Molecular Methods for Hybrid Rice Production

Molecular Methods for Hybrid Rice Production

A report for the Rural Industries Research and Development Corporation by Jorge E. Mayer Richard A. Jefferson

CAMBIA Canberra ACT

August 2004 RIRDC Publication No 04/003 RIRDC Project No CMB-1A

© 2004 Rural Industries Research and Development Corporation. All rights reserved.

ISBN 0 642 58713 2 ISSN 1440-6845 “Molecular Methods for Hybrid Rice Production’ Publication No. 04/003 Project No. CMB-1A The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report. This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.

Researcher Contact Details Dr. Richard Jefferson Center for the Application of Molecular Biology to International Agriculture (CAMBIA) GPO Box 3200 CANBERRA ACT 2601 Phone: (02) 6246 4502 Fax: (02) 6246 4501 In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Website:

02 6272 4819 02 6272 5877 [email protected] http://www.rirdc.gov.au

Published in August 2004 Printed on environmentally friendly paper by Canprint

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Foreword Corn has benefited from the higher productivity of hybrid seed production for quite some time now. In rice yield increase through traditional breeding was limited until recently by self-pollination. By capturing the heterosis effect hybrid rice varieties can produce 15-30% more grain than either parent. Manual methods for hybrid rice production so far are only economically practicable in countries with cheap manual labor and also result in non-homogenous progenies, available transgenic technologies still require a three-line system to maintain the male sterile parents. The main goal of this project was to develop molecular tools that will help solve the problems that prevent the use of hybrid seed technology in Australia. The rationale behind our two-line hybrid system is to create facultative male-sterile lines based on activatable gametocides using a transgenic rice plant producing the activator enzyme in any part of the male sexual organ. Plants would be fully fertile until treated with the compound. Upon treatment the plant would become a female parent for the breeding process. Another goal of this project in the long-term is the fixation of the heterotic effects obtained from hybridisation by introducing apomixis (parthenogenic seed development) into rice. We provide here additional tools to understand and manipulate flower development to this end. With these contributions we would like to provide the means to develop more efficient breeding programs, including targeted introgression of important traits, custom-taylored production of varieties adapted to micro-environments and increased genetic diversity in the field. This project was funded from industry revenue which is matched by funds provided by the Australian Government. This report, is an addition to RIRDC’s diverse range of over 1000 research publications, forms part of our RICE R&D program, which aims to improve the profitability and sustainability of the Australian Rice Industry. Most of our publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/fullreports/index.html • purchases at www.rirdc.gov.au/eshop

Simon Hearn Managing Director Rural Industries Research and Development Corporation

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Acknowledgments The authors wish to thank RIRDC for partially funding this project. We also wish to recognise the contributions of the Rockefeller Foundation for PhD grants to Mr Wei Yang (China), Mr Satya Nugrohu (Indonesia), and Mr Tuan Nguyen (Vietnam), as well as CAMBIA Biosystems LLC, the Maharashtra Hybrid Seed Company (Jalna, India) for supporting the project within the frame of a collaborative agreement which included long-term stays at CAMBIA of Dr Valasubramanian Ramaiah and Dr K.S. Ravi (the latter also suuported by a Rockefeller post-doctoral fellowship).

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Contents Foreword ............................................................................................................................................... iii Acknowledgments................................................................................................................................. iv Contents.................................................................................................................................................. v Executive Summary ............................................................................................................................. vi 1. Introduction ....................................................................................................................................... 1 2. Objectives ........................................................................................................................................... 2 3. Genetic transformation of rice: Development of high-throughput technology ........................... 3 3.1 Rice genetic transformation methodology ................................................................................... 3 3.2 Development of a kit of versatile transformation vectors (Roberts et al., 2000) ......................... 4 4. Isolation and characterization of an Egg Apparatus-Specific Enhancer Element (EASEE) ..... 6 4.1 Isolation and characterization of the EASEE-containing sequence ............................................. 6 4.2 Characterization of the DsE flanking region in ET253................................................................ 7 4.3 EASEE function analysis in rice and tobacco.............................................................................. 7 5. Inducible male sterility: Pollen-specific activation of progametocides......................................... 8 5.1 Cloning of anther-specific promoters........................................................................................... 8 5.2 Pro-gametocide activation............................................................................................................ 9 5.3 Development of a glucuronide-responsive promoter for field level control of transgene expression ......................................................................................................................................... 10 6. Tools for apomixis: Understanding meiosis in the embryo-sac................................................... 11 6.1 Isolation and characterization of a ScRIM11 rice homologue................................................... 11 6.2 Functional analysis of OsSK11.................................................................................................. 13 6.3 Identification of additional ScRIM11 homologues in rice......................................................... 17 6.4 OsSK11 gene function analysis in transgenic rice and in a yeast null-mutant........................... 20 7. Conclusions ...................................................................................................................................... 23 8. Publications derived from this project .......................................................................................... 24 9. Bibliography .................................................................................................................................... 25

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Executive Summary Self-pollination in rice poses a barrier to yield ceiling improvements using hybrid seed technology. While manual methods for hybrid rice production are economically impracticable in Australia and also result in non-homogenous progenies, available transgenic technologies are laborious in that a threeline system is required to maintain the male-sterile (MS) parents. The rationale of this project is to create facultative MS lines based on transgene activation of pro-gametocides. A long term goal of this project is to fix the heterotic effects obtained from hybridisation by introducing apomixis into rice. Here we provide some of the tools required to achieve this goal. Most milestones were met during the RIRDC funded phase of the project, while activities are still ongoing and will be carried on to fruition. Within the frame of this project, highly reproducible and efficient rice genetic transformation techniques developed for local rice varieties. Versatile vectors for rice transformation, now widely used by the research community, were developed and tested extensively. Thousands of transgenic rice plants were and are routinely being produced at CAMBIA to test genetic constructs belonging to this and to other, related projects. Stable inheritance of introduced traits was successfully tested over generations together with phenotypic assessment. Two rice genes with sequence homology to yeast early meiosis regulatory genes were isolated and partially characterised. Further homologues were identified. The promoter regions of one of the genes was used to drive the expression of a marker gene in rice, showing that gene expression was specific to nodal meristems and pollen cells. This together with a set of other pollen-specific promoters can now be used to direct the expression of a hydrolase capable of cleaving pro-gametocides in the pollen sac. An egg apparatus gene regulatory region was isolated from Arabidopsis thaliana. The corresponding enhancer sequence was identified and reintroduced into Arabidopsis, thereby confirming its function and orientation. Transgenic rice plants containing these constructs were produced and are being evaluated. In a different approach rice lines containing enhancer traps that display ovule-specific expression and other interesting expression patterns have been produced. Some of these regulatory regions will provide useful tools to the project. A secretable hydrolase (beta-glucuronidase) of microbial origin was identified and modified for optimal expression in plants. This enzyme is a critical tool required to make the two-line hybrid system work. A set of experimental gene switches designed to control the expression level of the hydrolase were constructed. Some preliminary experiments (susceptibility to certain plant growth regulators and herbicides) were done to test the suitability of these compounds as pro-gametocides. These experiments were complemented with measurements of uptake and transport of glucuronides in plants. Synthesis of pro-gametocides will be done through collaborations or by contracting out.

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1. Introduction Hybrid rice varieties could have a substantial agronomic and economic impact on rice in Australia. By capturing the heterosis effect hybrid varieties can produce 15-30% more grain than either parent. Thanks to the work of Yuan Longping, director of the National Hybrid Rice Research and Development Center in Changsha, Hunan Province, a male sterility trait was bred into indica rice varieties. This development was responsible for hybrid rice now covering more than 50% of China's rice acreage. An extensive hybrid rice breeding project launched by the Indian Council of Agricultural Research has boosted yields from less than 100 Kg per hectare to 1.5 metric tons, which has led to 150 thousand hectares now being planted to hybrid rice in India. Hybrid rice is also being grown in Vietnam and the Philippines while locally adapted varieties are being developed for Bangladesh, Sri Lanka and Indonesia. Currently available methods of hybrid rice seed production, such as those used in China and India, are cumbersome and subject to GxE interactions (photoperiod and temperature sensitivity). Genetic drag associated with introgression of existing male sterility traits often compromises the quality and speed of breeding programs. Additionally, manual labor costs would make the use of these technologies prohibitive in Australia. In the USA, RiceTec of Alvin, Texas, after acquiring the rights to the Chinese male sterile lines, has mechanized the seed production process and has thereby managed to eliminate most of the hand labor involved in hybrid seed production. Field trials show an average yield increase of 33%. The next step in the process of introduction of rice hybrid seed technology to developed countries will be the control of quality traits. The provision of a simpler alternative to hybrid rice production would open the avenue to capturing the heterotic effect in terms of absolute yield and also to the quick and efficient introgression of quality traits and resistance to abiotic (especially cold) and biotic stresses. The main objectives of this project were to develop a commercially viable two-line hybrid rice system as well as molecular tools geared toward the fixation of heterozygosity and epistasis through apomixis in rice. The concept utilized for producing inducible male-sterile lines is based on gene fusions between malespecific plant gene promoters and ß-glucuronidase (GUS). GUS is a hydrolytic enzyme not naturally present in rice. Its presence in a given tissue will make this tissue uniquely sensitive to otherwise innocuous conjugates of toxins or plant growth regulators with glucuronic acid. When the conjugate is applied GUS will release the active ingredients in a localized manner. In the case of plants expressing GUS in the anthers the plant will be converted into a female line only after treatment with the conjugate, which in this case we would call a progametocide. The use of GUS as a marker gene in many thousand plants belonging to many species has been widely reported in the literature, while no ill effects to the physiology or agronomic traits of the plant have been encountered. This makes GUS ideally suited as an activating agent for procompounds in delicate tissues, such as the pollen sac. The most important innovations to make hybrids both economically viable in inbreeding crops, and environmentally and biologically sound with regard to genetic and systems diversity will be methods that encourage (1) numerous and diverse parental materials to be used, and (2) immediate evaluation and propagation of desirable genotypes adapted to regional ecosystems. Existing molecular approaches to male sterility have been patented or filed for patent by all major multinationals. Among the most widely publicized is the PGS approach (Mariani et al., 1990), involving the expression of a transgenic RNase gene (barnase) in the tapetum, thereby destroying the pollen-forming ability in the plant. A transgenic restorer line producing an RNase inhibitor is required, making it a three-line system. The system is potentially risky, as environmental conditions could affect gene expression levels, changing the ratios of RNase expression and that of the inhibitor (Brandle et al., 1995; Register et al., 1994). 1

Nuclear male sterility in plants has been obtained using other recombinant DNA techniques, e.g. through transformation with antisense constructs in Petunia hybrida (Van de Meer et al., 1992) or using cosuppression (Jorgensen et al., 1996) . In tobacco, male sterility has been induced by the expression of an Agrobacterium rhizogenes rolC gene, restoration of fertiliy was achieved by the expression of an antisense RNA in the F1 hybrids (Schmulling,1993). As mentioned above fixation of heterozygosity and epistasis through apomixis could be of tremendous value to farmers (Grossniklaus, 1998; Jefferson & Bicknell, 1996; Koltunow et al., 1995). Part of this project was to capture genes expressed exclusively in the megaspore mother cell (or embryo mother cell) and to develop tools capable of producing apomixis in the field. Together with inducible or suppressible gene cassettes the project is geared toward the development of aposporous apomixis and restorable plant sexuality in rice in the field. As a by-product apomixis could have the added value of cold tolerance if autonomous endosperm formation rendered pollen formation unnecessary.

2. Objectives 1. To develop a viable two-line hybrid rice system by provision of a method for facultative, environment-independent nuclear male sterility in transgenic rice.

2. To provide a molecular toolkit of inducible rice genes specific to female heterotic effects and achieve new yield ceilings.

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3. Genetic transformation of rice: Development of high-throughput technology The development of an efficient high-throughput transformation protocol for the japonica rice varieties used in our studies (Millin and Nipponbare) was an absolutely necessary step for the success of the whole project. Until a few years ago it was believed that Agrobacterium tumefaciens could not be used for transformation of monocots. Based on that assumption particle bombardment protocols were developed (Christou, 1991, 1997; Chen et al., 1998a, 1998b). The downside of these protocols is that the borders of the segment of DNA to be inserted are ill-defined and also that in most cases multiple copies of the insert are found in the genome. After Hiei et al. (1994, 1997) developed an efficient method for Agrobacterium-mediated transformation of rice we decided to adapt this protocol to our needs, because with Agrobacterium the problems cited above are minimised. This group showed that cocultivation of calli derived from rice scutella, with Agrobacterium can produce rice transformants with an efficiency similar to that of transformation in dicotyledonous plants. When the protocol was first established at CAMBIA we were obtaining regeneration efficiencies of the order of 10% from hygromycin-resitant calli. By the end of the project we had improved the efficiency to 50%, covering a range from 30-80%, depending on the construct, Agrobacterium strain and rice variety used. More than 50% of transformants possess a single copy of the T-DNA, which is inherited in a Mendelian fashion without compromise to varietal characteristics. Several thousand transgenic rice plants have been produced for different purposes at CAMBIA using this protocol, around 1000 of these containing and expressing the GUSPlus gene within the frame of this project. GUSPlus is a secretable and sturdier β-glucuronidase which plays a central role in the activation of pro-toxins for the production of inducible male sterile plants (see there). Our present transformation protocol allows us to produce ca. 500 transgenic plants per month. Details of the protocol, including modifications to the original literature, are as follows:

3.1 Rice genetic transformation methodology Seed sterilization. Dehusked seeds were surface-sterilized by sequential treatment in 70% ethanol (1 min), rinsed twice with sterile deionized water followed by a solution containing bleach and a few drops of Tween-20 (30 min on a rotating wheel),and washed six times with sterile water. Sterilized seeds were placed on 2N6 medium (for media recipes please refer to the literature) containing 2,4-D and allowed to form callus (3-4 weeks) in darkness at 26°C. Scutellum-derived calli obtained from these seeds were used for transformation. Embryogenic callus induction and preparation. Calli thus obtained were cut into approximately 5 mm in diameter pieces and plated on fresh 2N6 medium and incubated at 25°C in the dark for 4-7 days before cocultivation. Cocultivation with Agrobacterium. Three days before cocultivation, A. tumefaciens containing the constructs to be transformed into rice was streaked onto AB solid medium containing antibiotics (depending on the pCAMBIA vector and Agrobacterium strain combination used the following concentrations of antibiotics were used: chloramphenicol, 100 µg/mL for AGL-1 and EHA105, 10 µg/mL for LBA4404; kanamycin, 50 µg/mL for all strains; hygromycin 50 µg/mL) and grown at 29°C. After 3 days incubation, the bacterial lawn was scraped from the AB plates, and resuspended in AAM liquid medium containing 100 µM acetosyringone (to induce vir genes), shaked well for one minute and left at room temperature for about one hour. An OD600 of approximately 1.0-1.5 should be obtained. The 4-day-old calli were then added to the bacterial suspension, mixed by swirling and left

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for another hour at room temperature to allow contact with the bacteria. The calli were then placed onto sterile filter paper to remove excess medium without allowing calli to become too dry. After drying, calli were transferred onto cocultivation medium 2N6AS. Cocultivation was done for 3 days at 25°C in the dark. Selection, subculturing and regeneration. After cocultivation, Agrobacterium was removed from the calli by washing in sterile deionized water containing 250 mg/L cefotaxime (Claforan, Hoechst Marion Roussel). Calli were left in the solution for one hour with gentle shaking. The washing was repeated two more times until the washes turned clear. Calli were then blotted dry on sterile filter paper. After drying, calli were transferred onto selection medium (2N6-CH + antibiotic or herbicide) plates containing timentin (Cheng et al., 1998) and cefotaxime (250 mg/L each) to kill the bacteria still attached to the calli, and incubated at 25°C in the dark. The calli were subcultured regularly onto fresh selection medium (2N6-CH) every 2 weeks, until good size proliferation of transgenic calli was obtained. Transient gusA expression can be tested by staining a few washed calli with X-GlcA while expression of green fluorescent proteins (GFP) can be viewed under the fluorescence microscope using a GFP filter. When proliferating, antibiotic resistant calli reached ca. 0.5-1 cm in diameter, they were transferred to regeneration medium (RGH6) and incubated in the dark at 25°C for 7 days or alternatively they were subcultured. Calli were transferred for regeneration to light at 25°C. Calli start turning green after 5-10 days and after 2-3 weeks shoots start to differentiate. Shoots were then transferred onto rooting medium and plantlets were produced. Plantlets were transferred to 1/2MS-H medium. After the roots were well developed the plants were hardened and transferred to the greenhouse in pots containing soil mix. Glasshouse conditions were set to 28-35°C during the day and 18°C minimal temperature during the night. Photoperiod was adjusted to 16h thoughout the life cycle of the plants using additional halogen lighting. Agrobacterium strains. LBA4404, EHA105 and AGL-1 harboring pCAMBIA vectors (Roberts et al., 2000; Genbank acc nrs AF234290-234316) were used for transformation. LBA4404 carries vir helper Ti plasmid pAL4404 derived from the octopine Ti plasmid pTiAch5 (Ooms et al., 1982). The vir helper Ti plasmids in strains EHA105 (Hood et al., 1993) and AGL-1 (Lazo et al., 1991) are derived from the leucinopine-type supervirulent Ti plasmid pTiBo542 (Hood et al., 1986).

3.2 Development of a kit of versatile transformation vectors (Roberts et al., 2000) The modular pCAMBIA vectors were designed having in mind both efficient transformation of dicots and monocots via Agrobacterium or particle bombardment. These vectors are based on the pPZP vector series developed by Hajdukiewicz et al. (1994). They show high stability in Agrobacterium, high copy number for routine DNA preparation in E. coli, a modular choice of selectable markers for both bacterial (chloramphenicol or kanamycin resistance genes) and plant (hygromycin and kanamycin resistance genes) selection, blue/white screening, various permutations with and without promoters, and various versions that use ß-glucuronidase (GUS) with a catalase intron, the Green Fluorescent Protein (GFP) of Aequoria victoria or GFP:GUS fusions as reporter genes. These vectors are fully sequenced, and portions which are restricted by intellectual property rights can be readily removed for particular purposes. Some of these vectors are designed to facilitate expression of foreign genes, others to analyse promoters. One set contains no foreign genes between the T-DNA borders to facilitate its use as a backbone for sophisticated constructions. The main features of the cassette structure are: (i) strong ribosome binding and efficient translation; (ii) choice of 5'-cloning sites; (iii) compatible sticky overhangs for easy gene re-arrangement / re-orientation; (iv) a carboxy-terminal hexa-histidine tag for immobilized metal affinity chromatography purification (or alternatively without, for untagged protein expression). Transgenic rice of the japonica, indica and javanica types have been generated with these vectors, typically giving copy numbers between one and three. The new pCAMBIA vector series features a convenient modular format for versatile manipulation (Fig. 1); the main features of the cassette structure are: (i) consensus sequences for Shine/Dalgarno

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and Kozak positions for strong ribosome binding and efficient translation; (ii) choice of 5'-cloning sites, the Nco I site contains start codon and second base of Kozak consensus; (iii) 5'-Spe I and 3'Nhe I sites have compatible sticky overhangs, allowing easy gene re-arrangement/ re-orientation; (iv) carboxy-terminal hexa-histidine tagging with incorporated stop codon for immobilized metal affinity chromatography purification if protein cloned into NheI site; (v) 3'-Bst EII site for untagged protein expression; stop codon required in this case. The pCAMBIA vectors can be grouped into the following families according to their potential uses: Do it yourself vectors. These vectors contain a range of restriction sites on either side of the pUC8 (0380) or pUC9 (0390) polylinker, making these vectors suitable for advanced construction purposes with the user inserting their own promoters, selection genes or reporter genes, etc. The only functional signals between the T-DNA borders are the start and stop codons, the histidine tag, and the Nos polyA signal. Minimal Selection Vectors. These vectors contain minimal heterologous sequences for plant transformation and selection of transformants; they allow insertion of any desired genes for transformation into plants but require all promoter and terminator sequences. They do not contain the start, stop, and histidine tag sequences of the modular design. This vector series is provided with one of three different CaMV35S driven and terminated plant selection genes: hptII encoding resistance to hygromycin; nptII encoding resistance to kanamycin; and bacterial resistance to either kanamycin or chloramphenicol. The pUC18 polylinker within the lacZα fragment allows blue/white screening of clones in E.coli cloning work. The full modular format is provided for convenient PCR cloning and gene expression. Polylinkers are proximal to the right T-DNA border, thus plants containing the selectable marker gene will also contain the gene of interest. Gus Intron Selection (GIS) Vectors. These vectors contain a fully functional gusA reporter construct for simple and sensitive analysis of gene function in regenerated plants by GUS assay. The construct uses gusA (N358Q) with a catalase intron (for eukaryote-specific expression) as the reporter gene, cloned in modular format. This allows use of these plasmids as carriers suitable for transformation of any gene of interest in plants. These vectors contain the same bacterial and plant selection genes and pUC18 polylinker-lacZα of the corresponding pCAMBIA Minimal Selection Vectors. GFP Selection Vectors. Similar in utility to the Gus Intron Selection (GIS) vectors, these vectors contain the mgfp5 version (Siemering et al., 1996) of the Aequorea victoria green fluorescent protein either alone or in translational fusion with gusA (N358Q) in both arrangements, either as a gusA-mgfp5 or as a mgfp5-gusA fusion. Fuse & Use Vectors. Designed to utilize gusA as a true reporter of gene expression by fusion construction, these vectors contain a promoterless, intronless gusA (N358Q) gene (without an initiation codon) in three reading frames, and with either pUC8 or pUC9 oriented polylinkers. This permits simple construction of carboxy-terminus protein fusions to gusA. Promoter Cloner Vectors. Designed for promoter testing in planta, these vectors feature a promoterless version of gusA (N358Q) with the catalase intron immediately downstream of a truncated lacZα containing either the pUC8 or pUC9 polylinker. All plasmids in this series have hygromycin as the plant selection gene, and bacterial selection is available with either chloramphenicol or kanamycin. The truncated lacZα is functional for blue/white screening of clones in suitable E.coli host strains. pCAMBIA vectors for Agrobacterium-mediated or direct plant transformation have been extensively tested yielding excellent results and demonstrating the usefulness of the constructs. These vectors are stable in Agrobacterium even under non-selective conditions. A large number of tobacco and rice transformants were produced by Agrobacterium-mediated and biolistic methods as described (Hajdukiewicz et al., 1994; Hiei et al., 1994) using pCAMBIA vectors.

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4. Isolation and characterization of an Egg Apparatus-Specific Enhancer Element (EASEE) (W. Yang 2001) Progress in the field of apomixis research has been impressive in the last few years because of the various promises it holds for farmers (Jefferson & Bicknell, 1996; Grossniklaus et al., 1998; Grossniklaus & Schneitz, 1998). Still many problems remain to be solved, e.g. parental imprinting (Haig & Westoby, 1991; Grossniklaus et al., 2001; Messing & Grossniklaus, 1999) and management of transgenic apomicts (van Dijk & van Damme, 2000). Some important contributions to understanding the underlying mechanisms of apomixis can be found in the most recent literature (Bicknell et al., 2000; Guerin et al., 2000; Luo et al., 1999; Vivian-Smith & Koltunow, 1999; Kindiger et al., 1996). Although techniques are available for the isolation of cDNAs from a few cells (Dresselhaus et al., 1994), in this case we chose to make use of existing Ds-based transposon insertion lines to isolate a gene involved in very early developmental processes of embryo sac formation (Sundaresan et al., 1995). Here we describe the isolation and characterization of an embryo-sac or megagametophytespecific regulatory element (EASEE) as a tool to generate apomixis in rice. The Ds-based transposon insertion lines in Arabidopsis contain a GUS reporter gene to signal the insertion of the transposon into an actively expressed gene. Line ET253, displaying an ovule-specific expression pattern was obtained from Dr U. Grossniklaus (Cold Spring Harbor Laboratory, NY; now at Zurich University, Switzerland), who has screened ca. 30-thousand lines searching for interesting expression patterns. A 5 Kb-long DNA fragment was obtained by inverse PCR from this line and sequenced. The EASEE was further narrowed down by deletion analysis of a 2.2 Kb candidate sequence.

4.1 Isolation and characterization of the EASEE-containing sequence To avoid activation of the minimal promoter driving gusA expression by a strong nearby CaMV35S promoter a new vector (pWY-K105.1) was constructed using the 1’promoter from the T-DNA 1'-gene to drive the Basta resistance selectable marker gene bar . GUS staining of transgenic Arabidopsis plants transformed with this vector showed no background. A 2195bp upstream flanking sequence of the DsE insertion site in ET253 possesses the EASEE activity. A series of deletions were used to narrow down the region to only 318bp directly upstream of the DsE insertion site. A series of 5’ and 3’ deletions of the 318bp fragment were then designed, PCR amplified and inserted into pWY-K105.1. Transformation of Arabidopsis with these deletion constructs showed that a 77bp sequence (5∆77, Fig. 2-1) was sufficient to confer EASEE activity. However, the deletion of 68bp from the 3’end abolished EASEE activity completely (3∆68), indicating that the EASEE element is within the 77 bp region. 5∆318 5∆ 259 5∆ 206 5∆181 5∆ 136 5∆ 77 HindIII

DsE insertion site

∆237 ∆169 ∆117 ∆68

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Figure 2-1. Deletion analysis of the 318bp upstream of the DsE flanking sequence from ET253

77bp fragment with EASEE activity DsE Insertion site MITE-like transposable element

EcoR I (2)

atpH21-2 gene

predicted protein SnaBI (11573) gene

atpH21-1

3.1kb

2.1kb

1.4kb

Figure 2-2. Genomic region (on Arabidopsis chromosome 4) covering the DsE insertion site in ET253.

Constructs containing the 318 or the 77 bp fragments in different positions and orientations and also as tandem repeats in pWY-K105.1 were transformed into Arabidopsis to test the EASEE function. Preliminary results showed that the EASEE had the following features: (1) position-independent but orientation-dependent; (2) tandem repeats (2×) give much stronger activity in driving embryo-sac specific GUS expression; (3) single copy present in the genome according to Southern analysis using the 318bp fragment as a probe.

4.2 Characterization of the DsE flanking region in ET253 The DsE insertion site in ET253 is located at the top arm of chromosome 4 of Arabidopsis. Since the whole chromosome 4 has been sequenced, it is possible to look at the flanking region of the DsE insertion site in ET253 to see if the EASEE element is associated with a certain gene. The nearby genes from the DsE insertion site are: two peroxidase genes (atpH21-1 and atpH21), gene coding for a predicted protein related to an EST tag, and a gene coding for formamidase (Fig. 2-2). Southern analysis showed that the gene related to the predicted protein is a multi-copy gene (at least 3 copies) but the region harboring the two peroxidase genes is unique in the genome. Northern blotting of mRNA from different tissues of showed that the predicted protein-related mRNA exists mainly in floral tissues (gynoecium and other floral tissues) and trace amounts can be detected in leaf tissue. However, the peroxidase gene atpH21-1 mRNA is present in all tissues examined (gynoecium, other floral tissues and leaves). These results suggested that both the peroxidase genes and the predicted protein gene are less likely to be associated with the EASEE. Interestingly, the 170 bp sequence directly downstream of the DsE insertion site (including the 8bp DsE duplication sequence GCCTTAAT) in ET253, is highly homologous to many such sequences in all five chromosomes at different locations and is quite likely to be a MITE-transposable-element related sequence. Therefore, the EASEE could also be associated with this MITE-like element.

4.3 EASEE function analysis in rice and tobacco Transgenic rice plants were obtained by transforming rice callus with pWY-F68. The 1’/bar construct did not work well for rice transformation. To solve this problem, a new vector pWY-O98.2 was constructed based on 1’/hyg selection for rice transformation. Constructs with the 77 bp fragment and tandem repeats thereof (4×) driving a CaMV35S minimal promoter/gusA gene fusion were then built into pWY-O98.2 and transformed into rice. Sixty hygromycin-resistant callus lines were obtained. In addition to rice, tobacco was also transformed and 55 transgenic plants were obtained. While the EASEE gave mebryo-sac-specific expression patterns in Arabidopsis, microspore-specific expression was obtained in rice.

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Using molecular tools directed elimination of certain tissues can be achieved (cell ablation), e.g. using the already mentiond barnase gene (Goldman et al., 1994). We used cell ablation to visualize embryosac-specific EASEE-dependent gene expression using the diphteria toxin (DTA) gene. Activation of gene expression by the binding of the GAL4/VP16 activator protein to the yeast Upstream Activating Sequence (UAS) offers a very sensitive testing system, therefore a construct containing the GAL4/VP16 activator gene under the control of an EASEE/CaMV35S minimal promoter fusion and a UAS-mp-gusA reporter was introduced into Arabidopsis plants. Various GUS expression patterns were obtained as well as lines with embryo-sac-specific expression. Transformants were then crossed with a UAS-DTA line obtained from Dr Jim Haseloff (Cambridge, UK). The cross produced shriveled seeds from normal siliques, indicating that GAL4/VP16 can be specifically expressed in the embryo-sac using EASEE, demonstrating that the element can be very useful to control gene expression in the megagametophyte for different purposes.

5. Inducible male sterility: Pollen-specific activation of progametocides (V. Ramaiah) Available two-line hybrid systems based on conditional male sterility suffer from various drawbacks. In cytoplasmic or conditional nuclear sterility trait penetrance might depend on environmental conditons, while chemical hybridising agents might also reduce female fertility. Varietal differences will also restrict access to a wide range of genotypes (Parmar et al., 1979; Goldberg et al., 1993). Promoter sequences were obtained from Genbank and other publications. As part of the freedom-tooperate strategy we looked for promoters published several years ago or with no issued patents so far. Transgenic tissue-specificity of these promoters has been reported in a number of publications. Several were introduced into rice and Arabidopsis using mainly pCAMBIA1301 as a backbone (promoters were inserted to drive the expression of gusA with catalase intron). Newer constructs contain the GUSPlus gene (Nguyen et al.; in preparation) and are being used in most of our transformation experiments. Anther-specific promoters and protoxins in combination with an enzyme capable of activating the protoxin are the main components of this conditional male sterility system. A library of anther-specific promoters was assembled and introduced into rice to be able to select the most suitable candidates to be used in the field. The other part of the work consists in selecting, synthesizing and evaluating appropriate progametocides.

5.1 Cloning of anther-specific promoters Sequence and literature databases were searched for potentially useful anther-specific promoters from a wide variety of plants including monocots and dicots. Because temporal and spatial dependence of expression of anther-specific genes it is necessary to try a range of promoters for best suitability (Koltunow et al., 1990). The selected promoters were PCR-amplified and cloned into the pCAMBIA1301 vector upstream of the gusA gene (by replacing the CaMV35S promoter). The constructs were transformed into rice (cv Nipponbare), tobacco and Arabidopsis to evaluate and verify the expression patterns produced by the promoters.

8

PROMOTER

ORIGIN

GENE

MS21 Apg 2

A. thaliana A. thaliana

Male Sterility 2 proline-rich protein

NTM193 Zmg134 Bgp15 Bp4a6 Olnb47 APT18 NTP3039 NPG110 Lat5211

N. tabacum Z. mays B. campestris B. napus B. napus A. thaliana N. tabacum N. tabacum L. esculentum

NTM19 Zmg13 Bgp1 Bp4A oleosin-like phosphate transporter

ubi312 UBF913

S. tuberosum Z. mays

Act114 Osg4B15 RTS216 Bnm117 Rha118 EASEE19

O. sativa O. sativa O. sativa B. napus A. thaliana A. thaliana

polygalacturonase pollen hydration/ germination ribosomal ubiquitin ubiquitin fusion protein Actin 1

SPECIFICITY

SIZE [bp]

tapetum, pollen anther, microspore development pollen, microspores pollen anther pollen tapetum roots pollen pollen late pollen development non-specific strong endosperm expression Actin 1 tapetum microspore microspore guard cells embryo sac, enhancer-trapderived

973 1500 817 400 794 313 1957 936 846 835

905 906

CONSTRUCTS

VALA73.2 pVALA58.1*, pVALB97.2+ pVALB83.2 pVALB57.5*+ pVALB101.2* pVALB8 pVALB108.2* pVALNTP303*

pVALB82.2*

1200 1651 1274 817 1000

pVALD15 pWY-5D77A (as)+, pWY-5D77S (s)+, pWY-O93.1+ (4x77S, mpEGFP)+, pWYH84*+

Table. 3-1. Tissue-specific promoters and constructs. *, transformed into rice; +, transformed into Arabidopsis; promoters without listed constructs are candidates for future cloning. (1) Aarts et al., 1997; (2) Roberts et al., 1993; (3) Oldenhof et al., 1996; (4) Guerrero et al., 1989; (5) Xu et. al., 1993, 1995; (6) Albani et al., 1990; (7) Hong et al., 1997; (8) Smith et al., 1997; (9) Weterings et al., 1995; (10) Tebbutt et al., 1994; (11) Twell et al., 1989; (12) Garbarino & Belknap, 1994; (13) Chen & Rubenstein, 1991; (14) McElroy et al., 1990; (15) Tsuchiya et al., 1992, 1994, 1995; (16) Lee & Hodges, Genbank acc nr U12171; (17) Treacy et al., 1997; (18) Terryn et al., 1993; (19) Yang, 2001.

5.2 Pro-gametocide activation ß-Glucuronides and sulfates are the most important detoxification mechanisms of xenobiotics in vertebrates, leading to secretion of these derivatives in the urine and bile (Dutton, 1966, 1980; Carrera et al., 1995; Fukai et al., 1995). With the help of ß-glucuronidase E. coli can set the glucuronic acid moiety (GlcA) free and utilize it as a carbon source. The usefulness of the E. coli GUS gene has been widely demonstrated in numerous publications, mostly as a reporter gene. Some interesting recent applications include the use of GUS as a positive selectable marker gene for the development of more efficient transformation protocols. The gene has been used to release cytokinin from a glucuronide conjugate (Okkels et al., 1997); only tissues expressing the gene were able to grow when supplied with the conjugate (Joersbo and Okkels, 1996). The great advantage of this method is that other tissues, not expressing the gene, do not become necrotic, their growth is only halted, thereby avoiding the production of growth-inhibiting tissue breakdown substances. Apart from phytohormone conjugates, positive selection can be achieved by supplying the plant tissues with glucuronide conjugates of nutrients. This principle has been demonstrated using the phosphomannose isomerase and the xylose isomerase genes to convert two non-metabolizable sugars into their metabolizable counterparts (Joersbo et al., 1998; Haldrup et al., 1998). CAMBIA has filed a patent that addresses a specific use of this technology, the liberation of glucose from cellobiouronic acid, which is a GlcA-Glc disaccharide (Jefferson, 1997).

9

Another example of procompound activation in planta was provided by Hsu et al. (1995): a transgenic plant expressing the glucuronidase gene in the root tips selectively released the active ingredient from a biologically inert nematicide [oxamyl]/glucuronic acid conjugate, thereby controlling root knot nematodes at the site of damage. One of the main features in such a system is the positive change of phloem mobility produced by glucuronidation (Tyree et al., 1979; Hsu et al., 1988; Kleier, 1988; Hsu & Kleier, 1996). Some of our glucuronide phloem mobility experiments are mentioned in section 3.3. This principle is applicable to hybrid seed production, male sterile plants can be selectively produced by application of the otherwise non-toxic glucuronide conjugate to plants expressing the activating gene in the male organ tissue. Active ingredients can totally lose their biological effect through minimal chemical modification, as was shown for acetylated phosphinothricin (PGS/Aventis proprietary technology). This unselective herbicide is totally innocuous to plants in its acetylated form, but plants expressing the corresponding deacetylase gene in the pollen sack become male sterile after application of this product, leaving the rest of the plant unharmed, moreover production of the deacetylase ceases after destruction of the tissue, thereby delimiting the effects with extreme accuracy (Kriete et al., 1996). The most common glucuronides have O-glucuronosidic bonds but also N, S and C-glucuronosidic bonds are possible. Although GUS has a wide substrate range different glucuronidases will not only discriminate between these bonds but also among them. Synthesis of glucuronides has been described for many compounds and several are available commercially because of their clinical implications. The synthesis of fluorouracil-GlcA (FU-GlcA), as an example, was described by Kanekoet al. (1977). FU liberated by GUS becomes toxic to cells expressing the cytosine deaminase gene, which could be one way to achieve localized cell ablation. Chemical synthetic procedures for the production of glucuronide conjugates were reviewed recently by Stachulski & Jenkins (1998). The glucuronosidic bond is more stable than the glycosidic bond, conversely bond formation is less efficient when using the popular Koenigs-Knorr reaction. Yu et al. (2000) have circumvented this problem by creating activated intermediates which behave like a normal glycosidic donors (6-phenylthio-glucopyranosides).

5.3 Development of a glucuronide-responsive promoter for field level control of transgene expression (K. S. Ravi) The ß-glucuronidase (GUS) gene from E.coli is part of a glucuronide-inducible operon. The GUS repressor (GUSR) --also encoded on the operon, binds to a palindromic operator sequence, thereby blocking gene expression. The repressor is released when a glucuronide binds to it. This part of the project deals with the generation of a chemically inducible promoter for transgene expression using the GUSR gene in combination with the operator sequence. The following experiments were conducted to this end: (i) The coding sequence of gusR was amplified by PCR and cloned into an E.coli expression vector; (ii) purified GUSR was used for binding studies with phenylthio-β-Dglucuronide and saccharolactone-agarose; (iii) gusR was cloned into plant and yeast expression vectors to investigate its expression in heterologous systems; Southern analysis showed that nearly 50% of transgenic plants had a single copy of the gene; (iv) for the generation of chemically induced gene expression systems, gusR was fused in frame with activation domains of the GAL4 protein from yeast and the VP16-derived activators from Herpes simplex virus, respectively; the precise DNAbinding properties of GUSR were determined by nitrocellulose filter binding and mobility shift assays; (v) constructions containing one or multiple copies of the gus operator for in vivo expression studies in yeast and plants using the β-galactosidase or β-glucuronidase reporter system were made; (vi) glucuronide uptake experiments showed fast uptake and mobility of radiolabeled phenylthioglucuronic acid across by cut shoots as well as by intact roots of Arabidopsis thaliana, rice (Oryza sativa) and swede (Brassica napus); the glucuronide was also taken up through the leaf lamina and transported systemically.

10

Future studies will focus on: (i) In vitro and in vivo evaluation of GUSR-binding properties to multiple gus operators; (ii) in vivo evaluation of GUSR-transcriptional activator fusions in yeast and plants to study the transcriptional regulation of target genes; (iii) identification of glucuronide conjugates with varying degrees of specificity, permeability, solubility and affinity for the induction system to increase the versatility of applications under varied environmental conditions.

6. Tools for apomixis: Understanding meiosis in the embryo-sac (S. Nugroho) Studies show that apomixis seems to occur by the combination of a failure to reduce the female gamete through meiosis and the ability to initiate embryogenesis without pollen-mediated fertilization. This shows that at a certain time during plant development, a determining event decides whether the progenitor of the megaspore mother cell is to undergo meiosis and then mitosis, or continue directly with mitosis. Because these early events are crucial in making the decision whether to reproduce sexually or apomictically, a greater understanding of the early meiotic gene functions in plants is required. As these processes are well characterized in yeast at the molecular level, we decided to search for homeologous genes in rice and establish their function, especially one protein kinase (RIM11) which is responsible for the activation of the meiosis initiation protein IME1 (Bowdish et al., 1994). The principal activities toward this goal were: (1) To isolate and characterize homeologous genes to Saccharomyces cerevisiae RIM11 (ScRIM11) and other members of the GSK-3 kinase family in rice, and (2) To investigate the function of the rice gene by promoter fusions with GUS, gene knockout, over-expression and gene replacement in yeast RIM11 null mutants (Jonak et al., 1995).

6.1 Isolation and characterization of a ScRIM11 rice homologue After screening BAC and YAC libraries from Pamela Ronald (UCD) and from the Japan Rice Genome Project, Tsukuba, a rice expressed sequence tag (EST) encoding a protein closely related to the S. cerevisiae RIM11 gene (ScRIM11) was identified. Thee gene is also related to other GSK-3 serine/threonine protein kinase family members from animals, including Rattus novergicus GSK-3 and Drosophila melanogaster zw3, which are involved in early development and segmentation processes. In yeast, the functions of the gene family members are generally restricted to meiosis. Genes related to ScRIM11 have also been isolated from various plant species including Arabidopsis thaliana, Nicotiana tabacum and Petunia hybrida. In plants, the function of the GSK-3 gene family members is still unknown. However the fact that in yeast and animals homeologous genes play critical regulatory roles, suggests that they may play a similarly central role in plants too. Homology database search using the ScRIM11 sequence pinpointed a 374 bp EST of a rice cDNA (Fig. 4-1). The rice partial gene sequence is 70% identical to that of ScRIM11. The putative amino acid sequence of the rice EST was determined to be 79% identical to ScRIM11, suggesting that the rice EST may be a fragment of a rice gene transcript coding for a protein functionally related to ScRIM11. The candidate rice gene was called OsSK11 (for Oryza sativa RIM11). PCR amplification of rice genomic DNA using primers OSK1 and OSK2 resulted in the amplification of a 854 bp DNA fragment. Sequence analysis revealed the presence of 3 exons and 2 introns (Fig. 4-2). emb|Y11527|OSSTTPK Oryza sativa mRNA for serine/threonine protein kinase, partial

11

Length = 1053 Identities = 265/375 (70%), Positives = 265/375 (70%)

RIM11: 953 GTCTGTCATAGAGACATTAAGCCTCAAAATTTATTAGTAGATCCTGAGACCTGGTCCTTA ||||| ||| |||| ||||| ||||||||| || | || OsSK11:

||||| | ||

|

4 GTCTGCCATCGAGATATTAAACCTCAAAATCTACTGGTCAATCCTCATACACATCAACTG OSK1

RIM11:1013 AAACTGTGCGATTTCGGCAGTGCAAAGCAATTGAAACCTACTGAACCTAACGTTTCCTAT || || || ||||| || ||||| ||| |||||

|||

|||||||||| | || ||

OsSK11: 64 AAGCTTTGTGATTTTGGAAGTGCTAAGAAATTGGTTCCTGGTGAACCTAACATATCATAC

RIM11:1073 ATTTGTTCACGGTACTATAGAGCACCAGAGCTAATCTTCGGCGCAACAAATTATACCAAC ||||| || ||||| ||||| || || || ||||| || || || ||| | ||||||| OsSK11:124 ATTTGCTCGCGGTATTATAGGGCTCCTGAACTAATATTTGGAGCTACAGAGTATACCACA

RIM11:1133 CAAATCGACATATGGTCCTCTGGCTGCGTAATGGCGGAACTGCTATTGGGCCAACCAATG ||| || || |||||

||| || ||| | || || || ||

| || || ||

||

OsSK11:184 GCAATTGATATCTGGTCTGTTGGTTGTGTACTAGCTGAGCTTCTGATTGGTCAGCCTCTG

RIM11:1193 TTCCCTGGAGAAAGTGGTATTGATCAACTAGTGGAAATCATTAAAATCTTAGGTACTCCA |||||||||||||||||| |||||||||| |||||||| || || || || ||||| ||| OsSK11:244 TTCCCTGGAGAAAGTGGTGTTGATCAACTGGTGGAAATAATAAAGATTTTGGGTACACCA OSK2

RIM11:1253 TCAAAGCAAGAAATTTGCTCTATGAATCCCAATTATATGGAGCATAAGTTCCCGCAAATT | |

|||||||

| |

|||||||| || |||

||

|| ||||| || ||

OsSK11:304 ACTAGAGAAGAAATCAGGTGCATGAATCCAAACTATTCTGAATTCAAATTCCCTCAGATA

RIM11:1313 AAACCAATACCATTG ||| |

|||| |

OsSK11:364 AAAGCTCATCCATGG

Figure. 4-1. Rice EST encoding a serine/threonine kinase was identified by similarity search with the ScRIM11 sequence. The rice EST was 70% identical to the ScRIM11 fragment. The two arrows indicate the primers (OSK1 and OSK2) used to perform PCR amplification of rice genomic DNA to isolate the putative rice gene homologue.

12

1 51 101 151 201 251

301 351 401 451

501 551 601 651 701 751 801 851

GTCTGCCATC GAGATATTAA V C H R D I K actcattttt atcaggcacc tttccctttt ttactatttg attgaaacat gcatttgcag tttcagctgt ttatgtgttg tgtctcattg caagttgatc HindII ACTGAAGCTT L K L cctgctagca gacactccca

TGTGATTTTG C D F G tgtttgattc tttcagGTTC V P CGCGGTATTA TAGGGCTCCT R Y Y R A P EcoRV ACAGCAATTG ATATCTGGTC T A I D I W S TGGTCAGgta tagaatcatt G Q atgacccgag gatatacttg atcttaagtc acaccattac catttgaatg caagtacttc tgctgtgctg caagactgtt ggacaattgc agCCTCTGTT P L F GGAA E

ACCTCAAAAT P Q N cctcctcagg ggtacttgta cttgtgcaaa tgatataagt gatttttcag

CTACTGgtat L L gcatgctttg aacagatgat agcatgattg aaacctgctg GTCAATCCTC V N P H

gtatcatgag

GAAGTGCTAA S A K tgttgtttgc CTGGTGAACC G E P GAACTAATAT E L I F

GAAATTGgta K L acatgttatt TAACATATCA N I S TTGGAGCTAC G A T

tgcttcttgt

caagtgttct ggagtttaag cctgcaatat tacaagcaat ATACACATCA T H Q

tgacaatgca TACATTTGCT Y I C S AGAGTATACC E Y T

TGTTGGTTGT GTACTAGCTG AGCTTCTGAT V G C V L A E L L I ctagcagcta gtttttttct agtcaagttc attgaataat caaaaaataa attttatggg tcctgctttg CCCTGGAGAA P G E

tttgtatcga ataacataac ggttgggggt gctgtctttt AGTGGTGTTG S G V D

acttactgtt tcagcatttc taataatgga ttccgaatat ATCAACTGGT Q L V

Figure 4-2. Genomic DNA fragment of the rice ScRIM11 homologue. The fragment was obtained by PCR amplification using primers OSK1 and OSK2. Exons, uppercase; introns, lowercase. The deduced amino acid sequence is 83% identical to ScRIM11.

Southern analysis of rice genomic DNA using the 854 bp PCR product as a probe showed that OsSK11 is a single-copy gene in Oryza sativa var. Millin. Rice genomic DNA flanking the 854 bp PCR fragment was further characterized. Using inverse PCR a 4,940 bp rice DNA fragment was isolated. The putative amino acid sequence of OsSK11 was predicted by analysis of the open reading frames and comparison with ScRIM11. The transcriptional start site was determined by cRACE; it is located at a G residue 92 bp upstream of the putative start codon (ATG). The stop codon (TGA) and a 399-bp 3' untranslated region (3'-UTR) were determined by 3' RACE. The putative open reading frame (ORF) of OsSK11 has a total length of 1428 bp encoding 475 amino acids (Fig. 5-3). The putative gene consists of 12 exons and 11 introns, which makes it a highly fragmented gene (longest exon 272 bp and the shortest only 50 bp).

6.2 Functional analysis of OsSK11 The rice gene possesses a conserved putative catalytic domain made up of two regions characteristic of serine/threonine protein kinases. The first region is the stretch of glycine-rich residues located in the N-terminal extremity of the catalytic domain in the vicinity of a lysine residue: VGTGSFGVVFQAKCLETGETVAIK KVLQD (Hanks and Hunter, 1995). The second region is located in the central part of the catalytic domain, characterized by the presence of a conserved aspartic acid, which is responsible for the catalytic transfer of phosphate to the protein substrate and a conserved lysine nearby which is a signature for a serine/threonine kinase: CHRDIKPQNLLV (Knighton et al., 1991).

13

A. GGGGGGGGGG GCTTTAATAA GAACATTTAT ATCTGCCAAT CTTTGTTGAG GCACTGGGCA GAGAATAAGG TCAGGATGGA

GGGGGAAGTG AGGTTAAAGG TCCCTGGTGT AATAGGTTTA CGTTGAACAG GTGTATATTG AAAGTAGGAT GCAGGAGATA

GGCATTGCAT CGACAGCTTG TCATGCTTCA TTGGATTGGG CTTTTCAGAA TAGAATGAAT TAGTTTCTCC TAGTCATTGA

TGGGTGAAGG TTTGCTTCTG TGGGCTCATG AAGTTATCAA GGATGTGATT TGGTGGAAGA TCCCTATTAT GCCACATCTA

GTGTCGCGCT CCCGACTATT AGGAGATTTG GGAAATGCTT TATGACGTAG GTTTGATGCA GAGCTTGAGT ACTGATGACA

GCATTATTGC TATTTTGTCT ATGCTGCATT TTCAATGTGT TTGATGTCTC CAATGTTAGC TGATAACCTA AACCTATGCG M R CTACTAGTAA GAATCCTGGT AGAACTGAAG AAGCAGGTGC GGATATTCTT CCAAAAGAAA T S K N P G R T E E A G A D I L P K E M gttattttga taccatttga cttgtcactt cttgacataa tgccacccta ctttcgttac GCAAATGGAA A N G T tctcatgcgt caccccccaa gttctcttgg

CAGAAACAGG E T G tctgctggat aaaaaaagaa atgtttgcta

TCAGATTATT Q I I ctgctggata aaaaacacac tactacattc

GTGACATCAA V T S I ttaatacaat tatgatgcaa tttacataca

TTGGAGGTCA G G Q gcatcagcac tgcattccat ggcagggtca

GTACTGGTTC ATTTGGTGTA GTTTTTCAGg T G S F G V V F Q tgaccagtta ttgatacaaa cagGCCAAGT A K C CAACTGCTTG ACCATCCTAA TGTGGTCCAG Q L L D H P N V V Q TTATCGTGTT GCTAAATACT ACAATCGGAT Y R V A K Y Y N R M tttcaaactg ttaaatccat gattctaaag TGCCATCGAG ATATTAAACC TCAAAATCTA C H R D I K P Q N L ctatttgggt acttgtaaac agatgatgga tataagtaaa cctgctgtac aagcaattgt ATTGgtatgc L CTCGCGGTAT S R Y tatagaatca accaaaaaat tggctgtctt

ttcttgtcct gctagcatgt TATAGGGCTC Y R A P ttctagcagc aaataacata ttttccgaat

CTGAACTAAT E L I tagttttttt actcagcatt atggacaatt

agacagtaat ttaggtctgt ggactgatcc TGAATTCAAA E F K caactgcagC L tttgagccat tgtttgtatc

TTCCCTCAGA F P Q I TTTTGGGTAA L G K tttaactaat tactaattag

TAAAAGCTCA K A H GCGCATGCCA R M P tattgaacca tcctagattg

AAATGGGAAG N G K tgaaaatatc cagtattgcg aacattaatg

tatattattt cacattttgc cattctgtat GCTTGGAAAC L E T CTAAAGCATC L K H H GAACCAGCGT N Q R aaattatttg

TGGAGAGACT G E T ACTTCTTTTC F F S GTGCCCATAT V P I L ttctaatgtg

GCTTTAATAA AGTTTGGTTC GGCGATGGCT AACTTTTCAT ATAATACTGA TAATAGGTAT TATTGTTCTT CGTAGACCAA V D Q TGAATGAGAT N E M ttatgtcgta

AGGTTAAAGG AAGAGGATAG GGACGGTGGT GATGGGTCTG GAACAAAACA AGTATGTGAC GTTAGGGTGG GAATCATCTT E S S S GACAATAAGT T I S gtttttctga

CGACAGCTTG CGTTTTTGTC ATAGCTGTCC CTTTGGTCAT TTGCTCACCA TTCTAGCGGA TGATTCTGGC CATCTCATAG S H R GATGATAAAG D D K V ttatagGAAT E S CCAAAGCAGg tgatgcatca acccattatg P K Q ttcatgccct gaaaactggg agattccttc ggtgttattt cttcctgatg gttgtgtatt tgtgctaatt gttttacagA AGGTATCATA K V S Y cgctactcct cgtcatgatg gaaatttaca

GTTGCCATTA V A I K GACGACTGAG T T E TGCATGTTAA H V K ttttactatt

AGAAGGTTCT GCAAGATAAA K V L Q D K AGGGGTGAAG TTTACCTCAA R G E V Y L N GCTGTATGCA TATCAGgtct L Y A Y Q tttcatagAT GTGCCGTGCC M C R A CTGgtatgta tcatgagact catttttatc aggcacccct cctcagggca L gtttaagatt gaaacatgca tttgcagctt gtgcaaaagc atgattgcct ctcattgcaa gttgatcgat ttttcagGTC AATCCTCATA CACATCAACT V N P H T H Q L tgattctgtt ttgcacatgt atttgacaat gcagacactc catttcagGT V ATTTGGAGCT ACAGAGTATA CCACAGCAAT TGATATCTGG TCTGTTGGTT F G A T E Y T T A I D I W S V G C ctagtcaagt tcatgacccg aggatatact tgattgaata attttgtatc tccatttgaa tgcaagtact tcattttatg ggggttgggg gttaataatg gcagCCTCTG TTCCCTGGAG AAAGTGGTGT TGATCAACTG GTGGAAATAA P L F P G E S G V D Q L V E atttcttgtt tttttttctt attatagATT TTGGGTACAC CAACTAGAGA I L G T P T R E TCCATGGCAC AAGgttggtg gatctctaat tccatagcac tttagttcta P W H K CCTGAAACTG TTGATCTTGT GTCAAGGTTG CTGCAATACT CGCCAAATTT P E T V D L V S R L L Q Y S P N L gatcggtatt aggtggatca tcatgagcac aataatctaa tttcctcata ggaaacaaag aaacaaaaaa aatcttgatt actgttctga ttaaacgtat

TCATCCATTC TTTGATGAAC TGCGGGATCC CAAGACCTGC TTGTCAAATG GACGATCATT ACCACCCTTG TTCGACTTCT H P F F D E L R D P K T C L S N G R S L P P L F D F S Aaagttgaat ttctattata acccttgtct tatccctcat ccaaccattg gaatggtggt ctacagAATT GGAAGGCCTT L E G L P GAACATATGA GGAAGTGAAA CGGAATGGCA GGAAAACACA ATTTCGCTGT TAGAACAGCG GATAGGTGTG AGTGAGATGG E H M R K * ATCAGGAGTA GACTAGATTC ACTGCCTTGC TAGATGTGGA AAGAGGGATG CTATAGAAGT TCCTCATCAT CACTTCACAT AATTCCAAGA GGTAATGAGC AACTGAGATT GGCACGGCAA CTCGCACATT TTCATCATGT GCCACCAGAG ACGTTTATCT GCTGAGTTTG CAGTTGTTTG TAGACACTTG TTTCCCTGAA CAAGCGATGA GAGAAATCCA GATTCCAAAG TGCTTGCTTT AATTTTGATT AGTGCACGCA ATGGCCTTTC CTTTATGTTT CAGAAGTCTA ATCAAATTCT CTCAATCATG GTGTCTTAGC TGTTTGGATG CTATACTAAG TTTGCCACCA TTCACCATAC TATTTGTACC ACATTTGCCT AAGTTGTGGC TTACAAAAAG TGTGCCACAC TTTGTTGAGT TGATGACATG TGGGGCCCAA ATAGATGAGA AAGGAATCTT GCCACAAGTG TGGCTATGAA CATAACATGA AAATGTGGTA ACCTTGGGCA AA

AGATATAAGA R Y K N CCTTGTACTT L V L gataagttat

TTTGCTTCTG TGCAGAGCTG GACATTACTA GTCCCTTAAT ATCACAATAA TGAATATGAA ACCAAAAGAC AGATGCTGAG D A E TTGATGGCCA D G H CTGAAGGTGT E G V tatttatctg

GCATTATTGC TAGCAATGTG GAAAAAGAGA CGTAGTCTTG AAAAAATTGG TTCTGTGCTT CAAAATTCGA GCAAGCACAT A S T S TAATGATAAG N D K TATTGTTAAT I V N caccattctc

cttcaacccc tttgcttagg CATGGCAGAA M A E atatttgggc

ccaccccccc aaaaagtagc CGTGTAGTT R V V G ataaagttat

ACAGAGAGCT CCAAACAATG R E L Q T M GAATATGTCT CAGAGACAGT E Y V S E T V aattttagct ttgtagatca

CTTGCATATA TCCATCGTGT TGTTGGTGTC L A Y I H R V V G V tgctttgcaa gtgttctttt ccctttttta gcaatatttt cagctgttta GAAGCTTTGT GATTTTGGAA K L C D F G S TCCTGGTGAA CCTAACATAT P G E P N I S GTGTACTAGC TGAGCTTCTG V L A E L L gaacttactg ttatcttaag gatgctgtgc tgcaagactg TAAAGgtttc tatctcctgt I I K AGAAATCAGG TGCATGAATC E I R C M N P cactgatgta accttctcac GCGTTGCACA R C T agaaagtgtt ttggctgttc V D CAGCTGCTGg A A E CCTGTTGAGC V E L TTGATGCAAA

GCTgtaagac A gtactacata ttccagGTTG A C A taagaatttt

AAAGAGCGAG ACTAGGCTGC GCTATCATGA TGATGGTTTC TGAGCCACAC CCAAACAATA

GCAGTGGCTC TGTTGTATAA AAGCCTGCCC ATCTTGCATG CTTAGCTAGC TGCTAACTTG

tgtgttgtga GTGCTAAGAA A K K CATACATTTG Y I C ATTGGTCAGg I G Q tcacaccatt tttcctgctt catccattag CAAACTATTC N Y S aaaatttttc taatattgga tgatatatta ATGCTTGTGC ctctacttga

TAGTCCACCG AATCATTCCT V H R I I P CATCCATAAC TTATGGATGG ATTCTGATGC TCAGGTACAT GGAACATGTC ACTCAGGGGC AAAATTGGAT GACAAACTTG

B.

5'-UTR

3'-UTR

Figure 4-3. A. Complete sequence of OsSK11. Amino acid sequence in bold capital letters; 5' and 3' untranslated regions (UTRs) underlined. B. Block diagram of OsSK11. Exons, checkered boxes and 5'- and 3'UTRs, empty boxes.

Transcript levels of OsSK11 were analysed in different organs by RT-PCR. Transcripts were detected in all organs but were slightly higher in generative tissues, represented by different stages of panicles, and in merismatic tissues, represented by nodes. This has also been reported for other members of the GSK-3 family. Although ScRIM11 is functional at meiosis initiation it is also present in non-meiotic, vegetative cells (Bowdish et al., 1994).

14

1.22 0 .4 2

0.97 0.84

0 .3 5

0.58 0 .1 3 0 .0 9 0 .0 4

R

L

I

N

P

PMP MP

POMP

Figure 4-4. Semi-quantitative RT-PCR analysis of OsSK11 transcript levels from different organs and floral stages. The numbers on top of the bars represent the ratio of OsSK11/Actin1 expression levels in the same organs (Actin1 expression levels are used as an internal reference standard). R, root; L, leaf; I, internode; N, node; P, panicle; PMP, pre-meitotic panicle; MP, meiotic panicle; POMP, postmeiotic panicle.

The deduced amino acid sequence of the complete rice gene is 50% identical and 70% similar to ScRIM11, while the putative catalytic domain of the rice protein is 58% identical and 74% similar to that of the ScRIM11. Further analysis revealed that OsSK11 also has high sequence identity to other known yeast meiotic genes. The rice putative amino acid sequence is 48% identical and 64% similar to the yeast MDS1 protein, which is important for the completion of meiosis and sporulation and has no apparent role in vegetative growth (Mitchell, 1994; Puziss et al., 1994). OsSK11 also has a high sequence identity (42% identity, 60% similarity) to MCK1, a yeast gene known to encode a positive regulator of meiosis and spore formation (Neigeborn and Mitchell, 1991). It also has high sequence identity (48% identical, 65% similar) to MRK1, another yeast serine/threonine kinase (He et al., 1995), whose function is still unknown. OsSK11 has also high sequence similarity to many plant and animal serine/threonine protein kinases, e.g. the maternal and embryonic forms of shaggy/zeste-white3 product from the fruitfly Drosophila melanogaster glycogen synthase kinase (GSK-3), also to the corresponding rabbit, frog and human products, and protein serine/threonine kinases from various plant species including Arabidopsis, Nicotiana, and Petunia.

15

OsSK11 ASKalpha MSK1 NtSK111 PSK4 Dmsgg39 RnGSK3beta ScRIM11

------------------------------------------MRVDQESSSSHRDAEASTSTSKNPGRTEEAG------ADILPKEMNEMTISDDKVD--GHNDKES --------------------------------------------------------MASVGIAPNPGARDSTG------VDKLPEEMNDMKIRDD---------KEM --------------------------------------------------------MASVGVAPTSGFREVLGDGEIGVDDILPEEMSDMKIRDD---------REM MNVMRRLKSIASGRSSVSDPGGDFSLKKVTVEQEVDHRVDGETQLEEQCTIAPKQDVTSTSEESTVCTLNVDTRPEKSRYEELPKEMNEMKIRDEKTNGHEDDIKDM ---------------------------------------------------------MASGIMPSAGGKHRTD---AMLVDKLPEEINEMKIRDDKAE------KEM ----------------------------------------------------------MSGRPRTSSFAEGNK------QSPS-LVLGGVKTCS----------------------------------------------------------------------MSGRPRTTSFAESCK------PVQQPSAFGSMKVSRD--------------------------------------------------------------------------------------------MNIQSNNSPNLSN-------------


EGVIVNANGTETGQIIVTSIGGQNGKPKQKVSYMAERVVGTGSFGVVFQAKCLETGETVAIKKVLQDKRYKNRELQTMQLLDHPNVVQLKHHFFSTTER-GEVYLNL EATVVDGNGTETGHIIVTTIGGRNGQPKQTISYMAERVVGHGSFGVVFQAKCLETGETVAIKKVLQDRRYKNRELQTMRLLDHPNVVSLKHCFFSTTEK-DELYLNL EATVVDGNGTETGHIIVTTIGGRNGQPKQTISYMAERVVGHGSFGVVFQAKCLETGETVAIKKVLQDKRYKNRELQTMRLLDHPNVVSLKHCFFSTTEK-DELYLNL EPAVVSGNGTETGQIIVTTLSGRNGRQKKTLSYMAERVVGTGSFGVVFQAKCLETGESVAIKKVLQDRRYKNRELQIVRMLDHPNVVHLRHCFYSTTEK-NEVYLNL EAAVVDGNGTEKGHIIVTTIGGKNGEPKQTISYMAERVVGQGSFGIVFQAKCLETGETVAIKKVLQDKRYKNRELQTIRLLDHPNVVALRHCFFSTTEK-DELYLNL ------RDGSKITTVVATPGQGTD--RVQEVSYTDTKVIGNGSFGVVFQAKLCDTGELVAIKKVLQDRRFKNRELQIMRKLEHCNIVKLLYFFYSSGEKRDEVFLNL ------KDGSKVTTVVATPGQGPD--RPQEVSYTDTKVIGNGSFGVVYQAKLCDSGELVAIKKVLQDKRFKNRELQIMRKLDHCNIVRLRYFFYSSGEKKDEVYLNL -------NIVSKQVYYAHPPPTIDPNDPVQISFPTTEVVGHGSFGVVFATVIQETNEKVAIKKVLQDKRFKNRELEIMKMLSHINIIDLKYFFYERDSQ-DEIYLNL .*. *.* ****.* . . ...* *********.*.*****. .. * * *.. * *. . *..***


VLEYVSETVYRVAKYYNRMNQRVPILHVKLYAYQMCRALAYIHRVVGVCHRDIKPQNLLVNPHTHQLKLCDFGSAKKLVPGEPNISYICSRYYRAPELIFGATEYTT VLEYVPETVHRVIKHYNKLNQRMPLIYVKLYTYQIFRALSYIHRCIGVCHRDIKPQNLLVNPHTHQVKLCDFGSAKVLVKGEPNISYICSRYYRAPELIFGATEYTT VLEYVPETVHRVIKHYSKLNQRMPMIYVKLYTYQIFRALSYIHRCIGVCHRDIKPQNLLVNPHTHQVKLCDFGSAKVLVKGEPNISYICSRYYRAPELIFGATEYTT VLEYMSETVYRVSRHYSRVNHHMPIIYVQLYTYQLCRALNYMHNVTGVCHRDIKPQNLLVNPHTHQLKICDFGSAKMLVPGEPNISYICSRYYRAPELIFGATEYTN VLEYVPETVYRVLRHYSKANQQMPMIYVKLYTYQIFRALAYIHG-IGVCHRDIKPQNLLVNPHTHQLKLCDFGSAKVLVKGEPNISYICSRYYRAPELIFGATEYTF VLEYIPETVYKVARQYAKTKQTIPINFIRLYMYQLFRSLAYIHS-LGICHRDIKPQNLLLDPETAVLKLCDFGSAKQLLHGEPNVSYICSRYYRAPELIFGAINYTT VLDYVPETVYRVARHYSRAKQTLPVIYVKLYMYQLFRSLAYIHS-FGICHRDIKPQNLLLDPDTAVLKLCDFGSAKQLVRGEPNVSYICSRYYRAPELIFGATDYTS ILEYMPQSLYQRLRHFVHQRTPMSRLEIKYYMFQLFKSLNYLHHFANVCHRDIKPQNLLVDPETWSLKLCDFGSAKQLKPTEPNVSYICSRYYRAPELIFGATNYTN .*.*. ... . . . . . .. * .*. .* *.* ..***********. * * .*.******* * ***.***************** **


AIDIWSVGCVLAELLIGQPLFPGESGVDQLVEIIKILGTPTREEIRCMNPNYSEFKFPQIKAHPWHKVLLGKRMPPETVDLVSRLLQYSPNLRCTAVDACAHPFFDE AIDVWSAGCVLAELLLGQPLFPGESGVDQLVHIIKVLGTPTREEIKCMNPNYTEFKFPQIKAHPWHKIFH-KRMPPEAVDLVSRLLQYSPNLRSAALDTLVHPFFDE AIDVWSVGCVLAELLLGQPLFPGERGVDQLVEIIKVLGTPTREEIKCMNPNYTEFKFPQIKAHPWHKIFH-KRMPAEAVDLVSRLLQYSPNLRCQALDCLTHPFFDE AIDIWSAGCVFAELLLGQPLFPGESGVDQLAEIIKILGTPTREEIRRMNPNYKEFKFPQMKAHPWHKIFN-RRIPPEAVDLASRLLQYSPTLRCTALEACAHPFFNA AIDIWSVGCVLAELLLGQPLFPGESGVDQLVEIIKVLGTPTREEIKSMNPNYTEFKFPQIKAHPWHKIFH-KRMPPEAVDLVSRLLQYSPNLRSTALEACTHTFFDE KIDVWSAGCVLAELLLGQPIFPGDSGVDQLVEVIKVLGTPTREQIREMNPNYTEFKFPQIKSHPWQKVFR-IRTPTEAINLVSLLLEYTPSARITPLKACAHPFFDE SIDMWSAGCVLAELLLGQPIFPGDSGVDQLVEIIKVLGTPTREQIREMNPNYTEFKFPQIKAHPWTKVFR-PRTPPEAIALCSRLLEYTPTARLTPLEACAHSFFDE QIDIWSSGCVMAELLLGQPMFPGESGIDQLVEIIKILGTPSKQEICSMNPNYMEHKFPQIKPIPLSRVFK--KEDDQTVEFLADVLKYDPLERFNALQCLCSPYFDE **.** *** ****.***.***. *.*** .**.****....* ***** * ****.* * .. . ... . .* * * * . . .*


LRDP-KTCLSNGRSLPPLFDFSAAELEGLPVELVHRIIPEHMRK--------------------------------------------------------------LRDP-NARLPNGRFLPPAFHFKPHELKGVPLEMVAKLVPEHARKQCPWLGL-------------------------------------------------------LRDP-NARLPTGRFLPPLFNFKPHELKGVPVETLMKLVPEHARKQCPFLGL-------------------------------------------------------LREP-NACLPNGRPLPPLFNFTPQELSGASTELRQRLIPEYMRK--------------------------------------------------------------LRDP-KTRLPNGRPLPPLFNFRPQELKGASADLLNKLIPEHAKKQCTFLGV-------------------------------------------------------LRMEGNHTLPNGRDMPPLFNFTEHELS-IQPSLVPQLLPKHLQNASGPGGNRPSAGGAASIAASGSTSVSSTGSGASVEGSAQPQSQGTAAAAGSGSGGATAGTGGA LRDP-NVKLPNGRDTPALFNFTTQELS-SNPPLATILIPPHAR----------------------------------IQAAASPPANATAASDTN-----------LKLDDGKINQITTDLKLLEFDENVELGHLSPDELSSVKKKLYPKSK------------------------------------------------------------*. ** .


--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------SAGGPGSGNNSSSGGASGAPSAVAAGGANAAVAGGAGGGGGAGAATAAATATGAIGATNAGGANVTGSQSNSALNSSGSGGSGNGEAAGSGSGSGSGSGGGNGGDND -AGDRGQTNNAASASASNST-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------


---------------------------------------------------------------------------------------------------------AGDSGAIASGGGAAETEAAASG -------------------------------------------

Figure 4-5. Alignment of OsSK11 with other related kinases. High density of asterisks and dots shows zones of high similarity.

Most related genes from the animal kingdom have been characterized and their functions have been determined. The Drosophila zw3 and the GSK-3 class genes are segment-polarity class genes required for the patterning during segmentation and have implications in cell-fate determination and differentiation (Dominguez et al., 1995; He et al., 1995; Siegfried et al., 1990; Stambolic and Woodgett, 1994). These genes are crucial in animal early embryonic development. The corresponding plant genes have not been characterized functionally so far except fo differential expression patterns in different tissues. When compared to known plant kinases OsSK11 was most similar to Nicotiana tabacum NtK-1, whose highest expression levels were detected during pollen development (Einzenberger et al., 1995). This may indicate a functional role in flower development. The high degree of similarity of OsSK11 to GSK-3 family members from different organisms indicates that the rice gene may play an important role in rice development.

16

ScMCK1 RnGSK3beta HsGSK3beta XGSK3beta XGSK3 RnGSK3alpha HsGSK3 Dmsgg39 Dmsgg10 Dmsgg46 ASKetha OsSK11 PSK6 NtSK91 NtSK6 PSK7 NtSK59 NtSK111 BnSKtheta ASKtheta PSK4 NtK-1 MSK1 ASKgamma ASKalpha MSK3 MSK2 ASKiota ASKdzeta ScMRK1 ScRIM11 ScMDS1

Figure 4-6. Phylogenetic tree of the complete protein sequence of different GSK-3 family members. The tree was generated using the neighbor-joining algorithm. Parsimony analysis essentially gave the same results. ScMCK1 was used as the outgroup.

Phylogenetic analysis positions OsSK11 in a cluster of other plant genes from the SGG/GSK3 gene family, which form two major subgroups. Examples of some of the gene products found in that cluster are, PSK6 and PSK7 from Petunia hybrida, NtSK6, NtSK59, NtSK91 and NtSK111 from Nicotiana tabacum, ASKtheta from Arabidopsis thaliana, and BnSKtheta from Brassica napus. Most often highest expression of these genes was found during flower development. Although their function remains unknown, the high similarity to many important fungal, animal and plant regulatory serine/threonine kinases involved in various crucial regulatory processes, such as signaling before and during meiosis in yeast and in early embryogenesis in animals suggests such a role for this gene family in plants as well.

6.3 Identification of additional ScRIM11 homologues in rice The GSK-3 protein kinase family is usually a multi-copy gene in other organisms. The huge progress made in the characterisation of the whole rice genome allowed us to search for additional family members in rice. We screened the high-density rice genomic DNA BAC library obtained from the Clemson University Genomics Institute (Mao et al., 2000) and we also screened the most recently available sequences deposited in various databases, which led us to 23 additional candidates using either the ScRIM11 or the rice OsSK11 probes.

17

Clone 10C18 contained an insert coding for another serine/threonine kinase of the GSK-3 family. The putative amino acid sequence of the incomplete gene is 50% identical to ScRIM11. The gene was called OsSK11B.

A.

1

2 3 4 5 6 7 8 9 10 11 12

2.4 Kb

1

13 14 15 16 17 18 19 20 21 22 23

2.4 Kb

EcoRI

2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 23

HindIII

2.8 Kb

2.8 Kb

B. Hind III (2827)

EcoR I (1081)

EcoR I (3522)

Hind III (46)

Figure 4-7. A. Identification of putatice ScRM11 homologues in rice. Southern analysis of the BAC clones digested with EcoRI and HindIII using OsSK11 cDNA fragment as probe gave 14 positive clones. Five clones (3, 9, 12, 16 and 23) have been characterized to represent OsSK11. The other nine positive clones (4, 6, 7, 8, 10, 13, 14, 21 and 22) have different digestion patterns from OsSK11 and might represent different gene(s). Clone 22 (10C18) has been partially analyzed.. B. OsSK11 DNA fragment. Boxes represented exons, checkered boxes represent the exons used as the OsSK11 cDNA fragment probe.

18

ccacatgtta tcagccccaa caactaagca cgatgaaacg gtttctttct agtcattgaa taagaaagca aaagacgtac ctctcatgca gttatcggta ctgaccggtc cgccgccggg ggtgtccaga gccgtttttt AGAACGGCGA N G E ctcgtaacaa ggaatcgcaa tgtttggtta cttcacttct cttaactgca tattgtacat GCGTTACAAG R Y K ATCTTGTTAT L V M tgtcagccac

ataaaaaatt ctttttatcc tctataaaat gacaaaatca gtcccaacct atatacaaat acaattacga cacatctcca aatcctctaa aaaccacttc aaaagcacac gccggagccc tctggggtgt tttttttttt

actacggtaa tgatacaacc tcatgctaaa cgtgttaaca tttatcgtaa gaagcattca tgaaaccgat ttcgtcatcc aattcctcct cccatctgtc caccaccatc atggagctgg cggggtggat tttttttttg

ataaatttca aaaccacatg attccggccc atacaaatta aacggataaa ctattcttct atataaccgc ccaaagactc cttcaaaaat gccacaccac cagcagccgc cggcggcggc tttcggtggt cgggggcgac T G GCCGAAGCAG ggtgagcttt tttcggggtc tttttggttt ctgttcttct ttgtgtacag P K Q tcacttggtt cgctaagnat ttgtgctctc ttaagccgtt tctaagctgt tggtagtgtt ttttgtgggt ttggggctga ctagatacag ttgtcgtttt gatttgtaaa caatttagca ttcaattact tgaggataat gtacatttta ggaagcatga ttttcttttt ctgtccgcta aattttagtt agtcatgata agctggaact ctgatgcaca acatagaatc cgtcaatctt gACAATTAGT TATATGGCGG AGCGAGTTGT AGGAACTGGT TCTTTCGGGA TTGTGTTCCA T I S Y M A E R V V G T G S F G I V F Q tttttgatat gtttgaatca aactgattta gttttcacat ttagGCTAAG TGCTTGGAGA A K C L E T AACCGTGAAC TGCAGCTTAT GCGCGCAATG GAACACCCCA ATGTCATCTG CCTGAAGCAC N R E L Q L M R A M E H P N V I C L K H GGAGTACGTC CCTGAGACAC TCTACCGTGT GCTCAAGCAC TACAGCAATG CTAACCAGCG E Y V P E T L Y R V L K H Y S N A N Q R catgttaaca cctctctgat tgccactatg cttctcattg cttctctaaa gatacttttg

CTGTTCCAGG AGTCTGCCAC AGGGATGTGA V P G V C H R D V K tctaattgct tgctgataca gGTTGATCCT V D P tcagtggttt gatgtatgct gaaattattc tgcctttcat gattgatttg ggataagaag atgatcaata ttttccaaat tggttttatc ttcctgagct tggattataa ctgttggcct

ccgtcgttga ttaagaaaga gatgtactac aaaatatttc tacaacctaa taaaattcat aaaaaccgta atacccaatt ataaaacatc caaactccaa cccgcaaagc acgcgccacc ctgggctggg gttggaggtg

actatacaga aacaatcatg atctctattc tacgtaaccg agaaagcaca gctaaagatg cgatgataaa ctttatccta ccagttcatg agccggcctc caagcaccaa gccccccgcc gctcggtgcc ctgcagaaga

ttaagcatcc atgaacagat ttctttattt tcattgaaat atcacgatga tacaacatct aaatatttct atcaatacca aaagaaaacc accggcagca cgctcagcag nccgtggcgg gatctgctgg aggaggaggg

tgctaaagac ttaatataaa aactttttat agcatttata aacataaaac tgacttattc catttgaaca cttctaaact atcatactga accacaccac ctccctcccc gaccgccggc cgttgggcta gccgtgACGG H I I tggaatgtgc

gtaccacatt tattactatg cctgatacaa aaaatcatgc catatgttaa tagcccgagc tacaaattaa aagaaactca gcacagttat aacaccccgc agctcagctc atcagcgaga atgctggttt GTCACATCAT S T T tagttttggc

tctattcctc taaccgttgt tcttaagaaa taaagacgta taaaaatatt tttttatccc gcatcaacaa agattttcac tagctggagg agccttgctg agctcccccc aggtaacccg tctctccggt CTCCACCACC I G G K gcccttcgct

ctgactattt tgaaatatac gcaacaatta ccgtatagtt tctacttaac gatacaacct aattcatgcc cttggttcat agtcagcaca cctcccctcc cgcatggagg tggggagggg gtctcgcggc ATCGGCGGCA

cgggctgatc tttgacttga tttcatagtc gagatattgt Ggtacgatgc

aagttagcta taaaattacg tatcctgttg gcgcgatttc ttggtcttat

tctatgtgct tagtgtattc tttctagggg tcatgcgatt ggaaatgcat

gattcgtcgg ctcaattaaa gtaataaatg tttttcttac gcttaccgtg

AACCACAAAA P Q N CTCACTCATC L T H Q tagtggtagg tttaaaattt atgtgctgtt ctcttcccta

GGTGGCCATC V A I CAACCACCAG T T S ATCTATGTGA I Y V K agcttTTCAG F R TGTTTTGGTA tgacttatgt gaacaaggct acattacttc tcttgcttgg V L V AAGTCAAGCT ATGTGACTTT GGAAGTGCAA AGGTTCTGgt atgttgattt V K L C D F G S A K V L ctagtcataa tgttgacagc ataagtggtt tatttcgtca tgttgctttt gcacttgtgg ctgggataga cttttttaag atagcttcaa taagttaatt attatgtcga aggattgtta agtttattaa gttggatgca aagttgctga gttctgttat ttcttcattt tgctatttta acaaatagat tgatgatctc

AAGAAGGTGT K K V L CAGGGATGAG R D E AGCTTTACAT L Y I AGGGTTAGCT G L A ttgtttgttt

TGCAGGACCG Q D R CTGTTCCTGA L F L N CTATCAGgtt Y Q TATATTCATA Y I H T tgaactggtc

tggatttgct ccttatggaa ctttcataca tctaaagcag

tattttagca gtagattgat gtcgcatgtg catgctggac

CGGGGGAAAC G E T TGCTTCTTCT C F F S GATGCCATTG M P L ctatgcttgc

tgcttccttg

ccacccttaa gagtaatttc

GCCATAGACC TTGCTTCCCG A I D L A S R

atgctgcttt tgcctaatca taatgatgaa tccaccagGT V CATACATTTG CTCTCGCTAT TATCGTGCTC CTGAGCTCAT ATTTGGTGCA ACCGAGTATA CAACTTCAAT TGACATATGG TCAGCTGGAT Y I C S R Y Y R A P E L I F G A T E Y T T S I D I W S A G C CTTGGTCAGg ttagtcacta tatctttatc taaggaactg caacattcag gttccttgat tagattatgc tgtgatgtta ttctgattat L G Q cagCCACTGT TTCCTGGAGA GAGTGCTGTT GACCAGCTAG TAGAGATAAT CAAGgtattg catgatgctg tgcaagctac attttttttt P L F P G E S A V D Q L V E I I K gttaactcga tattatggca gGTCCTTGGA ACTCCAACCC GTGAGGAAAT ACGATGCATG AACCCCAACT ATACTGAGTT CAAATTTCCT V L G T P T R E E I R C M N P N Y T E F K F P GCACAAGgta gccatgtttt cttatattgg atcaccttcc tcataccatt ttacttggng ggcattgnan gccacttcaa aaccagtttc H K ataaaaaaaa tgtatctgca gctgcttaac tgatccatct gttgttcttt cgcttaccac cagATTTTCC ACAAGAGAAT GCCTCCAGAA I F H K R M P P E TCTTCTCCAA TATTCACCAA GTCTACGCTG CACTGCTgtg agttttcttt cgtttgctta gttttatatt atctataaat ctattgcttt L L Q Y S P S L R C T A

cgctaagcgc aataaaccga gcgtgctggc cttatgcttt

attatttaca tgttgaggcc gtgagtgcaa cagtataatc

TCCTGGTGAA P G E GCGTTCTTGC V L A atttcataat

CCGAACATAT P N I S AGAGTTACTT E L L gtgtatttgt

ttgccacggg tatttcttgt CAAATTAAGG CTCACCCTTG Q I K A H P W tgcatcatgt ttaatttgaa

taggataatg aatggtttct tggctgaaga agctggaaaa

tttcatttca ggatccctgg cctgttaaat attttcaaag

agtgttgtta tcctgcatgg atagttgtta agcttagcat

TCTTTGATGA GCTACGAGAG CCCAATGCAC GCCTGCCAAA TGGTCGGCCA TTCCCTCCTC TGTTCAACTT F D E L R E P N A R L P N G R P F P P L F N F gctttttatg gtacatttat atttacctgt catggatgct tatcaatgct catttctggt atgcagCTAG L A TCATATCAGA CGGCAACATG GCCTCAACTT TGCGCATGCT GGGAGCTAAa gggcaccgcg acgccacccg H I R R Q H G L N F A H A G S * Ctagatgact ccattgtcct ggacttggac tccatctcag attgcaccat ccatgttcca tgatccatcc gaagtacaat tatgtaaatt atctaaccat ggagaaatgc gtgctgctgc tgctggtgtt aagtacgtga atgtagttgg tggagactga gagacatgtt aagctaggga accctgcttt ccttcttttt tttttctcct ccctacagta gtgtagctgc tccctgtttt tgtttgctct ttcatcatgt aaatgccaat cccaatcgca ttgattttgc agctttgttt gttgatgatc aagtagtgaa cctgaacatc tctgcacttc agtgcaagaa

acgtgtagat aaaagaaatt gctaggacta cttgtactat

gctgttacag tcattggaat agtgctccac ttggttgatt

tcaaatattc atagaccgtg tataatatcc agtggtccaa ttgaaaaagt tgctggtaat atatgcagCT TGACGCATGT L D A C CAAGCATGAA gtaagtaaac gagaacattt K H E CCAGCGCCTC TCCAGAACTC ATCCACAGGC S A S P E L I H R L ttttgttcgt ttatttttcc atgagctgga

tgtcaagaac aaccaaattg tgtttaaaag GCACATTCCT A H S F tcaaacagac

atccgtcaag cggggcatgc ttcattttgg atgaaccctc cacaagaatt

taccagagaa ttaaggataa cttaccattg actccctgtc

ccatttccgc tggttgagta tttcttccca tttccttcac cagca

aaggactcag gtaggtaagg ttttgtgatc atcctggtct

TCATACCGGA I P D cattgcaatc

Figure 4-8. Partial sequence of OsSK11B. The putative amino acid sequence of the incomplete gene is about 50% identical to S. cerevisiae RIM11. *, stop codon; 3' untranslated region (3'-UTR) underlined.

Three new rice protein sequences highly identical to ScRIM11 were obtained from database searches using the OsSK11 and ScRIM11 DNA and protein sequences as inputs. At least five serine/threonine protein kinase family members related to ScRIM11 are possibly present in the rice genome, based on both the library screening and database searches (Fig. 4-8). All of them have the conserved serine/threonine kinase catalytic domains. Phylogenetic analysis using the neighbor joining algorithm as well as parsimony analysis revealed that the rice genes may belong to three different subgroups within the plant GSK-3 group (Fig. 4-9).

19

BAA92214 BAA92966 T03777 OsSK11b OsSK11 ScRIM11

----------MGSVGVAPSGLKNSS-STSMGAEKLPDQMHDLKIRDD-------KEVEATIINGKGTETGHIIVTTTGGRNGQPKQTVSYMAERIVGQGSF -------------------MEAPPGPEPMELDAPPPPAAVAAAAAT---AGISEKVLQKKEEGGGDAVTGHIISTTIGGKNGEPKRTISYMAERVVGTGSF -------------------MAAMPGGPDLAGAGGAVAVAVDAMQVDDPPRASAEEKHGPTIMGGNDPVTGHIISTTIGGKNDEPKRTISYMAERVVGTGSF --------------------------------------------------------------------TGHIISTTIGGKNGEPKQTISYMAERVVGTGSF MRVDQESSSSHRDAEASTSTSKNPGRTEEAGADILPKEMNEMTISDDKVDGHNDKESEGVIVNANGTETGQIIVTSIGGQNGKPKQKVSYMAERVVGTGSF -----------------------------------------MNIQSN--------NSPNLSNNIVSKQVYYAHPPPTIDPN-DP-VQISFPTTEVVGHGSF * * .*. . .** ***


GIVFQAKCLETGETVAIKKVLQDKRYKNRELQTMRLLDHPNVVALKHCFFSTTEKDELYLNLVLEYVPETVHRVVKHYNKMNQRMPLIYVKLYMYQICRAL GIVFQAKCLETGETVAIKKVLQDRRYKNRELQLMRAMEHPNVICLKHCFFSTTSRDELFLNLVMEYVPETLYRVLKHYSNANQRMPLIYVKLYIYQLFRGL GVVFQAKCLETGETVAIKKVLQDKRYKNRELQIMRSMDHCNVISLKHCFFSTTSRDELFLNLVMEFVPESLYRVLKHYKDMKQRMPLIYVKLYMYQIFRGL GIVFQAKCLETGETVAIKKVLQDRRYKNRELQLMRAMEHPNVICLKHCFFSTTSRDELFLNLVMEYVPETLYRVLKHYSNANQRMPLIYVKLYIYQFR-GL GVVFQAKCLETGETVAIKKVLQDKRYKNRELQTMQLLDHPNVVQLKHHFFSTTERGEVYLNLVLEYVSETVYRVAKYYNRMNQRVPILHVKLYAYQMCRAL GVVFATVIQETNEKVAIKKVLQDKRFKNRELEIMKMLSHINIIDLKYFFYERDSQDEIYLNLILEYMPQSLYQRLRHFVHQRTPMSRLEIKYYMFQLFKSL *.** . **.* *********.*.*****. *. . * *.. ** *. . *..***..*.. ... . . . . . .* * .* *


AYIHNSIGVCHRDIKPQ-NLLVNPHTHQLKLCDFGSAKVLVKGEPNISYICSRYYRAPELIFGATEYTTAIDIWSAGCVLAELMLGQPLFPGESGVDQLVE AYIHTVPGVCHRDVKPQ-NVLVDPLTHQVKLCDFGSAKVLVPGEPNISYICSRYYRAPELIFGATEYTTSIDIWSAGCVLAELLLGQPLFPGESAVDQLVE AYIHTVPGVCHRDIKPQ-NILVDPLTHQVKLCDFGSAKMLIKGEANISYICSRYYRAPELIFGATEYTTSIDIWSAGCVLAELLLGQPLFPGESAVDQLVE AYIHTVPGVCHRDVKPQNVLVVDPLTHQVKLCDFGSAKVLVPGEPNISYICSRYYRAPELIFGATEYTTSIDIWSAGCVLAELLLGQPLFPGESAVDQLVE AYIHRVVGVCHRDIKPQ-NLLVNPHTHQLKLCDFGSAKKLVPGEPNISYICSRYYRAPELIFGATEYTTAIDIWSVGCVLAELLIGQPLFPGESGVDQLVE NYLHHFANVCHRDIKPQ-NLLVDPETWSLKLCDFGSAKQLKPTEPNVSYICSRYYRAPELIFGATNYTNQIDIWSSGCVMAELLLGQPMFPGESGIDQLVE *.* .*****.*** ..* * * .********* * * *.****************** **. ***** ***.***..***.***** .*****


IIKVLGTPTREEIKCMNPNYTEFKFPQIKAHPWHKVFH-KRLPPEAVDLVSRLLQYSPNLRCTAVEALVHPFFDELRDPNARLPNGRFLPPLFNFKP-HEL IIKVLGTPTREEIRCMNPNYTEFKFPQIKAHPWHKIFH-KRMPPEAIDLASRLLQYSPSLRCTALDACAHSFFDELREPNARLPNGRPFPPLFNFK--HEL IIKVLGTPTREEIRCMNPNYTEFKFPQIKACPWHKIFH-KRMPPEAIDLVSRLLQYSPNLRCTALEACAHSFFDELREPHAKLPNGRPFPPLFNFK--QEL IIKVLGTPTREEIRCMNPNYTEFKFPQIKAHPWHKIFH-KRMPPEAIDLASRLLQYSPSLRCTALDACAHSFFDELREPNARLPNGRPFPPLFNFK--HEL IIKILGTPTREEIRCMNPNYSEFKFPQIKAHPWHKVLLGKRMPPETVDLVSRLLQYSPNLRCTAVDACAHPFFDELRDPKTCLSNGRSLPPLFDFSA-AEL IIKILGTPSKQEICSMNPNYMEHKFPQIKPIPLSRVFK--KEDDQTVEFLADVLKYDPLERFNALQCLCSPYFDELKLDDGKINQITTDLKLLEFDENVEL ***.****...** .***** * ****** * .. . .... . .*.* * * .*. . .****. . . * * **


KGIPSDIMAKLIPEHVKKQCSYAGV---ASASPELIHRLIPDHIRRQHGLNFAHAGS ANTHPELVSRLLPEHAQRHSGF------ASASPELIHRLIPDHIRRQHGLNFAHAGS EGLPVELVHRIIPEHMRK----------GHLSPDELSSVKKKLYPKSK---------

Figure 4-9. Alignment of the five rice protein sequences related to ScRIM11.

6.4 OsSK11 gene function analysis in transgenic rice and in a yeast nullmutant Except for two Arabidopsis GSK-3 gene family-related genes (AtSK11 and AtSK12), which may play role in perianth and gynoecium development and AtGSK1, involved in salt stress signaling, the functions of most of the gene relatives in plants are unknown. To elucidate the role of OsSK11 in rice the promoter region was fused to the ß-glucuronidase reporter gene gusA and transformed into rice plants to determine its expression pattern along the life cycle of the plant. Active domains in the promoter were identified by deletion analysis. To make sure that the gusA expression patterns obtained from the transgenic rice obtained were not due to cis activation of the CaMV35S promoter, a cotransformation experiment with two separate plasmids was carried out (Komari et al., 1996). The first contained the OsSK11 promoter driving gusA and the second contained the CaMV 35S promoter driving the hptII gene. Results using this approach do not indicate cis activation by the strong CaMV35S promoter acting on the epxression of the 3 Kb OsSK11 promoter fragment. An alternative approach to elucidate the function of OsSK11 was the disruption of expression of the endogenous OsSK11 by overexpression or silencing the gene. Post-transcriptional gene silencing or cosuppression of homologous endogenous genes can be achieved by expression of sense and antisense constructs (Murfett et al., 1995; Green et al., 1986; Wagner & Simons, 1994; Bautista et al., 2000; Angell & Baulcombe, 1997). This kind of gene silencing has also been observed in yeast, Drosophila and Caenorhabditis elegans, probably as a mechanism of defense against invading DNA or RNA molecules. To silence the expression of OsSK11, constructs yielding nonfunctional sense and antisense RNA capable of forming pan-like structures according to Waterhouse et al. (1998) were prepared. These were transformed into rice along with sense-only and antisense-only constructs. Transgenic plants and their pollen (viability, starch deposition, the presence of three nuclear cells and the ability to germinate) were observed for phenotypical changes.

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ScMCK1 RnGSK3beta HsGSK3beta XGSK3beta XGSK3 RnGSK3alpha HsGSK3 Dmsgg39 Dmsgg10 Dmsgg46 ASKetha T03777 BAA92966 OsSK11 PSK6 NtSK91 NtSK6 PSK7 NtSK59 NtSK111 BnSKtheta ASKtheta PSK4 MSK1 ASKgamma ASKalpha BAA92214 NtK-1 MSK3 MSK2 ASKiota ASKdzeta ScMRK1 ScRIM11 ScMDS1

Figure 4-10. Phylogenetic tree of the complete protein sequences using the neighbor joining algorithm (1000x bootstrapped).

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ScMCK1 ASKetha OsSK11B BAA92966 T03777 ASKiota ASKdzeta OsSK11 PSK6 NtSK91 NtSK6 PSK7 NtSK59 NtSK111 BnSKtheta ASKtheta PSK4 BAA92214 MSK3 MSK2 NtK1 MSK1 ASKgamma ASKalpha Dmsgg46 Dmsgg10 Dmsgg39 HsGSK3 RnGSK3alpha HsGSK3beta RnGSK3beta XGSK3beta XGSK3 ScMRK1 ScRIM11 ScMDS1

Figure 4-10. Phylogenetic tree of the catalytic domains of different GSK-3 members. The neighbor-joining algorithm (1000x bootstrapped) was used.

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Eleven individual transgenic lines containing a 3 Kb OsSK11 promoter fusion to the gusA reporter gene were obtained. Expression patterns of gusA were analyzed in calli and in mature plants. Our results indicate that OsSK11 is expressed in every organ throughout the life cycle of the plant, in accordance with other experiments. Transgenic rice harboring the gusA reporter gene driven by a 1 Kb fragment of the OsSK11 promoter region showed higher gusA expression in pollen at the singlenucleus stage and in node areas at the early flowering stage, while no gusA expression was observed in other tissues or organs tested. These results indicate that the 1 Kb fragment probably does not represent the full promoter. The most striking observation was that tillers were formed from non-tillering nodes in plants transformed with pSAN-H75.4 and pSAN-H75.16, however the phenotype was not maintained in the next generation. All ten transformants with aberrant sense OsSK11 cDNA in pSAN-H75.4, and 1 out of 6 transformants with the antisense construct pSAN-H75.16 formed tillers from nodes which are normally non-tillering. In the wild-type tillering normally occurs on nodes V and below (nodes VI, VII and so on). However in the transformed plants tillering also occurred on nodes IV and III. These results were consistent with the strong gusA expression obtained in the analysis of the 1 Kb promoter region. However, similar phenotypes were not obtained from the transformants with full-length sense OsSK11 in pSAN-K37A or in the control plants containing pCAMBIA1301. Twenty plants of the T1 generation of each candidate mutants grown under the same conditions, however, did not show a similar phenotype. Multiple experiments designed to disrupt endogenous OsSK11 expression by overexpression and gene silencing did not produced unusual tillerings patterns, even though one plant seemed to be a complete knockout. It is still not clear how the unusual but non-inheritable nor reproducible tillering phenotype. Environmental stress effects could be involved.

7. Conclusions During the course of this project hybrid rice production has continued to grow in various Asian countries and management of seed production has improveed to the point that the technology is now more amenable to countries where manual labor is expensive. Breaking the yield ceiling of selfpollinating varieties has become an even more desirable goal than it was before. In this context a twoline hybrid system has a major role to play, even more so before apomixis can be tamed to our advantage. The best strategy to manage a two-line system, whether to have a parent line with inducible or with default male sterility and restorable sexuality remains open to discussion. The best strategy will depend on penetrance of the trait and on crop management issues. Whichever the strategic decision, the tools developed in the course of this project will be a valuable asset to this end. Some of the highlights derived from this project are, (i) a reproducible and efficient genetic transformation protocol for local rice varieties; (ii) a set of plant transformation vectors with a wide range of applications, freely available to the research community; (iii) some molecular tools for field level glucuronide-responsive egg apparatus and microspore-specific gene expression; (iv) tools for transgene-activated procompund activation; (v) molecular tools to generate a better understanding of early meiotic processes and apomixis to make these amenable to directed intervention. The ongoing "Rice Transgenomics" project, partially funded by RIRDC, represents a logical continuation of the project just described, it is designed to capture in the most efficient way traits of interest for breeders that they can incorporate into their programs utilising all of the above technologies.

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8. Publications derived from this project Jefferson RA, Nugroho S (1998) Molecular strategies for hybrid rice: male sterility and apomixis. In, Advances in Technology: Proceedings of the 3rd Hybrid Rice Symposium Nov 1997 India (Virmani SS et al., eds) IRRI Los Baños, Laguna, Philippines. Jefferson RA (1999) The Roving Reporter: Harnessing jumping genes to build a biotech arsenal for rice. Farmer's Newsletter, Rural Industries Research and Development Corporation (Australia) Mayer JE, Jefferson RA (2000) Extra-terrestrial rice? Farmer's Newsletter, Rural Industries Research and Development Corporation (Australia) No. 154:16-19 Mayer JE, Kilian A, Jefferson RA (2001) Gene Tweezers. Farmer's Newsletter, Rural Industries Research and Development Corporation (Australia) (in press) Nguyen TA, Rajagopal S, Badger M, Kilian A, Keese PK, Jefferson RA (1999) A synthetic reporter gene encoding a novel Bacillus ß-glucuronidase: design, construction and expression. Proceedings of the Rockefeller Foundation Meeting in Phuket, Thailand. Nguyen TA, Wenzl P, Rajagopal S, Badger M, Kilian A, Mayer JE, Jefferson RA (1999) Biochemical characterisation and preliminary secretion analysis of a novel Bacillus ß-glucuronidase. Proceedings of the Rockefeller Foundation Meeting in Phuket, Thailand. Nguyen TA, Wenzl P, Rajagopal S, Kilian A, Mayer JE, Keese P, Badger M, Jefferson RA. An improved reporter system based on a novel microbial β-glucuronidase (in preparation) Nugrohu S (2001) Physical and functional analysis of a kinase gene family from rice with high sequence homology to RIM11, the principal meiotic regulatory gene in yeast Saccharomyces cerevisiae. PhD thesis, ANU Australia Nugroho S, Keese PK, Desamero NV, Kilian A, Jefferson RA (1997) Molecular strategies for apomictic rice I: Isolation and analysis of genes controlling the meiotic process. Proceedings of the Rockefeller Foundation Meeting in Malacca, Malaysia. Nugroho S, Keese P, Kilian A, Desamero NV, Cohn PCL, Jefferson RA (1999) Molecular and cellular analysis of a rice gene related to a yeast meiotic regulatory gene, RIM11. Proceedings of the Rockefeller Foundation Meeting in Phuket, Thailand. Ravi KS, Jefferson RA (1997) Development of a glucuronide responsive promoter for field level control of transgene expression. Proceedings of the Rockefeller Foundation Meeting in Malacca, Malaysia. Roberts C, Rajagopal S, Smith LM, Nguyen TA, Yang W, Nugrohu S, Ravi KS, Vijayachandra K, Harcourt RL, Dransfield L, Desamero N, Slamet I, Hadjukiewicz P, Svab Z, Maliga P, Mayer JE, Keese PK, Kilian A and Jefferson RA (2000). pCAMBIA binary vectors: A comprehensive set of modular vectors for advanced manipulations and efficient transformation of plants. Genbank acc nrs AF234290-AF234316 Yang W (2001) Analysis of an egg-apparatus-specific regulatory element (enhancer) from Arabidopsis thaliana. PhD thesis ANU, Australia Yang W, Kilian A, Keese PK, Grossniklaus U, Jefferson RA (1997) Molecular strategies for apomictic rice II: Isolation and analysis of megagametophyte-specific regulatory elements and genes. Proceedings of the Rockefeller Foundation Meeting in Malacca, Malaysia. Yang W, Rajagopal S, Kilian A, Badger M, Grossniklaus U, Jefferson RA (1999) Characterisation of a megagametophyte-specific enhancer from Arabidopsis thaliana and its possible applications in rice. Proceedings of the Rockefeller Foundation Meeting in Phuket, Thailand.

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