by DMI2. PNAS,2005, 102:10375-10380
30. Madsen E B, Madsen L H, Radutoiu S, et al. A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature,2003,425:637-640
31. Messinese E, Mun J-H, Yeun L, et al. A novel nuclear protein interacts with the symbiotic DMI3 calcium and calmodulin dependent protein kinase of Medicago truncatula. Mo Plant-Microbe Interact,2007,20:912-921
32. Middleton PH, Jakab J, Penmetsa RV, et al. An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction. Plant Cell,2007, 19:1221-1234
33. Miller, J. H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1972
34. Mitra RM, Gleason CA, Edwards A, et al. A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development:Gene identification by transcript-based cloning. Proc Natl Acad Sci USA,2004,101:4701-4705
35. Miwa H, Sun J, Oldroyd GED, Downie JA. Analysis of nod-factorinduced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus. Mol Plant Microbe Interact,2006,19:914-923
36. Murakami Y, Miwa H, Imaizumi-Anraku H, et al. Positional cloning identifies Lotus japonicus NSP2, a putative transcription factor of the GRAS family, required for NIN and ENOD40 gene expression in nodule initiation. DNA Res,2006,13:255-265
37. Oldroyd G E D and Downie J A. Coordinating nodule morphogenesis with rhizobial infection in legumes. Annu Rev Plant Biol,2008,59:519-546
38. Oldroyd G E D and Long S R. Identification and characterization of NSP2, a gene of Medicago truncatula involved in Nod factor signaling. Plant Physiol,2003, 131:1027-1032
39. Oldroyd G E and Downie J A Nuclear calcium changes at the core of symbiosis signalling. Curr Opin Plant Biol,2006,9:351-357
40. Oldroyd, G E D and Downie, J A. Calcium, kinases and nodulation signalling in legumes. Nat Rev Mol Cell Biol,2004,5:566-576
41. Parniske M and Downie J A. Locks, keys and symbioses. Nature Publishing Group, 2003,425:569-570
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