Current Topics in Microbiology 197 and Immunology Editors A. Capron, Lille . R.W. Com pans, Atlanta/Georgia M. Cooper, Birmingham/Alabama· H. Koprowski, Philadelphia· I. McConnell, Edinburgh· F. Melchers, Basel M. Oldstone, La Jolla/California· S. Olsnes, Oslo M. Potter, Bethesda/Maryland· H. Saedler, Cologne P.K. Vogt, La Jolla/California· H. Wagner, Munich I. Wilson, La Jolla/California Gene Silencing in Higher Plants and Related Phenomena in Other Eukaryotes Edited by P. Meyer With 17 Figures Springer-Verlag Berlin Heidelberg New York London Paris Tokyo Hong Kong Barcelona Budapest DR. PETER MEYER Max-Delbruck-Laboratorium in der Max-Planck-Gesellschaft Carl-von-Linne-Weg 10 50829 Kbln Germany Cover Illustration: The front page shows three different examples of gene silencing phenomena in plants: (1) Silencing of a transgenic pigmentation marker in petunia flowers due to DNA methylation (background photo, provided by Iris Heidmann). See p. 15 for further details. (2) Inhibition of tomato fruit ripening by antisense technology (photos in the upper two panels, provided by Don Grierson). Wild-type tomatoes (right) and antisense transformants (left) are shown. See p. 77 for further details. (3) Silencing of anthocyanin pigmentation in maize anthers by paramutation at the PI locus (photos in the lower two panels, provided by Garth Patterson). The PI-Rh phenotype (right) and the P/'-mah phenotype (left) are shown. See p. 121 for further details. The photo on the back page shows examples of flower phenotypes derived from co-suppression of a gene of the pigmentation pathway in petunia (photo providedby Richard Flavell). See p. 43 for further details. Cover design: Kunkel + Lopka, IIvesheim ISSN 0070-217X ISBN-13: 978-3-642-79147-5 e-ISBN-13: 978-3-642-79145-1 001: 10.1007/978-3-642-79145-1 This work is subject to copyright. All rights are reserved. whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1995 Softcover reprint of the hardcover 1st edition 1995 The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Production: PRODUserv Springer Produktions-Gesellschaft, Berlin Typesetting: Thomson Press (India) Ltd, New Delhi SPIN: 10474049 27/3020-5432 1 0 - Printed on acid-free paper. Preface In March 1993, Richard Jorgensen, Amar Klar and Rob Martienssen organized a conference at the Bunbury Center in Cold Spring Harbor, where different epigenetic phenomena in bacteria and eukaryotes were discussed. At the Bunbury conference some scientists who worked on plant silencing phenomena discussed the initiation of a common research project. The idea to discuss gene silencing phenomena in a volume of the CTMI series was born at a symposium in Cologne that had been organized to establish a European research network on plant gene silencing. At this symposium, the participants presented their work to the members of the Max-Planck-Institute for Breeding Research. After the talks, Heinz Saedler convinced the participants of the gene silencing network to present the current state of the art of the different silencing systems in a CTMI volume and to complement this summary with reviews on related phenomena in other eukaryotes. The term 'gene silencing' refers to a complete or partial inactivation of gene activity. Silencing has been observed for endogenous plant genes and transposable elements,· but most frequently for recombinant genes introduced into transgenic plants. In transgenic plants, gene silencing can occur either in primary transformants or during further breeding and propagation of transgenic plants. Transgenes or endogenous genes can also become silenced under the influence of a second homologous copy. The silencing effect can be unidirectional (trans-inactivation) or bidirectional (co- suppression) and it can be influenced by developmental and environmental factors. Inactivation is either associated with a loss of transcription, often corresponding to hypermethylation within the promoter region, or attributed to post-transcriptional degradation of RNA. Certain silencing events show similarities with gene inactivation phenomena in other eukaryotes. In Drosophila the regulatory function of chromatin structure for gene VI Preface expression has been convincingly documented. Mechanisms such as position effect variegation and transvection that are mediated by modifications in chromatin structure might serve as a reference model to discuss trans-inactivation or paramutation in plants. In filamentous fungi, homology- dependent inactivation mechanisms are present, reminiscent of trans-inactivation in plants. DNA methylation, which is involved in the regulation of transcription of plant transgenes and transposable elements, also has an important role in developmental regulation in mammalian cells. Despite these similarities, however, the regulation and biological function of the common molecular mechanisms probably differ between plants and other eukaryotes. Also, gene silencing effects based on post-transcriptional regulation appear to be specific to plants. The identification of the underlying mechanisms for the different gene silencing phenomena in plants will not only improve the stability of gene expression in transgenic plants, a major prerequisite for the application of transgenic material. It should· also provide a better understanding of cellular mechanisms that control promoter activity, RNA transport, RNA stability or other steps involved in gene expression. The authors of the plant articles have tried to simpl ify the understanding of their very specialized research topics by providing some basic information on the common genetic and molecular tools used in plant molecular biology. I hope that this presentation especially encourages readers who are not working on plants to use this book to learn about a fascinating topic and to discover similarities with their own research systems. The four articles on gene silencing phenomena in Drosophila, filamentous fungi and mammalian systems should provide a basis for a comparative evaluation. Considering the common tendency in biological research to work on more and more specialized subjects, this book might stimulate some interdisciplinary research projects to compare gene regulation mechanisms in different eukaryotes. I am most grateful to the authors of this volume who provided excellent articles at short notice. I would like to thank Heinz Saedler for initiating this book, Sarah Grant and Bob Dietrich for helping me with the editing process, and Marga Botsch and Doris Walker for correspondence and technical editing. PETER MEYER List of Contents A.J.M. MATZKE and MA MATZKE: trans-Inactivation of Homologous Sequences in Nicotiana tabacum ............ . P. MEYER: DNA Methylation and Transgene Silencing in Petunia hybrida .......... .. 15 O. MITIELSTEN SCHEID: Transgene Inactivation in Arabidopsis thaliana 29 R.B. FLAVELL, M.O'DELL, M. METZLAFF, S. BONHOMME, and P.D. CLUSTER: Developmental Regulation of Co-suppression in Petunia hybrida . . . . . . . . . . .. . . . 43 P. DE LANGE, R. VAN BLOKLAND, J.M. KOOTER, and J.N.M. MOL: Suppression of Flavonoid Flower Pigmentation Genes in Petunia hybrida by the Introduction of Antisense and Sense Genes ...... . 57 A.J. HAMILTON, R.G. FRAY, and D. GRIERSON: Sense and Antisense Inactivation of Fruit Ripening Genes in Tomato . . . . . . . . . . .. . . . . . . 77 F. DE CARVALHO NIEBEL, P. FRENDO, D. INZE, M. CORNELISSEN, and M. VAN MONTAGU: Co-suppression of ~-1 ,3-Glucanase Genes in Nicotiana tabacum . . . . . . . . . . .. 91 F. MEINS JR. and C. KUNZ: Gene Silencing in Transgenic Plants: A Heuristic Autoregulation Model. . . . . . . . . . . . . 105 VIII List of Contents G.!. PATIERSON and V.L. CHANDLER: Paramutation in Maize and Related Allelic Interaction ......... 121 NV FEDOROFF: DNA Methylation and Activity of the Maize Spm Transposable Element. . . . . . . . . . . . . . . . . . . . . . . . . 143 M.J. SINGER and E.U. SELKER: Genetic and Epigenetic Inactivation of Repetitive Sequences in Neurospora crassa: RIP, DNA Methylation, and Quelling .................. 165 J.-L. ROSSIGNOL and G. FAUGERON: MIP: An Epigenetic Gene Silencing Process in Ascobolus immersus . . . . . . . . . . . . . . . . . . . . . . . . 179 S. HENIKOFF: Gene Silencing in Drosophila . . . . . . . . . . . . . . . . . . . . . 193 w. DOERFLER: Uptake of Foreign DNA by Mammalian Cells Via the Gastrointestinal Tract in Mice: Methylation of Foreign DNA- A Cellular Defense Mechanism 209 Subject Index . . . . . . ........ . . . . 225 List of Contributors (Their addresses can be found at the beginning of their respective chapters.) VAN BLOKLAND R.. ... 57 KOOTERJ.M. .......... 57 BON HOMME S. . . . . . .. 43 KUNZ C.. . . . . . . . . . . . .. 105 DE CARVALHO NIEBEL F. 91 DE LANGE P. .......... 57 CHANDLER V.L. ...... 121 MATZKE A.J.M ........ . CLUSTER P.o. ....... 43 MATZKE M.A. ......... . CORNELISSEN M. . . . . . 91 MEINSF.JR ............ 105 METZLAFF M. . . . . . . . . .. 43 DOERFLER W. . . . . . .. 209 MEYER P. . . . . . . . . . . . .. 15 MiTIELSTEN SCHEID O. . . .. 29 FAUGERON G. ....... 179 MOL J.N.M. .......... 57 FEDOROFF N.v. . . . . .. 143 FLAVELL R.B. ....... 43 O'DELLM. ...... . . . . .. 43 FRAY R.G. ......... 77 FRENDO P. ......... 91 PATIERSON G.!. .. . . . . . .. 121 GRIERSON D. . . . . . . .. 77 ROSSIGNOL J.-L. ........ 179 HAMILTON A.J. . . . . .. 77 SELKER E.U.. . . . . . . . . .. 165 HENIKOFF S. . . . . . . .. 193 SINGER M.J. . . . . . . . . . .. 165 INZE D. . . . . . . . . . . .. 91 VAN MONTAGU M. . . . . . .. 91 trans-Inactivation of Homologous Sequences in Nicotiana tabacum A.J.M. MATZKE and MA MATZKE Introduction ..... 2 Experimental System 2 3 Multiple Copies of Transgenes Are Often Poorly Expressed. 2 4 Region of Homology Important for Silencing ................. . 4 5 Characteristics of Epistatic Silencing Loci . 5 6 Susceptibility of Potential Target Transgene Loci to trans-Inactivation. 7 7 Mechanism of Epistatic trans-Inactivation 9 8 Genetic Implications of Delayed Recovery of Inactivated Target Loci 11 9 Conclusions. 12 References ............................ . 13 1 Introduction In this chapter, we focus on a specific class of homology-dependent gene silencing, epistatic trans-inactivation, in one plant species, Nicotiana tabacum (tobacco). Epistatic trans-inactivation is defined as a nonreciprocal interaction that occurs between homologous, or partially homologous, transgenes present at nonallelic (ectopic) chromosomal locations, and the outcome is that one trans- gene locus becomes inactivated in the presence of the second (reviewed in MATZKE et al. 1994b). This phenomenon possibly results from the unidirectional transmission of a hypermethylated state at one locus to a homologous unmeth- ylated region at the second locus. There are now several examples of epistatic trans-inactivation in transgenic tobacco. We will review these cases and contrast them with other classes of homology-dependent gene silencing such as paramu- tation (an allelic interaction) and co-suppression/sense suppression (a reciprocal ectopic interaction). Even though the consequences of co-suppression and epistatic trans-inactivation are similar, i.e., silencing of homologous genes, they can be distinguished by a number of features, thus pointing toward fundamentally Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, 5020 Salzburg, Austria 2 A.J.M. Matzke and MA Matzke different mechanisms for the two processes. In contrast, there are notable similarities between some paramutation systems and epistatic trans-inactiva- tion. 2 Experimental System Tobacco is a dicotyledonous plant that has been widely used in studies on plant gene expression, primarily because leaf cells can be transformed easily with foreign DNA and subsequently regenerated into whole, fertile plants. Indeed, this tractability is reflected in the fact that tobacco was one of the first plants to be engineered genetically (for example, see BARTON et al. 1983). Tobacco is a natural allotetraploid originating from two ancestral diploid species, N. sylvestris and either N. tomentosiformis or N. otophora (KENTON et al. 1993). Nevertheless, with respect to inheritance of foreign genes, tobacco behaves as a diploid. A common method for transforming tobacco uses vectors based on the transferred DNA (T-DNA) region of the Agrobacterium tumefaciens tumor- inducing (Ti) plasmid in conjunction with the so-called leaf disc method. In this, tobacco leaf pieces are incubated with a suspension of Agrobacterium cells containing a modified Ti plasmid that includes an antibiotic selection marker able to function in plant cells. Single leaf cells that have received the selectable marker gene (and any other genes positioned between the borders of the T-DNA) are able to divide and differentiate on a medium containing the appropriate antibiotic and a plant hormone to induce shoot regeneration. Although this procedure provides a simple way to produce transgenic tobac- co plants, achieving expression of transferred genes has not been so straightfor- ward. In any given experiment it was possible to find a subset of transformants that expressed strongly a given gene; nevertheless, there were almost invariably plants that exhibited low or unstable expression. Such cases were usually attributed to ill-defined position effects. However, with the recent realization that low (or no) expression was often coupled to the presence of multiple copies of homologous transgenes at both linked and unlinked chromosomal locations, a new framework has evolved for studying and understanding anomolous behavior of transgenes in plants. 3 Multiple Copies of Transgenes Are Often Poorly Expressed That multiple copies of transgenes could negatively affect expression was only discovered when investigators focused on plants exhibiting weak activity of transgenes. This was not initially a priority, since early studies were mainly