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Cis-Elements In Nematode-Responsive Promoters
Phytosfere'99 - Highlights in European Plant Biotechnology Gert E. de Vries and Karin Metzlaff (Editors). 9 Elsevier Science B.V. All rights reserved.
Cis-Elements In Nematode-Responsive Promoters
The problem Biotrophic plant pathogens that depend on living cells need to use subtle strategies to tame plant cells in way for these to suit the pathogen's needs while being wholly functional. Plant endoparasitic nematodes do so by obliging root cells to differentiate into "nurse cells" from which they feed, through still unraveled mechanisms [1]. Different nematodes induce different types of nurse cells, collectively termed Nematode Feeding Sites (NFSs) [2-4]. For a long time it has been believed that nematode secretions are responsible for NFS induction, but only recently this has received experimental support [5]. How do these secretions act to make a plant cell shift its developmental fate? (figure 1) Although the signal molecules of nematode secretions are still unknown, and so are their primary plant targets, it has now been well established that many genes change their expression patterns at different times during NFS development. For several of these genes, the change in expression occurs at the transcriptional level, and their promoter regions suffice to confer them nematode-responsiveness [6-9]. If promoters are turned on selectively, then nematode signal(s) must somehow influence the transcription transducers ] factor network that control promoter activity. Extensive changes in gene expression during cell differentiation are often due to modulation of transcriptional Transcription of regulatory genes regulation, brought about by signal transduction casActivation of regulatory proteins cades that are triggered by one or more key events. Such events, in the case of NFSs, may involve interactions of nematode secretions or surface molecules transducers with specific cell components of a signal transduction cascade in which transcriptional control is involved. Therefore, analysis of promoters differenTranscription tially regulated during NFS development should
iiiiiiiiili!iiiiiiiiiiililiiii i [iiiiiiiiiiiiiiiiiiiiiiiiiiiii ?!i;iiiiiiiiiiiiii)iliiiil)iiiiiiii iiiiiiiiii!iiiiiiii;iiiiiiiiiii!iiii !
Figure 1. Possible target points in the plant cell for nematode signals during NFS development.
of downstream genes (NFS-specific expression)
S. Sanz-Alf~rez, Departamento de Biologfa, Universidad Aut6noma de Madrid, Spain
177
Challenges of the Environment eventually lead us one step closer to the key events that initiate this developmental process. Finding the sequence elements responsible for nematode-inducibility and their counterpart transcription factors will guide us upstream within the signal transduction pathway linking nematode and plant genes. That will not just give insight into the subtle plant-nematode interaction, but it may also disclose important components of cell differentiation paths. In addition, precise knowledge on how nematodes trigger gene expression has a direct application in the construction of transgenic plants which either produce nematicidal proteins specifically at NFSs, or are unable to differentiate a competent NFS to sustain nematode development.
The question and the approaches Within the just said conceptual frame, the question can be simply formulated (figure 1): what is in there between the nematode signals and the transcription of plant responsive genes? Which are the genes that execute the nematode order to the root cell "acquire the NSF fate"? Which the proteins that regulate the expression of the adequate battery of downstream genes related to the building of NFSs? Which are, among the many a plant has, the signal transduction cascades involved in the process? And, will these cascades lead us to the primary plant targets for nematode signals? The approaches that our group is taking to start answering these questions involve: l) identification of nematode responsive plant genes and study of their promoters (regulatory and downstream genes); 2) identification in the promoters of putative target sites for transcription factors, both as nuclear protein binding regions and as nematode-responsive sequence elements (NREs); 3) cloning the relevant DNA binding proteins that act as direct transcriptional regulators; and 4) disclosing how such transcription factors are activated at NFSs
Nematode-responsive promoters and regulatory sequence elements The first requirement to characterize nematode-responsive promoters is the identification of such promoters. There are already a number of them known to be induced by root-knot and/ or cyst nematodes in which we are working. Some have been identified by others in Arabidopsis in a promoter-tagging effort, partly within the ARENA project [9]. Of the selected tagged sequences which were induced by nematode infection, at least one corresponds to the promoter of a real gene: RPE, which codes for the D-ribulose-5-phosphate 3- epimerase [10]. In collaboration with the groups that cloned the tagged sequences, we have initiated the search for elements involved in their transcriptional regulation, both in NFSs and elsewhere in the plant (Uribe, Herreros and Fenoll, unpublished data) In our laboratory, we have identified several promoters induced by root-knot nematodes, their induction being clearly observed when fused to GUS in transgenic plants [11,12]. Three are from Arabidopsis: the promoters of the HMG1 and HMG2 genes, which code for several isoforms of the hydroxymethyl-glutaryl-CoA Reductase [ 13] and the promoter of the H4A748 gene that codes for the S-phase specific Histone 4 [ 14]. We have also shown that the promoter of a sunflower gene that codes for one small heat shock protein (sHSP17 group) [15] is induced in tobacco plants by Meloidogyne spp. Several promoters from geminiviral genes have also been shown in our laboratory to be expressed at NFSs (unpublished results). 178
Cis-elements in nematode-responsive promoters Several deletions are available for all these promoters, and we have already been able to narrow down them to minimal regions that still confer nematode inducibility. We are presently analysing them in greater detail in terms of time course induction and sequence elements important for both nematode responsiveness and NFS specificity [ 12; unpublished results].
How are promoters activated at NFSs? They could respond to one or a few transcription factors (TFs) of a unique differentiation pathway, or, alternatively, they could be activated by diverse TFs belonging to separate differentiation paths. We have compared several nematode-inducible promoters (among them the said four plus several Arabidopsis tagged sequences) by using a number of programs available for the identification of putative cis elements (Sequence Interpretation Tools, TFSearch, TRANSFAC and TRADA). Such extensive sequence analysis reveals that there is very little in common among the available nematode-inducible promoters, and has failed to identify a conserved putative nematode-responsive element present in all promoters activated in NFSs. From these data, we conclude that the different genes must be regulated in NFSs by an array of different TFs, and that they respond to nematodes through (at least partially) different mechanisms. The hypothesis that we currently favour is depicted in figure 2. Nematodeborne factors will signal in the initial root cell either one or several primary event(s), from which branched transduction cascades, probably part of distinct cell differentiation pathways, carry on downstream the orders to develop a feeding site. Such cascades will certainly involve the activation and/or the de novo synthesis of batteries of tranN e m a t o d e signals scription activators that act (either individually or in a combined manTFa [] TFb 9 ner) on target sequences in the plant nematode-responsive promoters. The fact that some of these promoters are turned on only by [~iFiiJ cyst or by root-knot nematodes, while others respond to both types ~ of parasites, also suggests that multiple routes must be involved in feeding site formation. None of the nematode-inducible promoters tested so far is 100% specific for ~ l ~ NFSs; all are active somewhere else in the plant. This is not sur- Figure 2. The model explains how distinct cell differentiation pathways could mediate the activation of discrete sets of downstream prising, since NFS differentiation promoters (the nematode-responsive promoters described in the text) has to make use of pre-existent de- during NFS development. Different transcription factors (TFa, TFb) velopmental pathways in the ini- are activated, directly or indirectly, by signal molecules from the tial cell, most probably selecting nematode. Each one will, in turn, mediate the expression of other TFs. TF1, TF2 and TF3 directly activate groups of downstream the bits of each that better suit ac- genes, while R acts through additional cascades involving still other complishing the final goal: acquire TFs. For simplicity, direct activation of downstream genes by TFa NFS fate to nurture the nematode. and TFb is not shown. 179
Challenges of the Environment Because of these more or less broad expression profiles, the potential to engineer nematode resistance using the said promoters as they are is limited. Thus, it appears essential further analyses to characterise the NREs in each promoter and, eventually, to separate these from elements responsible for the expression in other tissues. We are currently undertaking these type of analyses, that can be exemplified in the LEMMI9 gene.
Analysis of the LEMMI9 promoter The LEMMI9 tomato gene was identified by differential screening of cDNA libraries by G. Gheysen's group as a highly abundant transcript in nematode-infected roots. Further, they showed by in situ hybridisation a high level of LEMMI9 transcript accumulation specifically in giant cells [ 16]. LEMMI9 codes for a homologue to LEA-14, a cotton late-embryogenesis abundant protein. The function proposed for this class of proteins is to protect the embryo during the dessication of the seed, but some LEAs are also induced during water stress responses in vegetative tissues. In a collaborative work with G. Gheysen's group within the ARENA project [ 17], a genomic clone was isolated from a tomato genomic library, that contained 3500 bp of the LEMMI9 promoter sequence, where the transcriptional start point and the putative TATA box were located. Most of the promoter region from the genomic clone was fused to GUS and used to produce transgenic potato plants. GUS staining revealed that the promoter fragment suffices to confer nematode inducibility. Through the analysis of DNAprotein interactions in electrophoretic mobility gel shift assays, we identified a region close to the putative TATA box that binds in a specific manner nuclear proteins from infected, but not from uninfected roots. Within this region we could map the protein-binding site to a 12-bp repeat [17]. The involvement of this 12-bp repeat in binding of proteins from nematodeinfected root sections and from other expressing tissues has now been confirmed in tomato plants by a ligation-mediated PCR version of in vivo footprinting of the endogenous LEMMI9 promoter (Uribe and Fenoll, unpublished results). We are presently analysing transgenic Arabidopsis plants carrying several deletion-mutation fusions to GUS, to assess the function of this element in the activation of the LEMMI9 promoter (Sanz-Alf6rez, Aristizfibat and Fenoll, unpublished data). An important goal in our studies is to identify the plant protein(s) that bind to the 12-bp sequence in a yeast one-hybrid system, to partially fill the gap between the modification of LEMMI9 expression and the unknown signal coming from the nematode. The results from this work on the LEMMI9 promoter demonstrate that, in spite of the technical difficulties for non-genetic molecular analysis of the plant-nematode interaction, putative promoter elements can be identified through the study of protein-DNA interactions. Our findings also substantiate our initial hypothesis: the existence of putative transcription factors absent (or inactive) in non-infected roots, that act specifically in feeding sites and/or the surrounding root cells.
Making models for further research We believe that the path leading from nematode to mature feeding site could be dissected taking promoter induction as the downstream effect, and walking upstream from this molecular event. The first step in this walk is the identification of the cis elements and the proteins that, upon binding to them, will turn promoters on. Since these promoters must lye down180
Cis-elements in nematode-responsive promoters stream in the cascade initiated by nematode signals (Figure 2), they and their activators will allow us to proceed upstream towards the regulatory molecule(s) that control the fate change of root cells to become NFSs. Our results in the LEMMI9 promoter could be taken as an example of how to tackle the problem from one end of the signal transduction cascade. Recently, we have started to approach the pathway from the other end. Nematode secretions are known to stimulate division in plant protoplasts and cultured animal cells [5], what strongly suggests that secretions are, at least in part, responsible for some of the molecular events in NFS induction. We are at present fractionating nematode secretions [ 18] and using the resulting fractions to induce plant responses in the absence of nematodes. The type of screening we are doing include following changes in gene expression by both testing promoter-GUS fusions, and by cDNA AFLPs. Once we link a secretion fraction to a molecular event, we could address the search for partner molecules in the plant cell, by combining yeast two-hybrid systems with anti-secretions antibody screening of nematode expression libraries. By proceeding simultaneously in the two senses (from plant promoter towards secretions, and vice versa), we hope to disclose elements that participate in this route. Since some of these elements may as well be part of better known signal transduction pathways involved in other differentiation events, we will be able to borrow pieces from those paths to help building the molecular puzzle of NFS differentiation.
Authors of this contribution S. Sanz-Alf6rez 1, X. Uribe 1,3,E A. Aristizfibal TM,E. Herreros 1, E F. del Campo l, C. Fenoll 1,2,g< 1 Departamento de Biologfa, Universidad Aut6noma de Madrid, E-28049 Madrid, Spain, 2Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, E-45071 Toledo, Spain, 3present address: Centro Nacional de Biotecnologfa, Cantoblanco, E-28049 Madrid, Spain, 4 Present address: Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia, Ciudad Universitaria, Santa F6 de Bogotfi, Colombia. *Corresponding author
Acknowledgements We want to thank the European Commission for funding this work through grants B IO4CT96 0318 and FAIR3-CT96 1714 to CF.
References 1. 2. 3. 4.
5.
C. Fenoll, EM.W. Grundler, S.A. Ohl, Cellular and molecular aspects of plant-nematode interactions, Kluwer Academic Publishers, The Netherlands (1997). EC. Sijmons, Plant-nematode interactions, Plant Mol. Biol. 23 (1993) 917-931. EC. Sijmons, H.J. Atkinson, U. Wyss, Parasitic strategies of root nematodes and associated host cell responses, Ann. Rev. Phytopathol. 32 (1994) 235-259. U. Wyss, Root Parasitic Nematodes: An overview, In: Cellular and molecular aspects of plant-nematode interactions, C. Fenoll, F.M.W. Grundler and S.A. Ohl, Eds., Kluwer Academic Publishers. The Netherlands, (1997) 5-22. A. Goverse, J. Rouppe van der Voort, C. Rouppe van der Voort, A. Kavelaars, G. Smant, A. Schots, J. Bakker, J. Helder, Naturally-induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells, Mol. Plant Microbe Interact.
181
Challenges of the Environment
6.
7. 8. 9.
10. 11.
12. 13.
14. 15.
16. 17.
18.
(1999) (in press). O.J.M. Goddijn, K. Lindsey, EM. van der Lee, J.C. Klap, P.C. Sijmons, Differential gene expression in nematode-induced feeding structures of transgenic plants harboring promoter-gusA fusion constructs, Plant J. 4 (1993) 863-873. C.H. Opperman, C.G. Taylor, M.A. Conkling, Root-knot nematode directed expression of a plant rootspecific gene, Science 263 (1994) 221-223. D.M. Bird, M.A. Wilson, DNA sequence and expression analysis of root-knot nematode-elicited giant cell transcripts, Mol. Plant Microbe Interact. 7 (1994) 419-424. N. Barthels, EM. Van der Lee, J.C. Klap, O.J.M. Goddijn, M. Karimi, R Puzio, EM.W. Grundler, S.A. Ohl, K. Lindsey, L. Robertson, W.M. Robertson, M. Van Montagu, G. Gheysen, RC. Sijmons, Regulatory sequences of Arabidopsis drive reporter gene expression in nematode feeding structures, Plant Cell 9 (1997) 2119-2134. B. Favery, R Lecomte, N. Gil, N. Bechtold, D. Bouchez, A. Dalmasso, R Abad, RPE, a plant gene involved in early developmental steps of nematode feeding cells, EMBO J. 17 (1998) 6799-6811. C. Fenoll, EA. Aristizfibal, S. Sanz-Alf6rez, EE Del Campo, Regulation of gene expression in feeding sites, In: Cellular and Molecular Aspects of Plant-Nematode Interactions, C. Fenoll, EM.W. Grundler, S.A. Ohl Eds., Kluwer Academic Publishers, The Netherlands, (1997) 133-149. EA. Aristizfibal, Identificaci6n y anfilisis de la expresi6n en plantas de genes regulados por nemfitodos endoparasfticos, Ph.D. Thesis, Universidad Aut6noma de Madrid, Spain, (1996). M. Enjuto, L. Balcell, N. Campos, C. Caelles, M. Arr6, A. Boronat, Arabidopsis thaliana contains two differentially expressed 3-hydroxy methylglutaryl-CoA reductase genes, which encode microsomal forms of the enzyme, Proc. Natl. Acad. Sci. USA 91 (1994) 927-931. R. Atanassova, N. Chaubet, C. Gigot, A 126 bp fragment of a plant histone gene promoter confers preferential expression in meristems of transgenic Arabidopsis, Plant J. 2 (1992) 291-300. M.A. Coca, C. Almoguera, T.L. Thomas, J. Jordano, Differential regulation of small heat-shock genes in plants: analysis of water-stress-inducible and developmentally activated sunflower promoter, Plant Mol. Biol. 31 (1996) 863-876. W. Van der Eycken, D.J. Engler, D. Inze, M. Van Montagu, G. Gheysen, Molecular study of root-knot nematode-induced feeding sites, Plant J. 9 (1996)45-54. C. Escobar, J. De Meutter, EA. Aristiz~ibal, S. Sanz-Alf6rez, RE Del Campo, N. Barthels, W. Van der Eycken, J. Seurinck, M. Van Montagu, G. Gheysen, C. Fenoll, Isolation of the Lemmi9 gene and promoter analysis during a compatible plant-nematode interaction, Mol. Plant-Microbe Interact. 12 (1999) 440-449. L. Robertson, W.M. Robertson, J.T. Jones, Direct analysis of the secretions of the potato cyst nematode Globodera rostochiensis, Parasitology 119 (1999) (in press).
182
Cis-Elements In Nematode-Responsive Promoters
The problem Biotrophic plant pathogens that depend on living cells need to use subtle strategies to tame plant cells in way for these to suit the pathogen's needs while being wholly functional. Plant endoparasitic nematodes do so by obliging root cells to differentiate into "nurse cells" from which they feed, through still unraveled mechanisms [1]. Different nematodes induce different types of nurse cells, collectively termed Nematode Feeding Sites (NFSs) [2-4]. For a long time it has been believed that nematode secretions are responsible for NFS induction, but only recently this has received experimental support [5]. How do these secretions act to make a plant cell shift its developmental fate? (figure 1) Although the signal molecules of nematode secretions are still unknown, and so are their primary plant targets, it has now been well established that many genes change their expression patterns at different times during NFS development. For several of these genes, the change in expression occurs at the transcriptional level, and their promoter regions suffice to confer them nematode-responsiveness [6-9]. If promoters are turned on selectively, then nematode signal(s) must somehow influence the transcription transducers ] factor network that control promoter activity. Extensive changes in gene expression during cell differentiation are often due to modulation of transcriptional Transcription of regulatory genes regulation, brought about by signal transduction casActivation of regulatory proteins cades that are triggered by one or more key events. Such events, in the case of NFSs, may involve interactions of nematode secretions or surface molecules transducers with specific cell components of a signal transduction cascade in which transcriptional control is involved. Therefore, analysis of promoters differenTranscription tially regulated during NFS development should
iiiiiiiiili!iiiiiiiiiiililiiii i [iiiiiiiiiiiiiiiiiiiiiiiiiiiii ?!i;iiiiiiiiiiiiii)iliiiil)iiiiiiii iiiiiiiiii!iiiiiiii;iiiiiiiiiii!iiii !
Figure 1. Possible target points in the plant cell for nematode signals during NFS development.
of downstream genes (NFS-specific expression)
S. Sanz-Alf~rez, Departamento de Biologfa, Universidad Aut6noma de Madrid, Spain
177
Challenges of the Environment eventually lead us one step closer to the key events that initiate this developmental process. Finding the sequence elements responsible for nematode-inducibility and their counterpart transcription factors will guide us upstream within the signal transduction pathway linking nematode and plant genes. That will not just give insight into the subtle plant-nematode interaction, but it may also disclose important components of cell differentiation paths. In addition, precise knowledge on how nematodes trigger gene expression has a direct application in the construction of transgenic plants which either produce nematicidal proteins specifically at NFSs, or are unable to differentiate a competent NFS to sustain nematode development.
The question and the approaches Within the just said conceptual frame, the question can be simply formulated (figure 1): what is in there between the nematode signals and the transcription of plant responsive genes? Which are the genes that execute the nematode order to the root cell "acquire the NSF fate"? Which the proteins that regulate the expression of the adequate battery of downstream genes related to the building of NFSs? Which are, among the many a plant has, the signal transduction cascades involved in the process? And, will these cascades lead us to the primary plant targets for nematode signals? The approaches that our group is taking to start answering these questions involve: l) identification of nematode responsive plant genes and study of their promoters (regulatory and downstream genes); 2) identification in the promoters of putative target sites for transcription factors, both as nuclear protein binding regions and as nematode-responsive sequence elements (NREs); 3) cloning the relevant DNA binding proteins that act as direct transcriptional regulators; and 4) disclosing how such transcription factors are activated at NFSs
Nematode-responsive promoters and regulatory sequence elements The first requirement to characterize nematode-responsive promoters is the identification of such promoters. There are already a number of them known to be induced by root-knot and/ or cyst nematodes in which we are working. Some have been identified by others in Arabidopsis in a promoter-tagging effort, partly within the ARENA project [9]. Of the selected tagged sequences which were induced by nematode infection, at least one corresponds to the promoter of a real gene: RPE, which codes for the D-ribulose-5-phosphate 3- epimerase [10]. In collaboration with the groups that cloned the tagged sequences, we have initiated the search for elements involved in their transcriptional regulation, both in NFSs and elsewhere in the plant (Uribe, Herreros and Fenoll, unpublished data) In our laboratory, we have identified several promoters induced by root-knot nematodes, their induction being clearly observed when fused to GUS in transgenic plants [11,12]. Three are from Arabidopsis: the promoters of the HMG1 and HMG2 genes, which code for several isoforms of the hydroxymethyl-glutaryl-CoA Reductase [ 13] and the promoter of the H4A748 gene that codes for the S-phase specific Histone 4 [ 14]. We have also shown that the promoter of a sunflower gene that codes for one small heat shock protein (sHSP17 group) [15] is induced in tobacco plants by Meloidogyne spp. Several promoters from geminiviral genes have also been shown in our laboratory to be expressed at NFSs (unpublished results). 178
Cis-elements in nematode-responsive promoters Several deletions are available for all these promoters, and we have already been able to narrow down them to minimal regions that still confer nematode inducibility. We are presently analysing them in greater detail in terms of time course induction and sequence elements important for both nematode responsiveness and NFS specificity [ 12; unpublished results].
How are promoters activated at NFSs? They could respond to one or a few transcription factors (TFs) of a unique differentiation pathway, or, alternatively, they could be activated by diverse TFs belonging to separate differentiation paths. We have compared several nematode-inducible promoters (among them the said four plus several Arabidopsis tagged sequences) by using a number of programs available for the identification of putative cis elements (Sequence Interpretation Tools, TFSearch, TRANSFAC and TRADA). Such extensive sequence analysis reveals that there is very little in common among the available nematode-inducible promoters, and has failed to identify a conserved putative nematode-responsive element present in all promoters activated in NFSs. From these data, we conclude that the different genes must be regulated in NFSs by an array of different TFs, and that they respond to nematodes through (at least partially) different mechanisms. The hypothesis that we currently favour is depicted in figure 2. Nematodeborne factors will signal in the initial root cell either one or several primary event(s), from which branched transduction cascades, probably part of distinct cell differentiation pathways, carry on downstream the orders to develop a feeding site. Such cascades will certainly involve the activation and/or the de novo synthesis of batteries of tranN e m a t o d e signals scription activators that act (either individually or in a combined manTFa [] TFb 9 ner) on target sequences in the plant nematode-responsive promoters. The fact that some of these promoters are turned on only by [~iFiiJ cyst or by root-knot nematodes, while others respond to both types ~ of parasites, also suggests that multiple routes must be involved in feeding site formation. None of the nematode-inducible promoters tested so far is 100% specific for ~ l ~ NFSs; all are active somewhere else in the plant. This is not sur- Figure 2. The model explains how distinct cell differentiation pathways could mediate the activation of discrete sets of downstream prising, since NFS differentiation promoters (the nematode-responsive promoters described in the text) has to make use of pre-existent de- during NFS development. Different transcription factors (TFa, TFb) velopmental pathways in the ini- are activated, directly or indirectly, by signal molecules from the tial cell, most probably selecting nematode. Each one will, in turn, mediate the expression of other TFs. TF1, TF2 and TF3 directly activate groups of downstream the bits of each that better suit ac- genes, while R acts through additional cascades involving still other complishing the final goal: acquire TFs. For simplicity, direct activation of downstream genes by TFa NFS fate to nurture the nematode. and TFb is not shown. 179
Challenges of the Environment Because of these more or less broad expression profiles, the potential to engineer nematode resistance using the said promoters as they are is limited. Thus, it appears essential further analyses to characterise the NREs in each promoter and, eventually, to separate these from elements responsible for the expression in other tissues. We are currently undertaking these type of analyses, that can be exemplified in the LEMMI9 gene.
Analysis of the LEMMI9 promoter The LEMMI9 tomato gene was identified by differential screening of cDNA libraries by G. Gheysen's group as a highly abundant transcript in nematode-infected roots. Further, they showed by in situ hybridisation a high level of LEMMI9 transcript accumulation specifically in giant cells [ 16]. LEMMI9 codes for a homologue to LEA-14, a cotton late-embryogenesis abundant protein. The function proposed for this class of proteins is to protect the embryo during the dessication of the seed, but some LEAs are also induced during water stress responses in vegetative tissues. In a collaborative work with G. Gheysen's group within the ARENA project [ 17], a genomic clone was isolated from a tomato genomic library, that contained 3500 bp of the LEMMI9 promoter sequence, where the transcriptional start point and the putative TATA box were located. Most of the promoter region from the genomic clone was fused to GUS and used to produce transgenic potato plants. GUS staining revealed that the promoter fragment suffices to confer nematode inducibility. Through the analysis of DNAprotein interactions in electrophoretic mobility gel shift assays, we identified a region close to the putative TATA box that binds in a specific manner nuclear proteins from infected, but not from uninfected roots. Within this region we could map the protein-binding site to a 12-bp repeat [17]. The involvement of this 12-bp repeat in binding of proteins from nematodeinfected root sections and from other expressing tissues has now been confirmed in tomato plants by a ligation-mediated PCR version of in vivo footprinting of the endogenous LEMMI9 promoter (Uribe and Fenoll, unpublished results). We are presently analysing transgenic Arabidopsis plants carrying several deletion-mutation fusions to GUS, to assess the function of this element in the activation of the LEMMI9 promoter (Sanz-Alf6rez, Aristizfibat and Fenoll, unpublished data). An important goal in our studies is to identify the plant protein(s) that bind to the 12-bp sequence in a yeast one-hybrid system, to partially fill the gap between the modification of LEMMI9 expression and the unknown signal coming from the nematode. The results from this work on the LEMMI9 promoter demonstrate that, in spite of the technical difficulties for non-genetic molecular analysis of the plant-nematode interaction, putative promoter elements can be identified through the study of protein-DNA interactions. Our findings also substantiate our initial hypothesis: the existence of putative transcription factors absent (or inactive) in non-infected roots, that act specifically in feeding sites and/or the surrounding root cells.
Making models for further research We believe that the path leading from nematode to mature feeding site could be dissected taking promoter induction as the downstream effect, and walking upstream from this molecular event. The first step in this walk is the identification of the cis elements and the proteins that, upon binding to them, will turn promoters on. Since these promoters must lye down180
Cis-elements in nematode-responsive promoters stream in the cascade initiated by nematode signals (Figure 2), they and their activators will allow us to proceed upstream towards the regulatory molecule(s) that control the fate change of root cells to become NFSs. Our results in the LEMMI9 promoter could be taken as an example of how to tackle the problem from one end of the signal transduction cascade. Recently, we have started to approach the pathway from the other end. Nematode secretions are known to stimulate division in plant protoplasts and cultured animal cells [5], what strongly suggests that secretions are, at least in part, responsible for some of the molecular events in NFS induction. We are at present fractionating nematode secretions [ 18] and using the resulting fractions to induce plant responses in the absence of nematodes. The type of screening we are doing include following changes in gene expression by both testing promoter-GUS fusions, and by cDNA AFLPs. Once we link a secretion fraction to a molecular event, we could address the search for partner molecules in the plant cell, by combining yeast two-hybrid systems with anti-secretions antibody screening of nematode expression libraries. By proceeding simultaneously in the two senses (from plant promoter towards secretions, and vice versa), we hope to disclose elements that participate in this route. Since some of these elements may as well be part of better known signal transduction pathways involved in other differentiation events, we will be able to borrow pieces from those paths to help building the molecular puzzle of NFS differentiation.
Authors of this contribution S. Sanz-Alf6rez 1, X. Uribe 1,3,E A. Aristizfibal TM,E. Herreros 1, E F. del Campo l, C. Fenoll 1,2,g< 1 Departamento de Biologfa, Universidad Aut6noma de Madrid, E-28049 Madrid, Spain, 2Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, E-45071 Toledo, Spain, 3present address: Centro Nacional de Biotecnologfa, Cantoblanco, E-28049 Madrid, Spain, 4 Present address: Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia, Ciudad Universitaria, Santa F6 de Bogotfi, Colombia. *Corresponding author
Acknowledgements We want to thank the European Commission for funding this work through grants B IO4CT96 0318 and FAIR3-CT96 1714 to CF.
References 1. 2. 3. 4.
5.
C. Fenoll, EM.W. Grundler, S.A. Ohl, Cellular and molecular aspects of plant-nematode interactions, Kluwer Academic Publishers, The Netherlands (1997). EC. Sijmons, Plant-nematode interactions, Plant Mol. Biol. 23 (1993) 917-931. EC. Sijmons, H.J. Atkinson, U. Wyss, Parasitic strategies of root nematodes and associated host cell responses, Ann. Rev. Phytopathol. 32 (1994) 235-259. U. Wyss, Root Parasitic Nematodes: An overview, In: Cellular and molecular aspects of plant-nematode interactions, C. Fenoll, F.M.W. Grundler and S.A. Ohl, Eds., Kluwer Academic Publishers. The Netherlands, (1997) 5-22. A. Goverse, J. Rouppe van der Voort, C. Rouppe van der Voort, A. Kavelaars, G. Smant, A. Schots, J. Bakker, J. Helder, Naturally-induced secretions of the potato cyst nematode co-stimulate the proliferation of both tobacco leaf protoplasts and human peripheral blood mononuclear cells, Mol. Plant Microbe Interact.
181
Challenges of the Environment
6.
7. 8. 9.
10. 11.
12. 13.
14. 15.
16. 17.
18.
(1999) (in press). O.J.M. Goddijn, K. Lindsey, EM. van der Lee, J.C. Klap, P.C. Sijmons, Differential gene expression in nematode-induced feeding structures of transgenic plants harboring promoter-gusA fusion constructs, Plant J. 4 (1993) 863-873. C.H. Opperman, C.G. Taylor, M.A. Conkling, Root-knot nematode directed expression of a plant rootspecific gene, Science 263 (1994) 221-223. D.M. Bird, M.A. Wilson, DNA sequence and expression analysis of root-knot nematode-elicited giant cell transcripts, Mol. Plant Microbe Interact. 7 (1994) 419-424. N. Barthels, EM. Van der Lee, J.C. Klap, O.J.M. Goddijn, M. Karimi, R Puzio, EM.W. Grundler, S.A. Ohl, K. Lindsey, L. Robertson, W.M. Robertson, M. Van Montagu, G. Gheysen, RC. Sijmons, Regulatory sequences of Arabidopsis drive reporter gene expression in nematode feeding structures, Plant Cell 9 (1997) 2119-2134. B. Favery, R Lecomte, N. Gil, N. Bechtold, D. Bouchez, A. Dalmasso, R Abad, RPE, a plant gene involved in early developmental steps of nematode feeding cells, EMBO J. 17 (1998) 6799-6811. C. Fenoll, EA. Aristizfibal, S. Sanz-Alf6rez, EE Del Campo, Regulation of gene expression in feeding sites, In: Cellular and Molecular Aspects of Plant-Nematode Interactions, C. Fenoll, EM.W. Grundler, S.A. Ohl Eds., Kluwer Academic Publishers, The Netherlands, (1997) 133-149. EA. Aristizfibal, Identificaci6n y anfilisis de la expresi6n en plantas de genes regulados por nemfitodos endoparasfticos, Ph.D. Thesis, Universidad Aut6noma de Madrid, Spain, (1996). M. Enjuto, L. Balcell, N. Campos, C. Caelles, M. Arr6, A. Boronat, Arabidopsis thaliana contains two differentially expressed 3-hydroxy methylglutaryl-CoA reductase genes, which encode microsomal forms of the enzyme, Proc. Natl. Acad. Sci. USA 91 (1994) 927-931. R. Atanassova, N. Chaubet, C. Gigot, A 126 bp fragment of a plant histone gene promoter confers preferential expression in meristems of transgenic Arabidopsis, Plant J. 2 (1992) 291-300. M.A. Coca, C. Almoguera, T.L. Thomas, J. Jordano, Differential regulation of small heat-shock genes in plants: analysis of water-stress-inducible and developmentally activated sunflower promoter, Plant Mol. Biol. 31 (1996) 863-876. W. Van der Eycken, D.J. Engler, D. Inze, M. Van Montagu, G. Gheysen, Molecular study of root-knot nematode-induced feeding sites, Plant J. 9 (1996)45-54. C. Escobar, J. De Meutter, EA. Aristiz~ibal, S. Sanz-Alf6rez, RE Del Campo, N. Barthels, W. Van der Eycken, J. Seurinck, M. Van Montagu, G. Gheysen, C. Fenoll, Isolation of the Lemmi9 gene and promoter analysis during a compatible plant-nematode interaction, Mol. Plant-Microbe Interact. 12 (1999) 440-449. L. Robertson, W.M. Robertson, J.T. Jones, Direct analysis of the secretions of the potato cyst nematode Globodera rostochiensis, Parasitology 119 (1999) (in press).
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