A NOVEL MECHANISM UNDERLYING PROGRAMMED CELL DEATH IN
PLANT DEFENSE SIGNALING
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of the Ohio State University
By
Li-Rong Zeng, M.S. (Biochemistry)
* * * * *
The Ohio State University
2005
Dissertation Committee:
Professor Guo-Liang Wang, Adviser Approved by
Professor Wolfgang D. Bauer Guo-Liang Wang
Professor Terrence L. Graham Adviser
Professor Jyan-chyun Jang Graduate program in Plant Pathology
ABSTRACT
During plant-microbe interactions, hypersensitive response (HR) cell death is intimately associated with plant disease resistance. In this study, rice lesion mimic mutant spotted leaf11 (spl11) was used to investigate the molecular mechanism underlying PCD in plant disease resistance. spl11 plants expressed constitutive cell death and showed enhanced, non-race specific resistance to both M. grisea and Xanthomonas oryza pv. oryza (Xoo) pathogens. Genome-wide gene expression profiling indicated that the spl11 mutation caused significant changes in rice transcriptome. Of the genes that were more than 3.5-fold induced in spl11, over 50 percent were either defense- or oxidative stress/cell death-related, indicating that cell death and defense signaling is activated by the spl11 mutation.
spl11 was identified from an ethyl methanesulfonate (EMS)-mutagenized IR68 population and was shown to be inherited in a recessive monogenic fashion. The Spl11 gene was isolated via a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection. The predicted SPL11 protein contains a U-box domain and an armadillo
(ARM) repeat domain, which were demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein–protein interactions, respectively. A single base
ii substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. Expression analysis indicated that Spl11 is induced in both incompatible and compatible rice-blast interactions. In vitro ubiquitination assays indicated that the SPL11 possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting the ubiquitination system plays a role in the control of plant cell death and defense.
Eight SPL11-interacting proteins (SPINs) were identified in the yeast two-hybrid screenings. Two of the SPINs were putative pre-mRNA processing-related proteins, suggesting a connection between alternative splicing and Spl11-mediated cell death and defense signaling. Seventy-seven and sixty-three U-box containing proteins were identified from rice and Arabidopsis genomes, respectively. Atspl11, a T-DNA insertion mutant of the Arabidopsis Spl11 ortholog displayed cell death and stunted growth phenotype analogous to that of rice spl11 mutant, suggesting that the function of the
Spl11 gene might be conserved between monocots and dicots.
iii
Dedicated to my parents
iv ACKNOWLEDGMENTS
I would like to thank my adviser, Dr. Guo-Liang Wang, for his patient guidance and continuous intellectual support throughout the program. I also want to express my gratitude to the members of my Student Advisory Committee, Dr. Wolfgang D. Bauer,
Dr. Terrence L. Graham, and Dr. Jyan-chyun Jang for many helpful discussions and suggestions on my doctoral research. They helped edit this manuscript. My very special thanks to Dr. Wolfgang D. Bauer for allowing me to use the equipment in his lab and for teaching me many things about academic life. I am indebted to my wife, Lisha, for her love, encouragement, and meticulous support of my study. I am grateful to my parents,
Xinfa Zeng and Yuhua Yang, my family and friends for their encouragement and moral support during this study.
Part of the research was funded by Ohio Agricultural Research and Development
Center (OARDC) Graduate Studies Enhancement Grant. I am grateful to Dr. Gurdev S.
Khush at International Rice Research Institute (IRRI) for providing us the spl11 mutant.
Some of the work presented in this dissertation was completed either in collaborations with other research groups or by the help of other people. I would like to thank all of them. For the genome-wide gene expression profiling, Dr. Tong Zhu and his group from the Syngenta Company helped me do the microarray hybridization and allowed me to use some of their software to analyze the hybridization data. The sequencing of the BAC v clone 78I19 during the map-based cloning of the Spl11 gene was conducted in Dr. Baek
Hie Nahm’s lab at Myongji University, Korea. The in vitro E3 ubiquitin ligase activity assay of SPL11 was conducted by Dr. Qi Xie’s group at Zhongshan University (now at
Institute of Genetics and Developmental Biology, CAS), China. Dr. Hei Leung at IRRI provided us the three IR64-background spl11 alleles and his group performed the allelic test of the IR64-backgroud spl11 mutants. Former postdoc Dr. Shaohong Qu and current lab technician Mrs. Maria Bellizzi helped me finish the rice transformation work in spl11 complementation test. Mrs. Ramya Rajappan helped me in the yeast two-hybrid screening for SPL11 interactors. Mr. Julian Gough from the Riken Institute, Japan helped me a lot in the identification of U-box genes from rice and Arabidopsis genomes.
vi VITA
1992 B. S., Agronomy, Hunan Agricultural University, China
1997 M. S., Biochemistry, Sun Yat-sen (Zhongshan) University, China
1998-1999 Junior Research Fellow, Institute of Molecular Agrobiology,
The National University of Singapore, Singapore
2000-2005 Graduate Research Associate, The Ohio State University,
Columbus, OH
Publications:
1. Zeng, L., Zhang, E-X., Lin, Z-F., Yu L-J. 1998. Purification and Characterization of Copper-Zinc Superoxide Dismutase from Ostrea rivularis Gould. Chinese J. Biochem. Mol. Biol.14: 583-587
2. Zeng, L, Zhang, E-X., Lin, Z-F., Yu, L-J., Xiao, X. 1999. Studies on stability of Cu, Zn-SOD in Grassostrea rivularis J. Oceanography Taiwan Strait. 18: 87-91
3. Wang, G-L., He, C.Z., Wu C.J., Yin, Z.C., Baraoidan, M., Zeng, L., Ronald, P.C., Khush, G.S. and Leung, H. 1999 Genetic Dissection of Disease Resistance Pathways in Rice. Adv. Rice Blast Res. eds: M.H. Lebrun, J.L. Notteghem, N.J.
4. Yin, Z., Chen, J., Zeng, L., Goh, M., Leung, H., Khush, G.S. and Wang, G-L (2000) Characterization of rice lesion mimic mutants and identification of a mutant vii with broad-spectrum resistance to rice blast and bacterial blight. Mol. Plant-Microbe Interact.13: 869-876
5. Liu, G., Lu G., Zeng, L., Wang, G-L. (2002) Two broad-spectrum blast resistance genes, Pi9(t) and Pi2(t), are physically linked on rice chromosome 6. Mol Genet Genomics. 267: 472-80
6. Zeng, L., Yin, Z., Chen, J., Leung, H. and Wang G.-L. (2002) Fine genetic mapping and physical delimitation of the lesion mimic gene Spl11 to a 160-kb DNA segment of the rice genome. Mol Genet Genomics. 268: 253-261
7. Wang, G-L, Wu, C., Zeng, L., C. He, M., Baraoidan, F., Silva, de Assis Goes da, Williams, C. E., Ronald, P. C. and Leung, H. (2004) Isolation and characterization of rice mutants compromised in Xa21 -mediated resistance to X. oryzae pv. Oryzae Theor. Appl. Genet. 108 379-3848
8. Zeng, L.R., Qu, S., Bordeos, A., Yang, C., Baraoidan, M., Yan, H., Xie, Q., Nahm, B.H., Leung, H., and Wang, G.-L. (2004). Spotted leaf11, a Negative Regulator of Plant Cell Death and Defense, Encodes a U-Box/Armadillo Repeat Protein Endowed with E3 Ubiquitin Ligase Activity. Plant Cell 16, 2795-2808.
FIELD OF STUDY
Major Field: Plant Pathology
Graduation Specialization: Plant Molecular Biology and Biotechnology
viii TABLE OF CONTENTS
Content Page
Abstract ...... ii
Dedication ...... iv
Acknowledgments...... v
Vita ...... vii
List of Tables ...... xi
List of Figures ...... xii
Abbreviations……………………………………………………………………….xiv
Chapters:
1. Introduction Programmed cell death (PCD): a general perspective………………………...1 PCD in plant development and defense ………………………………………5 Use of lesion mimic mutants (LMMs) to study plant defense-related PCD…..7 Rice lesion mimic mutant spotted leaf11 (spl11)……………………………..9 Significance and objectives of this study…………………………………….11
2 The spl11 mutation causes significant changes in rice transcriptome Introduction …………………………………………………………………..17 Materials and methods……………………………………………………....19 Results………………………………………………………………………...21 Discussion…………………………………………………………………….24
3 Fine genetic mapping and physical delimitation of the lesion mimic gene Spl11 to a 160 kb DNA segment of the rice genome Introduction……………………………………………………………….…..33 Materials and methods……………………………………..………………....36 ix Results………………………………………………………………………...39 Discussion…………………………………………………………………….44
4 Spl11 encodes a U-box/ARM repeat protein endowed with E3 ubiquitin ligase activity Introduction……………………………………………………………….…..56 Materials and methods……………………………………..………………....59 Results………………………………………………………………………...65 Discussion…………………………………………………………………….75
5 Identification of SPL11-interacting proteins Introduction……………………………………………………………….…..94 Materials and methods……………………………………..………………....95 Results………………………………………………………………………...99 Discussion……………………………………………………………………101
6 Genome-wide identification of rice and Arabidopsis U-box genes and isolation of a mutant for the Arabidopsis Spl11 ortholog Introduction……………………………………………………………….….111 Materials and methods……………………………………..………………...114 Results………………………………………………………………………..117 Discussion……………………………………………………………………122
7 A novel mechanism underlying programmed cell death in plant disease resistance……………………………………………………………………..139
Bibliography………………………………………………………………………….145
x LIST OF TABLES
Table Page
1 Genes ≥ 3.5-fold up-regulated at different leaf lesion development
stages in spl11 ………………………………………………………………..27
2 Genes ≥ 3.5-fold down-regulated at different leaf lesion development
stages in spl11………………………………………………………………....31
3 PCR-based markers for genetic and physical mapping of Spl11……………..49
4 Allelism tests between spl11 and three IR64 lesion mimic mutants…………79
5 U-box-ARM proteins identified in the rice full-length cDNA
database KOME……………………………………………………………….80
6 SPL11-interacting proteins identified in the yeast two-hybrid
screening……………………………………………………………………..104
7 Rice U-box gene names, their correspondence to sub-class, rice genome
locus, rice chromosome, GenBank protein IDs, and accessions for their
cDNA and cognate ESTs…………………………………………………….125
8 Domain organization of rice and Arabidopsis U-box proteins………………129
9 Arabidopsis U-box gene names, their correspondence to sub-class,
Arabidopsis genome locus, GenBank protein accession, and
accessions for their cDNA and cognate ESTs………………………………..130
xi LIST OF FIGURES
Figures Page
1 Hypothetical model of the common framework in plant PCD Signaling ………12
2 spl11 conferred enhanced resistance to rice blast and bacterial blight…………..14
3 Expression of defense-related genes in the spl11 mutant………………………..15
4 Number of genes with more than two-fold induced or suppressed in
the spl11 mutant………………………………………………………………….26
5 Screening for spl11-linked RAPD markers……………………………………...50
6 Mapping of spl11-linked RAPD markers to the rice genetic linkage
map using MAPMAKER 3.0……………………………………………………51
7 Genetic map and BAC contig of the Spl11 locus………………………………..52
8 Determination of the insert size of BAC68 and BAC78 using pulse
field gel electrophoresis (PFGE) analysis……………………………………….53
9 A NotI physical fine map of the BAC78 clone delimiting Spl11 on a DNA
interval of 160 kb……………………………………………………………..…54
10 Physical delineation of the spl11 mutation………………………………….…..81
11 Analysis of IR64 lesion mimic mutants allelic to spl11……………………….…..84
12 Functional complementation test of Spl11 candidate gene……………………..85
13 Transcript abundance of Spl11 in different tissues, gene structure, and
deduced amino acid sequence of Spl11………………………………………86 xii 14 Amino acid sequence alignments between SPL11 and U-box-ARM
proteins from other plant species……………………………………………….88
15 Sequence alignment between the ARM repeats of SPL11 and other
plant U-box-ARM repeat proteins………………………………………………90
16 E3 ubiquitin ligase activity of SPL11…………………………………………...91
17 Expression patterns of Spl11 in rice-blast interaction…………………………..92
18 Basis of the ProQuest Y2H system…………………………………….………105
19 Protein sequence alignments of SPIN1 rice homologs………………………...107
20 Expression patterns of SPIN1 in rice-blast interaction………………………...109
21 A putative conserved domain located closely to the C terminus of
Arabidopsis U-box-Leucine-rich type U-box proteins………………………...135
22 Phylogenetic relationship between rice and Arabidopsis U-box/ARM
repeat proteins………………………………………………………………....136
23 Atspl11 displays cell death and stunt growth phenotype……………………...138
xiii ABBREVIATIONS
Hypersensitive response: HR
Programmed cell death: PCD
Xanthomonas oryza pv. oryza: Xoo
Spotted leaf: Spl
Lesion mimic mutant: LMM
Yeast two-hybrid: Y2H
Salicylic acid: SA
xiv CHAPTER 1
INTRODUCTION
The main goal of my doctoral research was to study programmed cell death
(PCD) in the context of plant disease resistance. A lesion mimic mutant from rice, spotted leaf11 (spl11) was chosen for the investigation. Six chapters of this dissertation deal with various aspects of the molecular mechanisms underlying cell death and enhanced disease resistance in spl11. Global transcriptional changes detected in the spl11 mutant are presented in Chapter 2. Molecular cloning of the Spl11 gene and functional characterization of SPL11 are presented in Chapters 3 and 4. Screening for SPL11- interacting proteins (SPINs) via the Y2H and preliminary analysis of some of these
SPINs are presented in Chapter 5. Genome-wide analysis of rice and Arabidopsis U-box gene family and identification of a T-DNA insertion mutant of Arabidopsis Spl11 homolog that displays cell death phenotype are presented in Chapter 6. Relevant background information of this research is included in this chapter.
Programmed cell death: a general perspective
Cell death, like cell division and cell differentiation, is integral to the life of multicellular organisms. Generally, cell death could occur by one of two mechanisms, 1 physiological cell death or non-physiological cell death (Vaux and Korsmeyer, 1999).
Physiological cell death, also termed as PCD, refers to an active process encoded by the organism to kill its own cells on purpose. The great majority of metazoan and plant cells are destined to die by such a mechanism. Only relatively few die by non-physiological cell death, which occurs when a cell is injured by external events, such as poisons or lack of nutrients, or when intrinsic defects lead to the inability of a cell to sustain its own viability. The term “programmed cell death” was first coined in 1965 to describe cell death in insect metamorphosis (Lockshin and Williams, 1965). This occurred over 100 years after the first recognition that cell died in a predictable “programmed” fashion under physiological circumstance in neuronal system of developing toad embryos (see review of Vaux and Korsmeyer, 1999). In the early 1970s, Kerr and colleagues discovered that the morphology of dying liver cells in response to pathogenic stimuli is same as those in response to physiologic stimuli in embryo development and termed such type of cell death as apoptosis (Kerr et al., 1972). This pioneer work in characterizing apoptosis as “a basic biological phenomenon with wide-ranging implication in tissues kinetics” opened an active field of study from which significant insights have been gained into the components and signaling network implicated in PCD (Strasser et al.,
2000). Now it is well accepted that PCD is a common cellular mechanism that is triggered to sculpt of structure, to delete unneeded structure, to control cell numbers, to eliminate abnormal or harmful cells and to produce differential cells without organelles.
In metazoans, apoptosis is the type of PCD that received most attention in the past several decades. The word apoptosis was derived from two Greek roots: apo (away) and ptōsis (to fall) that was used to describe the falling of petals from flowers or leaves from
2 trees. Cells undergoing apoptosis display a series of characteristic morphological and biochemical changes, including cytoplasmic shrinkage, nuclear condensation, plasma membrane blebbing, loss of cell-to-cell contact in organized tissues, exposure of phosphotidyl serine, activation of cysteine proteases and DNA fragmentation, first to 50-
Kb fragments and then to nucleosomal ladders (Strasser et al., 2000). Some of these changes, such as DNA fragmentation, have been frequently used as markers in determining if a cell is undergoing apoptosis. Interestingly, most, if not all the above- mentioned morphological and biochemical characteristics have also been detected in plant PCD, though not all of the events were demonstrated in the same plant system
(Levine et al., 1996; Wang et al., 1996; Young and Gallie, 1999). Apoptosis is involved in both normal animal development and defense against pathogen infection (Gilchrist,
1998; Vaux and Korsmeyer, 1999). Abnormalities in apoptosis regulation can cause diseases such as cancer, autoimmunity and neurodegenerative disorders including
1Alzheimer’s, Parkinson’s and retinitis pigmentosa (Thompson, 1995; Meier et al., 2000;
Green and Kroemer, 2004), indicative of the significant impact of apoptosis on cell production and cell homeostasis. Multiple signaling pathways that are initiated either from triggering events within the cell or from outside the cell have been identified in apoptosis (Hengartner, 2000). Distinct pathways converge at the activation of caspases, cysteine proteases that recognize tetrapeptide motifs and cleave at the carboxyl side of an aspartate residue. Caspases are highly conserved through evolution and can be found from nematodes, insects to humans (Budihardjo et al., 1999). Nevertheless, homologues of caspase genes have not been identified in Arabidopsis genomes (Koonin and Aravind,
2002), even though several functionally caspase-like genes were identified recently
3 (Vercammen et al., 2004; Watanabe and Lam, 2005). Plant proteases that are structurally non-orthologous but functionally equivalent to caspases from metozoans, therefore are likely to be involved in plant PCD (Solomon et al., 1999; Zhao et al., 1999). In addition to caspases, other major functional groups of molecules involved in triggering and affecting the apoptotic process include the adaptor proteins, which control the activation of initiator caspases, members of the tumor necrosis factor receptor (TNF-R) super family, and members of the Bcl-2 family of proteins (Strasser et al., 2000). Proteins of the Bcl-2 family possess either pro- or anti-apoptotic activity and play an important role in modulating apoptosis (Kelekar and Thompson, 1998).
Whether apoptotic machinery exists in plants remains elusive. Besides caspases, homologues of most members in the Bcl-2 family have yet been identified in plants. The absence of two core components of animal apoptotic machinery in plants makes it a favorable assumption that such cell death machinery might not really exit in plants. The report of caspase-independent PCD in Dictyostelium suggested the existence of cell death program(s) other than apoptosis (Olie et al., 1998). Indeed apoptosis is even not the only type of PCD in animals (Schwartz et al., 1993). The difference between plant and animal cell structure, including cell wall, chloroplast, and vacuolar that are unique to plant cells and the difference in plant and animal life styles reasonably assure different mechanisms in plant cellular suicide to get adapted to specific features of plant development and defense. A unique role of a large lytic vacuole in the execution of plant PCD, especially in the development of tracheary elements (TEs) and the wide involvement of plant hormones in mediating plant PCD provided evidences in supporting such concept
(Kuriyama and Fukuda, 2002; Hoeberichts and Woltering, 2003).
4 PCD in plant development and defense
Despite divergent mechanisms underlining plant and animal PCD, the universal involvement of PCD in development and defense against pathogen infection is similar in plants and animals (Greenberg, 1996). Examples of plant developmental PCD include leaf and petal senescence, PCD during seed production and germination (such as the removal of embryonic suspensors and aleurone layers), ontogenesis of xylem of vascular bundles (vessels and tracheids), depletion of tissues of reproductive organs (such as stomium, tapetum, and ovaries, the reproductive organ primordia of dioecious plants),
PCD in root caps, and in cortex that is forming aerenchyma (Pennell and Lamb, 1997).
Studies from these systems indicated that the vacuole plays a central role in plant developmental PCD, and plant hormone signaling pathways were critical in transducing of PCD signals (Yamamoto et al., 1997; Gilchrist, 1998; Young and Gallie, 2000; Fath et al., 2001; Jones, 2001; Woo et al., 2001). In most cases, more than one plant hormone act in conjunction in the same developmental PCD system. It is believed that plant hormones may exert their respective effects on plant PCD through the regulation of reactive oxygen species (ROS) accumulation (Hoeberichts and Woltering, 2003). Other components that are important in signaling developmental PCD include calcium fluxes (Wu and Cheun,
2000) and ROS (Fath et al., 2000; Young and Gallie, 2000).
During plant-microbe interactions, cell death occurs both in the Hypersensitive
Response (HR) of resistant host and non-host plants and in susceptible plants under virulent pathogen attack (Greenberg, 1997). The HR occurs at the site of pathogen entry and involves cell death in and around the infection site. HR requires active plant cell metabolism and is defined as a type of PCD (Heath, 2000). The role of HR cell death in
5 plant defense against pathogen attack is inconclusive. Correlation between HR and plant disease resistance has been observed in most incompatible plant-pathogen interactions
(Richberg et al., 1998; Shirasu and Schulze-Lefert, 2000). In several studies, however, the HR cell death could be separated from defense gene induction and disease resistance
(Jakobek and Lindgren, 1993; del Pozo and Lam, 1998; Bendahmane et al., 1999; Shirasu and Schulze-Lefert, 2000). It is now generally accepted that the function of HR cell death in plant-microbe interaction may vary depending on the nature of plant-pathogen system.
Upon the infection by biotrophic pathogens, HR cell death can limit the supply of nutrients to the pathogens thus arrest the growth and development of the pathogen at the infection site (Greenberg, 1997; Delledonne et al., 2001). In case of necrotrophic pathogens, however, occurrence of cell death may be used by the pathogen to aid in their invasion of the plant (Govrin and Levine, 2000). Nevertheless, in both cases, HR could act as a signal to the rest of the plant to activate systemic defense rather than simply as a localized direct defense mechanism. Studies indicated that HR is closely associated with the conditioning adjacent cells to become responsive to pathogen elicitors (Graham and
Graham, 1999) and with the activation of systemic resistance throughout the plant (Hunt et al., 1996; Molina et al., 1999; Costet and Kauffmann, 2000). Molecular events, including ion fluxes, generation of ROS (Laloi et al., 2004) and nitric oxide (NO)
(Delledonne et al., 1998; Delledonne et al., 2001), expression of defense-related genes, production of anti-microbal molecules, and formation of cell wall apposition, have been shown to be correlated with the HR in disease resistance (Yang et al., 1997; Heath,
2000). The consistent association with the induction of local and systemic defense responses clearly defines a major difference between HR and developmental PCD.
6 However, the conservancy of certain cellular events occurred in these two types of plant
PCD suggested a common framework in signaling and execution of plant PCD (Jones,
2001; Hoeberichts and Woltering, 2003) (Figure 1).
Besides resistant plant-pathogen interaction, cell death also occurred in susceptible plants in which the pathogen can replicate well (Greenberg and Yao, 2004).
Cell death under such circumstances could function to promote pathogen growth, which is especially true for pathogens that secrete toxins to kill host cells (Stone et al., 2000;
Lincoln et al., 2002). During the cell death of susceptible plant-microbe interactions, some events occurred are encoded by the host. However, whether such cell death is a type of PCD remains elusive.
Use of lesion mimic mutants (LMMs) to study plant defense-related PCD
Various approaches at histological, cytological, genetic, molecular and biochemical levels have been employed in the study of plant PCD. The early years of plant PCD research were mainly focused on histological and cytological description
(Jones, 2001). These provided essential background for later studies aimed at understanding the mechanisms underlying plant PCD. In genetic analysis of plant defense-related PCD, a widely used method is to identify and characterize mutants displaying cell death phenotype in the absence of pathogen challenge. These mutants are collectively designated as “lesion mimic” based on their spontaneous lesion phenotypes similar to HR or disease symptoms (Hoisington et al., 1982; Dangl et al., 1996). The hypothesis behind this method is that genes defined by some of these mutations may function in PCD control in wild-type plants.
7 A large number of lesion mimic mutants have been identified from Arabidopsis
(Dietrich et al., 1994; Greenberg et al., 1994; Weymann et al., 1995; Bowling et al.,
1997; Mou et al., 2000), barley (Wolter et al., 1993), maize (Walbot et al., 1983; Johal et al., 1995), and rice (Kiyosawa, 1970; Leung et al., 2001; Takahashi et al., 1999; Yin et al., 2000). Only part of them display altered disease resistance, suggesting that some of the mutations either reside in the developmental cell death pathway or simply represent certain physiological perturbations irrelevant to plant defense. Different LMMs could possess different lesion phenotypes with respect to the color, size, timing, and conditions of lesion appearance albeit they collectively share the same name. In the early 1980s, it was assumed that two different mechanisms were in charge of controlling cell death in these mutants, pathways for initiation or suppression of PCD. Based on this assumption,
LMMs were classified into two groups, initiation mutants and propagation mutants: initiation mutants form localized necrotic spots of determinate size whereas propagation mutants are unable to control the rate and extent of the lesions (Walbot et al., 1983). In
Arabidopsis, nearly 40 LMMs that are grouped as either initiation mutants or propagation mutants have been identified (Lorrain et al., 2003). Among them, over a dozen genes were cloned. The proteins encoded by these genes are predicated to be involved in various cellular processes. Genetic analysis of these mutations with known components in plant defense signaling has gained knowledge of cross-talks between multiple plant defense pathways that could not be achieved in studying the wild-type plants. In addition to LMMs from Arabidopsis, LMM genes from other plant species were also cloned. The barley mlo mutant confers non-race-specific resistance to the powdery mildew fungus before the occurrence of the lesion mimics. Mlo is predicted to be a membrane-associated
8 protein with seven integral transmembrane-spanning helixes (Büschges et al., 1997). It is speculated that Mlo may represent a novel molecular mechanism by which the production of reactive oxygen intermediates is negatively regulated in plants (von Ropenack et al.,
1998; Devoto et al., 1999). The maize Lls1 is the third cloned lesion mimic gene that displays elevated resistance to fungal pathogens (Gray et al., 1997; Simmons et al.,
1998). The predicted Lls1 protein contains two conserved motifs resembling those found in aromatic ring-hydroxylating dioxygenases. Thus, it is thought that Lls1 functions to degrade a toxic phenolic compound or a phenolic signaling molecule that promotes PCD in certain circumstances. In addition to genes affecting the putative signaling components, disruption of certain metabolic processes also induces cell death in plants.
Disruption of the maize Les22 gene, which encodes uroporphyrinogen decarboxylase in the plant chlorophyll and heme biosynthetic pathway produces minute necrotic spots on the leaves (Hu et al., 1998).
Rice lesion mimic mutant spotted leaf11 (spl11)
In rice, lesion mimic mutations are manifested as spotted leaves; hence, most of the lesion mutants (but not all) are called spotted leaf (spl). Other rice LMMs include cdr
(cell death and resistance) mutants that exhibited significant resistance to the blast fungus, M. grisea (Takahashi et al., 1999). Two of the cdr mutants, cdr1 and cdr2 produced higher levels of H2O2 than the wild type when treated with calyculin A, an inhibitor of protein phosphatase 1, suggesting the involvement of these genes in regulating ROS production. Proteomic analysis indicated that defense-related proteins were induced in the cdr2 mutant (Tsunezuka et al., 2005). Recently, more LMMs
9 displaying various cell death phenotypes were identified in an IR64 deletion mutant collection (Leung et al., 2001).
The intimate involvement of cell death in plant-microbe interaction guarantees that an in-depth comprehension of cell death in the context of plant defense will help control plant diseases eventually. In light of this principle, we have characterized nine rice spl mutants (spl1, spl2, spl3, spl4, spl5, spl6, spl7, spl9, spl11) regarding their disease reactions to rice bacterial and fungal pathogen attack (Yin et al., 2000). Among the nine mutants, spl11 was the only one that conferred enhanced, non-race specific resistance to both rice blast and bacterial blight diseases. When inoculated with blast isolates from Philippines (PO6-6, 9232-5, C9214-29, and CBN9219-25), China (97-55-2,
97-104-2, 97-5-1, and 54-04), Portugal (PR-3), and Colombia (CL6), the average number of disease lesions varied from 1 to 7 per plant in the spl11 (LM+) mutants and from 7 to
34 in wild-type (LM-) plants, depending on the isolates examined (Figure 2A). Compared to the wild-type plants, the size of disease lesions on the mutants was smaller and they formed only at the tip of the newly developed leaves where no visible lesion mimic had developed. The attenuation in disease development was also observed in the mutants after bacterial pathogen treatment. The average lesion length ranged from 1.0 to 6.6 cm in lesion mimic mutants and from 4.0 to 25.2 cm in wild-type plants for the different Xoo isolates (Figure 2B). Moreover, The enhanced resistance of the spl11 mutant to the two pathogens correlates with the constitutive expression of several defense-related genes and lesion development on the leaves (Figure 3). These results suggested that the Spl11 gene might be involved in suppression of both cell death and induction of defense in rice.
10 Significance and objectives of this study
The wide involvement of PCD in plant development and defense against pathogen infections necessitates the study of plant PCD from the viewpoint of both pure and applied plant science. The intimate association of HR cell death to plant disease resistance suggests that an in-depth comprehension of PCD in the context of plant defense will help control plant diseases eventually. Rice disease is one of the main constraints on stable rice production each year worldwide (Baker et al., 1997). Rice is the staple food resource for more than half of the world population. Understanding the mechanism underlying rice disease resistance and engineering rice varieties resistant to pathogen infections is, therefore vital to feed the increasing human population.
The spl11 mutant used in this study expressed constitutive cell death and conferred enhanced resistance to both fungal and bacterial rice pathogen infections (Yin et al., 2000). It is hypothesized that the wild-type Spl11 gene might be a component of the PCD pathway that is associated with plant disease resistance. The main goal of this study is to understand the molecular mechanism underlying Spl11-mediated cell death and defense signaling. Specifically, this doctoral research attempted to answer two questions; 1) what the Spl11 gene encodes for?, and 2) why mutation of the Spl11 gene results in the constitutive expression of cell death and enhanced disease resistance in rice?
11
Figure 1. Hypothetical model of the common framework in plant PCD Signaling
(modified from Hoeberichts and Woltering, 2003). Cells integrate various signals to decide whether to die and subsequently how the corpse will be managed. Upon perceiving death signal(s) from either within or outside the cell, a signaling cascade cumulating at the collapse of the vacuole is initiated. Among the events in transducing death signal, calcium flux, production of ROS, and collapse of vacuole are shared by most plant cell deaths. Other divergent events in signal transduction include protein phosphorylation and dephosphorylation, change of mitochondrial membrane potential and release of pro-death factors (Yao et al., 2004), protease cleavages, ubiquitination 12 (Woo et al., 2001) and plant hormone modulations etc. The participated molecular events and the timing, location, and interaction of these events differ among cell death programs.
MAPK, mitogen-activated protein kinase; CDPK, calmodulin-like domain protein kinase;
ROS, reactive oxygen species; NO, nitric oxide; ABA, abscisic acid; GA, gibberellin; JA, jasmonic acid; SA, salicylic acid; ET: ethylene.
13
Figure 2. spl11 conferred enhanced resistance to rice blast and bacterial blight. A,
Average blast lesion number per plant on leaves of LM+ plants and LM- plants after they were inoculated with 10 blast isolates. B, Average lesion length of bacterial blight disease
(from 15 leaves) on leaves of LM+ plants and LM- plants after they were inoculated with four bacterial blight races. Standard errors are indicated (Yin et al., 2000). LM+, lesion mimic plant, LM-, wild-type plant.
14
Figure 3. Expression of defense-related genes in the spl11 mutant. Expression of defense-related genes during lesion mimic development. Total RNAs were extracted from newly developed young leaves (less than 1 week old) without visible lesion mimic
(YL), fully expanded leaves (2 weeks old) with few lesion mimics (FL), and old leaves (4 weeks old) with many lesion mimics (OL) of spl11 and leaves of wild-type IR68 at the corresponding developmental stage. PR1, pathogenesis-related 1; PBZ1, probenazole (3- allyloxy-1, 2-benzisothiazole-1,1-dioxide) induced 1; HvOxOa, barley (Hordeum vulgare) oxalate oxidase gene; HvOxOLP , barley oxalate oxidase-like gene; POX22.3 and POX8.1, peroxidase genes (Yin et al., 2000).
15 CHAPTER 2
THE spl11 MUTATION CAUSES SIGNIFICANT CHANGES IN RICE
TRANSCRIPTOME
ABSTRACT
spl11 plants conferred increased resistance to the infections by M. grisea and Xoo, the pathogens that cause rice blast and bacterial blight diseases, respectively. Northern blot analysis indicated that several defense related genes were induced in the mutant, suggesting that defense signaling was activated in spl11. In this study we performed an array-based expression profiling using the rice Genechips that harbor nearly half of all rice genes to analyze the impact of spl11 mutation on rice global gene expression. Rice leaves at three different stages with respect to lesion development were examined.
Compared to wild-type plant, significant changes in the rice transcriptome were detected in spl11 mutant. Among the various genes induced in the mutant most were oxidative stress /cell death- or defense-related genes. No well-defined groups of target genes for other hormonal or cellular responses were identified, suggesting that the Spl11 might not be a physiological homeostasis disturber but a component in the plant PCD pathway.
16 INTRODUCTION
The mutations manifested in LMMs could simply cause physiological perturbation in the cell rather than be a component of a programmed pathway leading to cell death. Unfortunately, we are unable to distinguish clearly between these two possibilities based simply on cell death phenotype. This question is pertinent to all lesion mimic mutants and has not been rigorously addressed in any case (Brodersen et al.,
2002). In multicellular organisms, non-physiological cell death could occur under various circumstances, such as cells injured by poisons or lack of nutrients, or a mutation in an essential enzyme or expression of an altered gene product that is toxic to the cells (Vaux and Korsmeyer, 1999). In this sense, the cell death caused by some, if not most lesion mimic mutations could be classified as non-physiological cell death and the gene they encoded in the wild-type plants may not really reside in the PCD pathways (Mou et al.,
2000; Lorrain et al., 2003). It is surmised that such mutation could cause significant transcriptional changes not only in cell death-related genes but also genes related to the physiological process in which the LMM gene resides. A global examination of the transcriptional status in the LMMs and the wild type plants therefore will facilitate not only in determining if a LMM gene is a PCD-related gene but in gaining information related to the putative function the LMM gene serves in the cell (Brodersen et al., 2002).
To date, several signaling molecules, such as salicylic acid (SA), jasmonic acid
(JA), ethylene, ROS, and NO (nitric oxide) are known to play critical roles in HR and disease resistance (Dong, 1998; Thomma et al., 2001; Laloi et al., 2004). By means of genetic crosses of LMMs to mutants altered in these signaling pathways, information of
17 the cross-talks between different pathways in HR and disease resistance have been obtained (Lorrain et al., 2003). In addition to the genetic method, genome-wide gene expression profiling could provide helpful knowledge as well. Even for those lesion mimic mutations that affect plant cell physiology but are unrelated to disease defense responses, this global transcriptional profiling could also be a useful tool in identifying novel components of other important developmental or physiological processes in plants.
Microarray technology provides biologists with the ability to measure the expression levels of thousands of genes in a single experiment (Reymond et al., 2000).
Thus, the array technologies offer the possibilities to study the impacts of a mutation on global gene expression. To compare the expression of all the arrayed DNA sequences, the cDNA probes of two samples from the mutant and wildtype are labeled using different fluorescent dyes. A mixture of the equal amount of labeled cDNAs is used in hybridization with the arrayed DNA or oligonucleotide spots on the slides. After hybridization, the slides are scanned with a laser microscopic scanner to measure the fluorescence of each spot for the determination of relative abundance of each specific gene in the two samples. This technology has been successfully used in the genome-wide analysis of gene expression in various plant systems (Himanen et al., 2004; Leonhardt et al., 2004; Schnable et al., 2004; Weber et al., 2004; Yamauchi et al., 2004).
As discussed in Chapter 1, spl11 plants conferred increased resistance to the infections by M. grisea and Xoo, the pathogens that cause rice blast and bacterial blight diseases, respectively. Northern blot analysis indicated that several defense related genes were induced in the mutant. These data suggested the activation of defense signaling alone with the induction of cell death in the mutant. However, what cell death and
18 defense-related genes are induced in spl11 and whether the signaling for other cellular processes is also affected in the spl11 mutant is unclear. In this study we performed an array-based expression profiling using the rice Genechips that harbor nearly half of all rice genes to analyze the impact of the spl11 mutation on rice global gene expression.
Rice leaves at three different stages with respect to lesion development were examined.
Significant changes in rice transcriptome were detected in spl11 mutant. Among the various genes induced in the mutant most were oxidative stress /cell death- or defense- related genes. No well-defined groups of target genes for other hormonal or cellular responses were identified, suggesting that the Spl11 gene might be a component in the plant PCD pathway.
MATERIALS AND METHODS
Plant growth, sample preparation, RNA extraction, and array hybridization
The design of the rice Genechip genome microarray was as described previously
(Zhu et al., 2003). IR68 and spl11 plants were grown in autoclaved soil in a controlled environment chamber at 26°C, 85% relative humidity, and a 12-h light (200 µE.m-2.sec-1 light intensity) and 12-h dark photoperiod cycle. Three types of leaf tissues with different lesion development phenotypes were collected from the same spl11 plant. Young leaf
(YL) is the newly expanded leaf (less than one week old) without any visible lesion developed on the leaf blade. Fully expanded leaf (FL) (two-week old) refers to the leaf that is fully expanded with 10-20 visible lesions developed most at the tip of the leaf blade (< 30% area of the whole leaf). The old leaf (4-week old) (OL) has developed
19 lesions on over 60% of the leaf area with numerous lesions enlarged and coalesced. For
IR68, three types of leaf with the same age as their spl11 counterparts were collected.
Leaf tissues at the same growth stage were collected from at least five 47-day-old individual plants and pooled for each sample. The samples were homogenized in liquid nitrogen prior to RNA isolation. Total RNA was isolated using RNAwizTM reagent
(Ambion, Austin, TX) and examined by gel electrophoresis for integrity and by spectrometry for purity. To ensure RNA quality, all samples were confirmed to have
A260/A280 ratios of 1.9-2.1 before subjected to subsequent experiments. The purification of mRNA, synthesis of cDNA, preparation of biotinylated complementary
RNAs (cRNAs), probe labeling, hybridization, washing of the array, array staining and scanning, and scanned signal intensity normalization were conducted as previously described (Zhu et al., 2003). All experiments were technically duplicated.
Data analysis
Stringent control measures were applied for the data analysis to ensure the reproducibility and reliability of the results on gene induction and repression presented.
The average intensity of all probe sets of each array was scaled to 100 so that the hybridization intensity of all arrays was equivalent. The hybridization data were then imported into GeneSpring (Silicon Genetics, Redwood, CA) and the data of the two replicas for each sample were averaged. To eliminate genes with signals beyond detection limit, probe sets was filtered so that only those with signal greater than 25 in all
12 hybridizations were subjected to subsequent analysis. An induction or repression ratio cutoff of at least 2.0 and 3.5 was used for comparison of spl11 and IR68 at each leaf type,
20 respectively. To rule out the effect of developmental programs on the analysis, probe sets showing significant differential expression between different IR68 leaf types were excluded from the final results.
RESULTS
The spl11 mutation causes significant changes in rice transcriptome
The abundance of transcripts corresponding to 23,780 genes that presumably constitute nearly one-half of all rice genes were examined in the test. Of the 23,780 probe sets presented on the Genechip, 23,530 showed hybridization signal greater than the noise level (25, as determined by the median of the distribution of noise from all negative control probe sets in 100 Genechip experiments) in all 12 hybridizations and were subjected to subsequent analysis. Welch’s approximate t-tests with the 23,530 probe sets identified 1126, 1836, and 2151 probe sets that were statistically significant (P < 0.1) in terms of their differential expressions between IR68 and the spl11 mutant at the YL, FL, and OL leaf stage, respectively. Among these genes, 88, 336, and 276 genes were more than 2 fold induced, and 16, 113, and 203 genes were over 2 fold suppressed at the YL,
FL and OL leaf stage respectively (Figure 4). This indicated that the spl11 mutation significantly affect the global gene expression in rice. It is worthy to note that as more lesions developed on the leaf, there were more genes showing differential expression between the spl11 mutant and IR68.
21 Most genes with significant expression changes are cell death and/or defense-related
To know what types of genes are affected in the spl11 mutant, genes that were over 3.5 folds induced (Table 1) or suppressed (Table 2) at single or multiple leaf stages in spl11 were then grouped according to their known or putative functions. For induced genes, the first group contains genes that are implicated in the oxidative burst and PCD or hypersensitive response. The oxidative burst may be followed by activation of antioxidant genes in the tissues surrounding the initial pathogen infection site during HR
(Lamb and Dixon, 1997). Accordingly, mRNAs encoding antioxidant enzymes such as glutathione S-transferase also accumulate in spl11. The induction of the rice Bax inhibitor-1 gene (OsBI-1) at the spl11 young leaf stage is notable. OsBI-1 was found to suppress Bax-mediated cell death in yeast (Kawai et al., 1999). Plant Bax inhibitor gene was rapidly induced when it is challenged by wound or pathogen (Sanchez et al., 2000).
Whether the up-regulation of OsBI-1 in spl11 has a similar function as that in response to external abiotic or biotic stress remains elusive. The second group includes defense- related transcripts that usually accumulate rapidly in resistant plant-microbe interactions.
Nearly 30% of all genes with significantly induced expression belong to this group, indicating an intimate association of the induction of defense responses to cell death in spl11. mRNAs of pathogenesis-related genes such as PR-1, PR-2, PR-3, PR-5 and PR-10
(PBZ1) are highly accumulated at FL and OL stage. In addition, genes required for the synthesis of phytoalexins such as phenylalanine ammonia-lyase (PAL) and anthranilate synthase (ASA) also are up-regulated at the FL stage (Niyogi and Fink, 1992; Yamada et al., 1994). Most genes in this group are significantly induced only in the FL and/or OL, with few are up-regulated at the YL. In this respect, the accumulation of the extensin
22 precursor transcript in the YL is notable. Extensins are hydroxyproline-rich proteins that are synthesized as soluble precursor but are immobilized in the cell wall as hydroxyproline-rich glycoproteins (HRGPs) later. It is believed that the enhanced deposition and rapid cross-linking of extensins functions as a rapid protection at the initial stages of the HR or wounding (Brisson et al., 1994; Merkouropoulos and Shirsat,
2003). Elevated expression of the extensin precursor gene in the spl11 YL therefore may suggest the activation of defense pathway(s) at the early stage of the cell death signaling.
The third group includes genes with putative signal transduction or regulation functions.
It includes the receptor protein kinase, serine/threonine protein kinase, calmodulins, and methyl tranferases. Overexpression of the calmodulin may function to transduce the elevated Ca2+ signal that has previously been implicated in HR signaling (Xu and Heath,
1998). The fourth group includes genes playing roles in basic cellular maintenance and development. The sugar carrier protein C, for example, is significantly induced in FL, consistent with the findings in a previous report (Schenk et al., 2000). Adenosine kinase
(ADK) catalyzes the salvage synthesis of adenine monophosphate from adenosine and
ATP and deficient of ADK activity result developmental abnormalities in plant (Moffatt et al., 2002). While adenosine has been shown capable to induce apoptosis in animals, role of ADK in plant PCD has not been reported before (Kulkarni et al., 1998).
Transcripts that are significantly reduced in spl11 are shown in Table 2. Most mRNAs that have a considerably reduced accumulation in spl11 includes genes involved in photosynthesis, which is similar to what is observed in the Arabidopsis mutant acd11
(Brodersen et al., 2002). Given the lesions spread on the leaf blade, this reduced
23 expression of photosynthesis genes seems not unexpected. The importance of the down- regulated genes in the other groups remains to be determined.
DISCUSSION
The clear cell death phenotype and the intimate association of cell death to the induction of several defense-related genes in the spl11 mutant suggested that the activation of a cell death pathway that is coordinated with the induction of defense responses leads to the spl11 phenotype. In the plant-pathogen system, coordination of
PCD and defense responses occurs both in incompatible and compatible plant-pathogen interactions. The activation of cell death and defense responses in these two types of disease reactions is dissimilar in terms of the time, location, and magnitude of their occurrence as demonstrated in Arabidopsis (Tao et al., 2003; Wu et al., 2003b). In spl11, the two processes are difficult to distinguish due to the constitutive activation of both of them. Nevertheless the enhanced resistance of spl11 to rice blast and bacterial blight diseases argues that the cell death pathway activated in spl11 is likely similar to what occurs during resistant interactions.
To test the above hypothesis, microarray hybridizations were performed to elucidate the genome-wide transcriptional reprogramming during cell death and defense activation. As expected, the microarray hybridization showed that numerous well-known
PCD and defense-related genes were significantly up-regulated, whereas photosynthesis- associated transcripts were repressed in spl11, corroborating with enhanced resistance phenotype and reduced photosynthesis areas observed in the mutant. The up-regulation of
24 some well-recognized PCD and defense-associated genes, such as the Bax inhibitor, calmodulin, and extensin in the young leaf when no lesions are formed reflects the constitutive manner of spl11 mutation-triggered cell death and defense signaling. In contrast to fully expanded and old leaf, few significantly altered transcripts were identified in the young leaf in the microarray hybridization. This might partially be attributed to the scarce number of early signaling genes in PCD and defense pathways identified from the plants and incorporated into the first version of Syngenta’s rice
Genchips. On the other hand, this might suggest that the activation of PCD and defense is augmented by the signals emanating from the dying cells during lesion formation or enhanced by environmental factors. It is also possible that genes with smaller changes that are involved in these processes are not include in Table 1 and Table 2 due to the stringent cutoff. The higher expression of chlorophyll a/b binding protein 21 in the YL suggests that spl11-mediated cell death signaling may has some adverse impact on the biosynthesis or function of the chloroplast, as such proteins are implicated in the assembly or repair of the photosynthetic machinery during early chloroplast development and abiotic stress (Binyamin et al., 2001).
Over three dozens genes with unknown function were found differentially expressed in spl11. It is unclear how these genes are connected to PCD and defense, either causally or symptomatically. Functional studies of these genes by targeted gene disruption may lead to the identification of novel components in cell death and/or defense signaling pathway.
25
Figure 4. Number of genes with more than two-fold induced or suppressed in the spl11 mutant. Total RNA was extracted from rice leaves of both spl11 and wild-type
IR68 plants and used for microarray hybridizations. Expression of 23,780 genes in the leaves of spl11 or IR68 plants at three different lesion development stages, Young leaf
(YL), fully expanded leaf (FL), and old leaf (OL) were examined, respectively. Numbers of genes with more than two-fold induced or suppressed than that of IR68 were indicated.
The microarray hybridizations were technically replicated twice for each sample. YL, newly expanded leaf without any visible lesion developed on the leaf blade; FL, the leaf that is fully expanded with 10-20 visible lesions developed most at the tip of the leaf blade (< 30% area of the whole leaf); OL, old leaf has developed lesions on over 60% of the leaf area with numerous lesions enlarged and coalesced.
26 Fold up-regulation Probe sets Protein or homolog YL FL OL Oxidiative stress/burst or cell death-related OS_ORF009142_at 1.0 2.5 4.9 L-ascorbate oxidase OS_ORF014847_at 1.1 8.5 17.4 Subtilisin-like protease OS001035_at 1.3 5.1 6.7 Alcohol dehydrogenase v OS001045_F_at 1.2 4.3 1.9 Peroxidase 2 precursor OS001069_at 0.7 2.8 5.2 Catalase isozyme a OS001240_at 1.3 3.7 4.0 Peroxidase 2 OS002264.1_F_at 1.4 5.1 8.8 Alcohol dehydrogenase short chain OS002995_I_at 0.8 2.9 5.1 Alcohol dehydrogenase iii OS004263.1_at 1.0 3.7 3.4 Ferredoxin--NADP reductase OS004902_at 1.2 5.2 4.6 Glutathione-s-transferase OS006912.1_at 1.1 3.8 2.4 NADH-ubiquinone oxireductase OS007966.1_at 1.4 5.1 5.2 Flavanone 3-hydroxylase-like protein OS010415.1_I_at 1.1 27.4 82.2 Putative alcohol dehydrogenase OS011795_at 0.8 2.3 4.1 Alcohol dehydrogenase 3 OS013624.1_F_at 1.4 6.0 10.0 Short chain of alcohol dehydrogenase-like OS010304_at 1.0 7.2 5.7 Potassium transporter OS017116_at 1.1 4.9 4.5 Potassium transporter - like OS019008.1_at 3.6 1.9 2.0 Bax inhibitor-1 OS004010.1_at 1.6 5.4 2.9 AAA type ATPase (yeast bcs1-like) OS013212_at 1.3 3.4 3.9 Nucellin -like OS002059.1_at 3.8 2.6 3.1 Bowman-birk type bran trypsin-like OS000404_at 1.4 7.5 21.1 Bowman-birk type bran trypsin-like OS006324.1_at 1.1 3.7 4.6 Germin-like protein 1 precursor Defense-related OS_ORF008199_at 5.4 8.1 5.5 Extensin precursor OS_ORF011125_at 2.6 5.7 2.5 Endochitinase 1 precursor OS_ORF015958_at 5.2 26.4 7.2 Beta-1, 3-glucanase OS_ORF015975_at 1.0 2.2 3.8 Yeast suppressor of sporidesmin-toxicity- like OS_ORF018465_at 2.2 3.0 4.1 R gene RPM1-like OS000314_R_at 3.0 28.8 20.5 Rice PR-5 (continued)
Table 1. Genes ≥ 3.5-fold up-regulated at different leaf lesion development stages in spl11. YL: young leaf that is newly expanded (less than one week old) and no any visible lesion has developed on the leaf blade. FL: fully expanded leaf (two-week old) with 10-
20 visible lesions developed most at the tip of the leaf blade (< 30% area of the whole leaf). OL: old leaf (4-week old) that has developed lesions on over 60% of the leaf area with numerous lesions enlarged and coalesced. 27 Table 1: Continued OS000349_at 1.8 9.4 7.4 PR-3 OS000452_at 1.2 9.3 9.5 Soybean p21 (thamatin-like) OS000536_S_at 1.1 22.9 17.3 PR-1-like OS000590_F_at 1.6 6.2 6.8 Endochitinase precursor OS000608.1_at 1.3 13.9 13.1 Hevein-like protein procursor OS000639_I_at 3.4 37.2 18.1 Beta-1,3-glucanase precursor OS000674.1_at 5.0 1.6 3.1 Salt-stress induced protein-like OS000733_F_at 2.2 7.6 4.1 Phenylalanine ammonia-lyase (PAL) OS000793_F_at 1.6 10.4 7.1 PAL-like OS000816_I_at 1.3 11.0 7.9 PAL-like OS000818_R_at 1.8 10.3 6.5 PR-2 OS000915_at 1.2 6.4 6.7 PR-1-like OS000920_S_at 1.2 6.9 4.4 Beta-1, 3-glucanase OS000959.1_at 2.9 10.5 2.0 Beta-1,3-glucanase (isoenzyme V) OS001127_at 1.8 4.4 3.0 Acidic endochitinase precursor OS001213_at 1.1 3.3 3.7 PR-1b OS001265_F_at 1.2 11.5 7.9 PR-1 precursor OS001563.1_at 2.3 21.2 30.1 PBZ1 OS001792_F_at 0.8 2.8 4.2 Metallothionein 2b OS002484_F_at 4.5 7.2 5.3 Extensin precursor-like OS002683_at 1.1 35.2 19.6 Subtilisin-chymotrypsin inhibitor-2-like OS002953.1_at 2.0 5.8 3.6 Anthranilate synthase OS003151_F_at 1.0 5.1 6.8 Class IV chitinase OS003187_at 1.1 2.6 4.1 Pectinase OS003764_F_at 0.9 3.9 3.6 Metallothionein-like protein type 1 OS003773_at 1.7 6.9 4.2 PR-3 (type Q) OS005401_at 1.2 3.7 2.1 R gene RPS2-like OS006332.1_at 0.9 3.2 4.2 Hevein-like protein procursor 2 OS008091_R_at 1.1 1.8 9.1 Basic endochitinase 2 precursor OS010488_at 1.2 4.5 2.7 Receptor-like protein kinase-type R gene OS011227.1_at 1.3 8.3 2.6 Dehydration-responsive protein RD22-like OS012858_S_at 2.0 5.3 3.6 Anthranilate synthase component II OS013927_at 0.9 3.8 4.1 Anthranilate synthase beta chain OS015102_at 1.2 3.9 2.3 Anthranilate synthase, alpha subunit OS015603_S_at 1.1 4.3 4.4 Anthranilate phosphoribosyltransferase OS015959_at 1.1 4.9 2.8 Beta-glucosidase-like OS016332_I_at 1.0 6.9 11.0 Subtilisin-like OS016724_I_at 1.5 4.1 4.6 Zeamatin OS017465_at 1.1 5.7 8.4 Beta-glucosidase OS020915.1_at 1.4 4.5 4.5 Pathogen-induced 1(PI1)-like OS024957.1_at 6.2 1.0 0.6 Salt-stress induced protein-like Signaling transduction OS_ORF015862_at 1.1 2.5 4.1 Receptor protein kinase OS_ORF019686_at 1.2 2.4 3.5 Receptor protein kinase-like OS_ORF020721_at 0.9 3.3 5.0 Receptor protein kinase-like OS001486_I_at 2.1 6.2 5.3 Serine/threonine-protein kinase-like OS002924_at 4.6 1.6 1.4 Calmodulin (continued) 28 Table 1: continued OS004243_at 3.5 0.8 0.8 NAM(no-apical-meristem)-like OS005717_R_at 1.8 8.8 13.3 Abscisic stress ripening protein OS005987_F_at 1.1 3.6 1.6 Maize cortical cell delineating protein-like OS007597.1_at 1.5 3.3 3.7 Calmodulin-like OS009478_at 1.2 3.4 6.6 Homobox protein-like OS010132.1_at 1.0 3.1 3.7 Cytochrome p450 OS010410_at 1.0 1.3 4.4 Cytochrome p450 81E1 OS012786.1_at 1.4 5.3 3.4 Calmodulin 1 (CAM1) OS013425.1_at 1.1 4.0 5.0 Chloroplast nucleoid DNA-binding protein OS024999.1_at 1.3 3.7 3.2 Calcium-binding protein OS001855_at 1.6 9.9 7.7 Methyltransferase House-keeping OS_ORF003709_F_at 2.1 3.9 4.2 Bran trypsin OS000213_R_at 5.3 0.8 1.0 Chlorophyll a/b binding protein 21 OS000716_at 0.9 6.2 6.5 DOPA decarboxylase OS000759.1_at 1.5 3.2 3.5 Sucrose synthase 2 OS004858.1_at 1.3 6.9 4.2 5,10 methylenetetrahydrofolate reductase OS005195_at 1.5 5.5 2.8 O-methyltransferase OS005484.1_at 1.4 3.7 6.1 DXP synthase OS006971.1_at 1.3 4.0 3.3 Diacylglycerol kinase OS018216.1_at 1.3 4.4 2.9 Adenosine kinase-like OS012817.1_at 0.9 3.2 4.2 Uricase (urate oxidase)-like OS013966.1_at 1.1 5.8 4.3 Indole-3-glycerol phosphate synthase OS014904_at 1.9 4.8 3.6 Phospho-2-dehydro-3-deoxyheptonate aldolase 1 precursor OS015078_at 1.0 4.4 3.5 EPSP synthase 2 OS015333_at 1.2 4.5 3.2 Adenosine kinase (rat)-like OS015423_at 1.0 2.4 3.8 Acyl-CoA synthase - like OS016556_S_at 1.2 2.0 5.2 Carboxypeptidase OS016581_I_at 0.9 4.0 2.4 Glucose-6-phosphate 1-dehydrogenase-like OS020639.1_at 1.1 5.0 3.7 EPSP synthase OS012567_at 1.3 14.9 29.0 Cytochrome p450 GA3 Unknown OS_ORF001749_at 0.9 4.2 5.0 Proline-rich protein OS003819_I_at 2.0 3.6 1.9 Unknown OS_ORF004743_at 1.2 3.9 3.9 Unknown OS_ORF009955_at 1.1 5.3 13.4 Unknown OS_ORF012127_at 1.8 8.8 4.7 Human multidrug resistance protein 1-like OS_ORF019129_at 0.9 4.2 8.9 Unknown OS001692.1_at 1.1 2.2 3.8 Unknown OS001733.1_at 0.7 4.7 4.0 Unknown OS002721_at 1.9 3.6 1.8 Unknown OS003145.1_I_at 2.7 2.6 3.6 Unknown OS005138_at 1.1 3.8 3.9 Unknown OS006777_R_at 1.2 3.9 5.2 Unknown OS006957_at 1.3 12.1 16.9 Unknown (continued)
29 Table 1: continued OS007065_at 3.8 1.6 1.1 Unknown OS008622.1_at 1.0 3.6 6.8 Unknown OS009475_R_at 2.0 8.8 10.6 Unknown OS009506_F_at 0.9 7.5 21.4 Unknown OS011299_at 2.2 3.6 2.1 Unknown OS011390_I_at 1.2 6.2 4.1 Unknown OS011597.1_I_at 5.2 1.2 1.1 Jasmonate induced protein OS017399_at 1.0 2.4 4.1 Unknown OS011187.1_S_at 1.3 3.6 1.3 DYW7 protein-like OS017630_at 0.7 2.7 11.4 Unknown OS019331_at 0.9 3.5 4.7 Unknown OS019378_at 0.9 3.7 3.2 Unknown OS020945_at 1.3 6.2 1.5 Unknown OS020951_at 4.4 2.2 2.2 Unknown OS024662_at 4.1 0.8 1.1 Unknown
30
Probe sets Fold down-regulation Protein or homolog YL FL OL Photosynthesis-related OS000017_s_at 0.9 2.5 3.7 Chloroplast 30s ribosomal protein OS000074_at 0.8 2.4 3.7 Heme binding protein (putative) OS000079_s_at 0.8 3.5 3.9 Photosystem iron-sulfer center OS000803_s_at 1.0 2.4 4.0 Chloroplast 30s ribosomal protein OS001209_s_at 0.8 2.7 3.7 Heme-binding protein (HBP) OS005776_at 1.0 1.6 3.7 Cytochrome B6-F complex 3.5 kD OS010669_i_at 0.9 3.3 3.9 Wheat photosystem I iron-sulfur center- like OS021845_at 1.3 3.3 3.8 NADH-plastoquinone oxidoreductase OS013747_i_at 1.1 3.9 4.3 Maize NADH-plastoquinone oxidoreductase-like OS000192_s_at 1.1 3.2 4.0 NADH-plastoquinone oxidoreductase 49 OS000610_i_at 1.6 3.8 4.1 Putative NADH-plastoquinone oxidoreductase House-keeping OS000076_at 0.7 3.0 4.4 Translation initiation factor1 like OS000276_s_at 0.8 2.5 3.6 DNA-directed RNA polymerase alpha OS000548_at 1.0 2.4 3.6 DNA-directed RNA polymerase beta OS001107_at 1.4 2.2 3.7 Fructose-bisphosphate aldolase OS005673_at 1.9 3.5 4.9 Thiamine biosynthesis protein OS011745_at 1.7 3.0 4.1 Putative thiamine biosynthesis protein OS005633.1_at 1.8 1.6 3.5 Aspartate transcarbamylase OS016778.1_at 1.7 2.2 4.4 Inorganic ion transport-related protein Signal transduction OS012138.1_at 1.7 4.5 5.2 Cytochrome p450 (CYP78A9) OS006429_s_at 1.1 2.5 3.7 Acyl CoA-binding protein Unknown OS001608.1_at 1.1 2.3 4.8 Unknown OS001615.1_at 2.3 2.1 4.2 Unknown OS002879_f_at 2.0 3.7 4.8 Unknown OS006005_at 1.3 3.6 3.3 Unknown OS006066_at 1.8 3.8 5.0 Unknown OS007120.1_at 0.9 2.0 4.7 Unknown OS008171.1_at 0.9 2.5 4.7 Unknown OS010680.1_f_at 1.3 2.0 4.4 Unknown OS012112.1_at 2.8 3.7 5.3 Unknown OS014326.1_at 1.6 4.1 3.6 Unknown OS016769_r_at 0.7 3.2 3.6 Unknown
Table 2. Genes ≥ 3.5-fold down-regulated at different leaf lesion development stages in spl11
31 CHAPTER 3
FINE GENETIC MAPPING AND PHYSICAL DELIMITATION OF THE
LESION MIMIC GENE SPL11 TO A 160 KB DNA SEGMENT OF THE RICE
GENOME
ABSTRACT
The rice lesion mimic mutant spl11 was previously found to confer broad- spectrum disease resistance to both M. grisea and Xoo. To better understand the molecular basis underlying cell death and disease resistance in rice, a map-based cloning strategy has been employed to isolate Spl11. Five Spl11-linked RAPD markers were developed and four of them were mapped to rice chromosome 12. A high-resolution genetic map was developed using a segregating population consisting of 1138 lesion mimic individuals. Recombination suppression was observed in the vicinity of Spl11.
Three molecular markers tightly linked to Spl11 were identified and were used to screen a
BAC library. A contig spanning the Spl11 locus was constructed and physical mapping delimited Spl11 to a 160-kb DNA segment within a single BAC clone. These results provide the essential information for the final isolation of this important gene in the rice defense pathway.
32 INTRODUCTION
Programmed cell death (PCD) is a basic biological process in the life of multi- cellular eukaryotes and it has become one of the emerging active research areas of plant biology in the past few years (Richberg et al., 1998). The hypersensitive response (HR) results in rapid localized death of host plant cells immediately at the initial site of infection by a pathogen. HR is defined as a type of PCD (Heath, 2000). Correlation between HR and plant disease resistance was observed in several plant species (Shirasu and Schulze-Lefert, 2000). It was speculated that HR serves to arrest the growth and development of the pathogen at the infection site, particularly in the case of biotrophic pathogens (Greenberg, 1997; Delledonne et al., 2001). Recent data showed that HR is also associated with systemic acquired resistance (SAR) (Hunt et al., 1996; Molina et al.,
1999), characterized by an elevated broad-spectrum resistance against subsequent infections in distal plant tissues (Hunt et al., 1996). Several events, including ion fluxes, a transient oxidative burst, expression of defense-related genes, production of anti-microbal molecules, and formation of cell wall apposition, have been shown to be correlated with the HR in disease resistance (Yang et al., 1997). However, the molecular basis behind the control and regulation of hypersensitive cell death remains largely unknown.
An important approach to studying the molecular mechanisms of cell death and its relationship with disease resistance is to use mutants with a cell death phenotype in the absence of challenge by the pathogens. These mutants are collectively designated as
“lesion mimic” based on their spontaneous lesion phenotypes akin to HR or disease symptoms (Hoisington et al., 1982; Dangl et al., 1996). A large number of lesion mimic
33 mutants have been identified from Arabidopsis (Dietrich et al., 1994; Greenberg et al.,
1994; Weymann et al., 1995; Bowling et al., 1997; Mou et al., 2000), barley (Wolter et al., 1993), maize (Walbot et al., 1983; Johal et al., 1995), and rice (Kiyosawa, 1970;
Takahashi et al., 1999; Yin et al., 2000). Only a few of them display altered disease resistance, suggesting that most of the mutations either reside in the developmental cell death pathway or represent certain physiological disorders irrelevant to defense responses. The earliest rice lesion mimic mutant, called Sekiguchi lesion (sl) mutant, was reported in the 1970’s (Kiyosawa, 1970). From then on, lesion mimic mutants have been identified by various research groups and have been mapped on different rice chromosome (Yin et al., 2000). Three cdr (cell death and resistance) mutants displaying significant resistance to rice blast isolate were characterized by a Japanese group
(Takahashi et al., 1999). Recently, more LMMs displaying various cell death phenotypes were identified in an IR64 deletion mutant collection (Leung et al., 2001).
At present, two main approaches are employed for identification of plant genes behind phenotypes: map-based cloning (also called positional cloning) and gene discovery by transposon or T-DNA tagging. Until recently, no active endogenous transposons have been reported and introduction of maize transposon Ac/Ds into heterologous host rice is still beyond the stage of application (Izawa and Shimamoto,
1999; Kim et al., 2004). The successfully application of rice transposon Tos17 to rice functional genomics in the past few years provided a new tool for the rice research community (Hirochika, 2001; Miyao et al., 2003; Kaneko et al., 2004). In addition to transposon insertion, T-DNA mediated gene knockout or activation tagging has also been applied in rice gene functional analysis (Jeon et al., 2000; An et al., 2003; Wu et al.,
34 2003a). However, certain limitations are inherent to transposon or T-DNA tagging, such as parts of the genome may not be accessible to insertion tagging, thus preventing complete genome coverage and some tagging line will not give detectable phenotype.
Thus positional cloning remains as the main choice in forward genetics in rice. Moreover, using the gene-tagging method to identify Spl11 is hampered by the lack of essential information about its gene product and lots of gene will cause cell death phenotype when it is disrupted. The positional gene cloning approach involves identification of linked markers, high-resolution mapping, construction of large-insert genomic library, screening the genomic library to identify the candidate gene and complementation test or allele references to confirm the candidate gene.
The spl11 mutant was originally identified in an ethyl methanesulfonate (EMS)- mutagenized population of IR68. It was shown that the lesion mimic phenotype of spl11 was controlled by a single recessive gene (Singh et al., 1995) and that spl11 conferred non-race-specific resistance to both M. grisea and Xoo (Yin et al., 2000), the causal pathogens of blast and bacterial blight, respectively. The enhanced resistance of the spl11 mutant to the two pathogens correlates with the constitutive expression of several defense-related genes and lesion development on the leaves. The same lesion mimic phenotype was observed in progeny from backcrossing the spl11 mutation into two japonica varieties, TP309 and Nipponbare, indicating that the spl11 mutant allele is expressed in both indica and japonica background (Zeng and Wang, unpublished data).
Furthermore, in a mutational screen for reversion and suppressor mutations, we have isolated putative suppressor mutations that reduced the severity of lesion mimics
35 (unpublished data). The phenotypic characteristics and genetic information available make spl11 an interesting candidate for detailed molecular analysis.
As a first step toward the molecular characterization of the spl11 mutation, we employed a map-based cloning strategy to isolate the Spl11 gene. Here we report (1) the identification of five randomly amplified polymorphic DNA (RAPD) markers linked to spl11 and mapping of the gene to rice chromosome 12, (2) development of a fine genetic map of the spl11 region using both RAPD and restriction fragment length polymorphism
(RFLP) markers; (3) screening of a bacterial artificial chromosome (BAC) library and establishment of a BAC contig spanning the Spl11 locus, and (4) delimitation of the target gene within a specific BAC clone.
MATERIALS AND METHODS
Plant materials
Plants were grown in the greenhouse with a temperature range of 26 °C (night) to
32 °C (day) during the summer season in Singapore. In total, 1138 lesion mimic plants from either the F6 or F7 generation of a cross between spl11 and the indica cultivar CO39 were sampled and used as the segregating population for the fine mapping of DNA markers linked to the spl11 region.
Mapping the RAPD markers on the rice linkage map
The mapping population used in this study contains 111 doubled-haploid (DH) lines. The population was developed from a cross between the indica variety IR64 and
36 the japonica variety Azucena (Huang et al., 1994). The genetic linkage map constructed in the DH mapping population contains 270 RFLP markers. Chromosomal localization of the identified RAPD markers was performed using the program MAPMAKER 3.0
(Lander et al., 1987; Lincoln et al., 1992).
DNA extraction and Southern hybridization
Genomic DNA from young leaves was extracted and purified according to the method described (Dellaporta et al., 1984). Approximately 2 µg of rice genomic DNA was restricted with an appropriate enzyme and fractionated on a 1.0% agarose gel by electrophoresis. For Southern blot analysis, the gel was first soaked in 0.25 M HCl for
10- 20 min, rinsed with distilled water, and then soaked in 0.4 M NaOH for 10 min. The fractionated DNA was then transferred to a Hybond N+ nylon filter under alkaline conditions (0.4 M NaOH). The pre-hybridization, hybridization, and washing of the filter were conducted using standard procedures (Sambrook et al., 1989).
Bulk segregant analysis and RAPD marker identification
To allow the evaluation of the effect of spl11 on disease resistance, we previously produced homozygous spl11/spl11 lines in which the major resistance genes against blast and bacterial blight in IR68 were removed by phenotypic selection (Yin et al., 2000).
From the F6 generation of the cross between spl11 and CO39, 10 lesion mimic plants
(designated as L+) and 10 wild-type plants (designated as L−) were used for bulk
+ − segregant analysis. Equal amounts of genomic DNA from each F6 L or L plant were mixed and diluted (10 ng/µl) to form an L+ and L− bulk, respectively. Approximately 10
37 ng of the bulk DNA was used for each RAPD reaction. Random primers were purchased from Operon Technologies (Alameda, CA). The reaction mixture was cycled through the following temperature profiles: 94°C for 120 s for 1 cycle, followed by 94°C for 45 s,
37°C for 45 s, and 72°C for 60 s for 35 cycles. Polymerase chain reaction (PCR) was terminated at 72°C for 5 min. The PCR product was denatured and then separated on
4.5% polyacrylamide gel using the Sequi-Gen® sequencing cell from Bio-Rad (Hercules,
CA). Dried gel was exposed to X-ray film for 2-3 days. Those primers that revealed polymorphisms between bulks were further tested using the 20 individual DNA samples included in the bulk. The primers and conditions for PCR amplification of the sequence characterized amplification region (SCAR) marker CG16 (Paran and Michelmore, 1993) are listed in Table 1.
BAC library screening and insert size estimation of the positive BAC clones
A large-insert HindIII BAC library constructed from the rice cultivar Nipponbare
(Presting et al., 2001) was used to develop a contig covering the Spl11 locus. The BAC
DNA was isolated according to Wang et al. (1995) and digested with NotI for 5 h before loading on a 1.0% agarose gel. The running conditions for PFGE were: initial switch time
15 s, final switch time 25 s, with voltage at 4.5 v/cm, running 16 h in 0.5X TBE buffer.
The fractionated DNA was soaked in 1.0 µg/ml ethidium bromide for 20 min and visualized under UV light.
38 Identification of RFLP and PCR markers from BAC78 for fine mapping
DNA from BAC78 was digested with a 4-bp restriction enzyme Sau3AI and fractionated on a 1.2% agarose gel. Fragments with sizes ranging from 0.5 kb to 1.5 kb were recovered from the gel and subcloned into the pBluescript II KS vector (Stratagen,
USA). The inserts of the subclones were released using PstI/SacI, blotted to Hybond N+ nylon membrane (Amersham, USA) and hybridized to different BAC78/NotI fragments.
Subclones from different BAC78/NotI fragments were then subjected to RFLP analysis.
Those showing polymorphism between spl11 and CO39 were selected.
BAC78 was partially sequenced using the shotgun sequencing strategy. Primers were produced based on the BAC78 sequence and the sequence information available in the public database (http://www.ncbi.nlm.nih.gov/). Two kinds of PCR markers were obtained: that produced polymorphic bands between spl11 and CO39 directly or that produced polymorphic bands after the PCR product was cut with certain restriction enzymes. Primer and PCR conditions used for fine mapping are listed in Table 3.
RESULTS
Identification of spl11-linked RAPD markers
To locate spl11 in the rice genome, we applied bulk segregant analysis
(Michelmore et al., 1991) and RAPD markers (Welsh and McClelland, 1990; Williams et al., 1990) to screen for markers linked to spl11. The detection of polymorphic amplifications was greatly facilitated by the application of 33P dCTP nucleotide to each
PCR reaction for labeling and the separation of the PCR products on a sequencing gel.
39 About 25 bands with sizes from 100 to 1500 bp could be detected for each primer. In total, 1200 Operon random primers were screened and five of them (OpE04, OpY07,
OpH10, OpR16, and OpW19) were found to produce reproducible polymorphism between L+ and L− DNA bulks. These five RAPD markers were further tested by using the 20 individual DNA samples that were included in the two bulks. Primer OpR16 produced a specific band from wild-type plants with no corresponding band from lesion mimic plants (Figure 5A). The other four primers yielded specific polymorphism bands only from lesion mimic plants (data not shown).
The reproducible polymorphic bands generated by the five primers from individual DNA samples were excised from the dried polyacrylamide gel and soaked in
1X TE buffer overnight. The eluted DNA then served as the template for a second-round amplification using the same primer. The re-amplified PCR products were cloned into the pGEM-T vector (Promega) and the recombinant clones were designated as pG04, pG07, pG10, pG16, and pG19. The inserts of these clones were sequenced, with sizes ranging from 283 to 916 bp (data not shown). The SCAR marker CG16 based on the sequence of marker pG16 was produced and used to screen 78 F6 lesion mimic plants in the genetic mapping analysis. This SCAR marker was not used in the succeeding experiments because of its unstable PCR amplification. The five RAPD markers were then developed into the corresponding RFLP probe using a method similar to that described previously
(Fang et al., 1998). The hybridization patterns of the five markers showed that all but pG04 have a single copy in the rice genome (data not shown). The markers pG10 and pG16 hybridized only with either genomic DNA from spl11 mutant (pG10) or CO39
(pG16), suggesting that they may be cultivar-unique sequences. The other three markers
40 hybridized with both spl11 and CO39. Southern analysis with the marker pG16 confirmed that only wild-type plants displayed a positive band, while no band was detected for the lesion mimic plants, even after extended exposure of the X-ray films
(Figure 5B). Absence of the corresponding hybridization band in the wild type plant IR68 confirmed pG16 as a DNA sequence unique to CO39.
Integration of the spl11-linked RAPD markers into the rice linkage map
The successful identification of five spl11-linked RAPD markers enabled us to map the spl11 gene into the rice linkage map. For this purpose, genomic DNA from the two parents, IR64 and Azucena, of the DH mapping population (Huang et al., 1994) was digested and blotted for parental survey as described before (Wang et al., 2001). Marker pG04 was found to be a repetitive sequence in the parental survey and therefore was not used in subsequent experiments. The other four markers showed detectable polymorphism for at least one of the 18 enzymes used. All four markers were found to map on rice chromosome 12 (Figure 6). Three markers, pG19, pG10, and pG16, were found within an 18.4 cM region between RFLP markers Sdh-1 (Causse et al., 1994) and
1F5 (Wang et al., 2001) in the long arm of chromosome 12. Marker pG07, however, was located more distant from spl11.
Construction of a high-resolution genetic map of the Spl11 region
A prerequisite for genetic mapping is the identification of polymorphisms between the parental genotypes of a segregating population. RFLP analysis was used to search for possible polymorphism for each marker mapped. Because pG10 and pG16 can
41 hybridize with only one of the parents of the segregating population and because of the recessive nature of the spl11 mutant, it was assumed that recombination events at pG10 and pG16 could be ignored in RFLP analysis with homozygous wild-type plants and heterozygous wild-type plants, respectively. We therefore collected lesion mimic individuals only from the segregating population. Genomic DNA from the 1138 sampled lesion mimic plants was then extracted and restricted by an appropriate enzyme for
Southern blot analysis with each corresponding marker.
Sixteen and ten recombinants were identified for pG10 and pG16, respectively, in an initial survey of 648 samples, suggesting that the Spl11 gene is closer to marker pG16.
Based on this information and the linkage map, markers pG19 and pG07 were not included in subsequent analyses due to their distant location in the opposite direction from Spl11. Instead, three single-copy RFLP markers, CDO459, R1709, and 1F5, mapped to the same region on the rice linkage map (Heun et al., 1991; Harushima et al.,
1998; Wang et al., 2001) were selected for the construction of a detailed genetic map spanning the Spl11 locus (Figure 7A). Marker CDO459 was found to co-segregate with
Spl11. One and two recombinants were recovered from crossing over in the intervals of
R1709-Spl11 and Spl11-1F5 among the 1138 individual plants of the mapping population, indicating a genetic distance of 0.044 and 0.088 cM in the respective intervals. Thus, the Spl11 locus was within a 0.13 cM region bracketed by R1709 and
1F5.
42 BAC contig at the Spl11 locus
The fine genetic map of the Spl11 region provides a foundation for the establishment of a contiguous array of BAC clones covering the locus. A large-insert
HindIII BAC library constructed from the rice cultivar Nipponbare was screened with markers 1F5, CDO459, and R1709, which detected nine, six, and four positive BAC clones, respectively. The information on the markers and their corresponding positive clones was sent to the Clemson University Genome Institute (CUGI) and a contig of these BAC clones was established based on BAC fingerprinting and BAC end sequence information available at CUGI (http://www.genome.clemson.edu/projects/rice/) (Figure
7B). Insert size estimation of the overlapped positive BAC clones suggested approximately 320 kb between marker 1F5 and R1709 (data not shown). Of the BACs in this contig, BAC78 contained both marker R1709 and CDO459, whereas BAC68 with an insert of 150 kb was found overlapped within BAC78 (Figure 4).
Spl11 gene is located on BAC78
It is speculated that the Spl11 gene may be located on BAC78 based on the data that both marker R1709 and marker CDO459 are included in the same clone as described above. PFGE analysis indicated that BAC78 digested with NotI yielded at least seven fragments, with sizes of 60, 32.5, 28.6 , 19.5 , 13.5 , 12.9 , and 12.3 kb (Figure 8). To locate the two markers within the BAC, the PFGE gel of BAC78 digested with NotI
(Figure 8) was blotted and hybridized to R1709 and CDO459 separately. The results indicated that R1709 and CDO459 were positive to the 60 kb and 28.5 kb NotI fragments, respectively (data not shown). Further Southern blot analysis of the BAC78/NotI blot
43 using the Sp6 and T7 end sequences of the BAC vector pBeloBAC11 (Kim et al., 1996) positioned the 60 kb and 12.9 kb fragments at the two ends of BAC78. Sequence alignment between R1709 and BAC78 placed R1709 7.4 kb inward from the T7 end of
BAC78. A cleaved amplification polymorphism sequence marker CG16C-7 was produced from the 12.9 kb BAC78/NotI fragment sequence. When the 13 recombinants identified by marker pG16 were screened by CG16C-7, only one recombinant was found at this site (Figure 5), suggesting that Spl11 was flanked by marker R1709 and CG16C-7 on BAC78. All the other molecular markers identified via either RFLP or PCR analysis as described in “Materials and Methods” co-segregated with Spl11 (Figure 9). Spl11 was thus physically delimited to a DNA segment of approximately 160 kb on BAC78.
DISCUSSION
Positioning Spl11 on rice chromosome 12
Knowledge of the location of a gene on the genetic linkage map provides the starting point for map-based cloning. Accordingly, the RAPD technique and bulk segregant analysis were used to develop Spl11-linked markers. The probability of identifying a target gene–linked RAPD marker within a certain distance is affected by the genome size of the organism, the number of primers screened, and the degree of sequence divergence between the two parents of the segregating population in the target region
(Martin et al., 1991). Bulk DNA from the progeny of a cross between two genetically diverged parents, spl11 and CO39, instead of a backcross population of the cross between
IR68 and spl11 was used to improve the chance of identifying Spl11-linked markers.
44 Separation of PCR products on polyacrylamide gels helped us screen a large number of
DNA fragments with sizes ranging from 100 to 1500 bp. Five spl11-linked markers were identified after analysis of more than 30,000 RAPD loci. The integration of four RAPD markers into the rice linkage map positioned the Spl11 locus on chromosome 12. The closest RAPD marker pG16 was about 0.57 cM from the Spl11 locus (Figure 7A).
The localization of spl11 mutant allele on rice chromosome 12 is inconsistent with previous genetic analysis (Singh et al., 1995), which indicated that spl11 was linked to another lesion mimic mutation spl5 located on chromosome 7. It is possible that the ratio of wild-type plants to lesion mimic individuals in the F2 of the cross spl11/spl5 was overestimated in the genetic study due to the difficulty in distinguishing the lesion mimic plants from the wild type, as described in the previous study (Yin et al., 2000). Another possibility is that epistatic interaction between spl11 and spl5 might make it difficult to distinguish single and double mutants in the progeny.
Fine mapping of the Spl11 region
Development of a high-resolution linkage map in the target gene region provides the basis for subsequent physical mapping and candidate gene identification in positional cloning. Both RAPD and RFLP markers in the spl11 region were used to construct the fine genetic map. The use of three RFLP markers mapped previously to the same region significantly increased the resolution of the genetic map, allowing us to identify a marker
CDO459 that co-segregated perfectly with Spl11 (Figure 7A). Furthermore, the fact that the Spl11 locus is flanked by two tightly linked markers, 1F5 and R1709, enabled us to
“land” on a narrow region of the chromosome in the physical mapping experiments
45 (Tanksley et al., 1995) (Figure 7B). The presence of Spl11 alleles in both rice indica and japonica cultivars allowed us to use a large-insert BAC library constructed from the japonica cultivar Nipponbare to assemble a contig encompassing the target locus. The assembly of the BAC contig spanning the Spl11 locus was facilitated by the physical map of the rice genome publicly available at CUGI. The positive BAC clones containing markers 1F5, CDO459, and R1709 were used to anchor other BAC clones based on
BAC-end sequences.
The results on physical and genetic maps of the Spl11 locus suggest a region of reduced recombination. The genetic distance between the two delimiting loci, 1F5 and
R1079, of the target locus is 0.13 cM (Figure 8A). The corresponding physical size of the same region is estimated to be 320 kb. Therefore, the average genetic to physical (G-P) distance ratio in the target interval is 2.46 Mb/cM. This is much higher than the estimated
G-P distance ratio of 0.28 Mb/cM for the whole rice genome (Arumuganathan and Earle,
1991; Harushima et al., 1998). The same phenomenon was also observed in the rice blast resistance gene Pib locus (Wang et al., 1999), which showed a G-P distance ratio of 1.34
Mb/cM. In our study, using plants from the F6 or F7 generation for the mapping analysis might account for the apparent reduced recombination. The partial sterility characteristic of the spl11 mutant makes it difficult to set up a large segregating population in the F2 or
F3 generation. The 1138 lesion mimic plants of the mapping population originated from a few F5 and F6 families, which may not reflect the full extent of recombination events that could occur in this chromosomal region. Alternatively, recombination suppression could be caused by proximity to the centromere region (van Daelen et al., 1993; Haupt et al.,
2001) or by a high degree of sequence divergence between the two parents (Ganal and
46 Tanksley, 1996; Chetelat et al., 2000). Based on the published genetic linkage map
(Harushima et al., 1998) and our genetic data, Spl11 is located at the middle of the long arm of chromosome 12 at least 40 cM from the centromere; thus, centromeric influence seems unlikely. Amplification of different loci inside BAC78, however, did reveal a high degree of polymorphism between the parents spl11 and CO39 (data not shown). The divergence between the chromosome regions of the two parental genotypes may explain, in part, the observed low recombination around the Spl11 locus.
Physical delimitation of the Spl11 gene
Our physical mapping data localized Spl11 within a 160 kb DNA segment. The number of genes in this region is unknown. Based on a 340 kb stretch of genomic DNA sequence on rice chromosome 11, Tarchini et al (2000) estimated a gene density of approximately one gene per 10 kb genomic sequence. Thus, the 160 kb region may contain approximately 16 genes. Currently, detailed sequence information in this specific region of chromosome 12 is not yet available in the public databases. The complete sequence of BAC78 will provide us with clues to the possible genes present in the clone.
The identification of a candidate gene for Spl11 using this method, however, might be hampered by the lack of essential information about its gene product. Furthermore, the
EMS-induced spl11 mutation may not change its expression pattern in the mutant plant, rendering the identification of candidate genes by northern blot analysis a daunting challenge. Genetic complementation of the spl11 mutant using subclones of BAC78 could thus be one option for the ultimate molecular isolation of the target gene. Another possible solution to these problems is to identify a series of spl11 alleles to establish a
47 perfect association between genomic changes at the locus and phenotypes, an approach used successfully in the molecular cloning of the Mlo gene in barley (Buschges et al
1997), which will be discussed in the next chapter.
48
Marker Primer sequence PCR Restriction conditionsa Endonucleaseb CG16 5'-GGAGATTTCTACCTATGTGG-3' 94°C 45 s 5'-GATTCCTCGTTGCTGTTTT-3' 55°C 45 s 72°C 60 s CG07 5'-CCCCCAAAAGGCACACTCAT-3' 94°C 45 s HinfI 5'-AAAGAAACTGAACTGAAGAA-3' 56°C 45 s 72°C 90 s CG43 5'-TAGCATTCAGCCTCCTCACA-3' 94°C 45 s 5'-CGGAAAGAACAAGCCAAAAC-3' 60°C 45 s 72°C 90 s CG16C-7 5'-CCAAAGTGCTAAACGGTGTA-3' 94°C 45 s NcoI 5'-ATGTCGTGCGGTGGAAGTT-3' 57°C 45 s 72°C 120 s
a All PCRs began with a denaturing step of 3 min at 94 °C, followed by 35 cycles of amplification performed under the conditions indicated. PCR was terminated at 72°C for
5 min. b Markers CG07 and CG16C-7 will show polymorphism between IR68 (spl11) and CO39 only after the PCR product was cleaved by the enzyme indicated herein.
Table 3. PCR-based markers for genetic and physical mapping of Spl11
49
Figure 5. Screening for spl11-linked RAPD markers. A representative example of 5 spl11-linked markers identified from more than 30,000 tested RAPD loci is shown. +: lesion mimic samples, -: wild-type samples. A. Random primer OpR16 amplifies a specific, reproducible polymorphic band (arrow) between two bulks and 20 individuals included in the bulk; B. Marker pG16 hybridizes with wild type progeny of the cross spl11/CO39 only. Genomic DNA from individual samples was digested with HindIII, fractionated on 1.0% agrose gel and blotted to a Hybond N+ membrane. Note that pG16 does not hybridize with IR68.
50
Figure 6 . Mapping of spl11-linked RAPD markers to the rice genetic linkage map using MAPMAKER 3.0 (Lander et al. 1987). Four markers identified in this study are indicated by arrows on rice chromosome 12. Other markers on the map are RFLP markers from S. McCouch at Cornell University. Numbers at the left are recombination fraction and genetic distance (cM), respectively. The maximum-likelihood map order for markers was determined with a LOD score threshold of 3.0 and all map distances (cM) are reported in Kosambi units.
51
Figure 7. Genetic map and BAC contig of the Spl11 locus. A. High-resolution genetic map at the Spl11 region. Genetic distances are given in cM and the number of recombinants/number of segregants tested is indicated. Vertical lines display the position of the respective marker or gene. B. BAC contig spanning the Spl11 locus. BAC inserts are overlapped to scale as bars. Vertical dotted lines mark the relative position of the corresponding markers. The Spl11-containing BAC insert (BAC78) is highlighted in gray.
52
Figure 8. Determination of the insert size of BAC68 and BAC78 using pulse field gel electrophoresis (PFGE) analysis. BAC DNA was digested with NotI for 5 h before it was fractionated on the gel. The estimated insert sizes of BAC78 and BAC68 are 179.2 and 154 kb, respectively. λ PFGE size marker (lane 1) and λ/HindIII ladder (lane 4) were run together with BAC68 (lane 2) and BAC78 ( lane 3) for size determination.
Numbers at both left and right sides are the positions of the λ PFGE size marker and
λ/HindIII size ladder (New England Biolabs ). BAC vector position is indicated by the arrow.
53
Figure 9. A NotI physical fine map of the BAC78 clone delimiting Spl11 on a DNA interval of 160 kb. The solid line represents the BAC insert. T7 and Sp6 indicate the orientation of the insert cloned into the BAC vector pBeloBAC11. NotI restriction site is indicated by the arrows. The numbers above the solid line indicate the size of each NotI- cut fragment. The dotted line marks the position of the corresponding markers. Marker names beginning with pG indicate an RFLP marker and CG a PCR marker. The genetic distances of the flanking markers R1709 (0.044 cM) and CG16C-7 (0.044 cM) are indicated. Based on the the preliminary sequence data (Zeng, L., unpublished), R1709 and CG16C-7 are 7.3 kb to the T7 end and 8.2 kb to the Sp6 end, respectively.
54 CHAPTER 4
SPL11 ENCODES A U-BOX/ARM REPEAT PROTEIN ENDOWED WITH E3
UBIQUITIN LIGASE ACTIVITY
ABSTRACT
The rice spl11 mutant was identified from an ethyl methanesulfonate-mutagenized indica cultivar IR68 population and was previously shown to display spontaneous cell death phenotype and enhanced resistance to rice fungal and bacterial pathogens. Here we have isolated Spl11 via a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection. The predicted SPL11 protein contains both a U-box domain and an armadillo repeat (ARM) domain, which was demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein-protein interactions, respectively. Amino acid sequence comparison indicated that the similarity between SPL11 and other plant U-box-
ARM proteins is mostly restricted to the U-box and ARM repeat regions. A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. Expression analysis indicated that Spl11 is induced in
55 both incompatible and compatible rice-blast interactions. In vitro ubiquitination assay indicated that the SPL11 protein possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting a role of the ubiquitination system in the control of plant cell death and defense
INTRODUCTION
In multicellular organisms, cell death can occur as either a physiological cell death or a non-physiological cell death (Vaux and Korsmeyer, 1999). Physiological cell death, also coined as PCD, refers to a process programmed by the organism to kill its own cells in purpose. The most predominant form of PCD in animals is apoptosis, which is morphologically characterized by membrane blebbing, cell volume loss, nuclear condensation, and DNA fragmentation (Kerr et al., 1972). In plants, PCD occurs during both normal development and in response to pathogen infection. Prominent examples of developmentally PCD include the degeneration of cereal aleurone cells, the development of tracheary elements in xylogenesis, leaf senescence, and cell death in plant reproduction
(Kuriyama and Fukuda, 2002). In plant-microbe interactions, PCD occurs during both plant hypersensitive response (HR) to avirulent pathogen infection and plant disease susceptibility under virulent pathogen attack (Greenberg, 1997).
HR cell death is characterized by the rapid localized cell death that occurs at the site of infection caused by avirulent pathogens. This response appears to be triggered through the recognition of an avirulent factor by a corresponding resistance (R) protein in the plant. A large number of mutants characterized by misregulated cell death phenotypes
56 mimicking the HR have been identified in maize (Walbot et al., 1983), Arabidopsis
(Lorrain et al., 2003), barley (Wolter et al., 1993), and rice (Yin et al., 2000). The constitutive activation of cell death and defense pathways in some of the mutants suggests that these mutations might define genes involved in the regulation of HR in wild-type plants. These mutants are collectively called lesion mimics based on their spontaneous lesion formation in the absence of pathogen infection. More than a dozen genes controlling lesion mimics have been isolated to date. The proteins encoded by these genes fall into various functional groups, including membrane associated protein
(Büschges et al., 1997), ion channel (Balagué et al., 2003), zinc-finger protein (Dietrich et al., 1997), heat stress transcription factor (Yamanouchi et al., 2002), and components involved in the biosynthesis/metabolic pathways of fatty acid/lipids (Kachroo et al.,
2001), porphyrin (Hu et al., 1998), and phenolics (Gray et al., 1997). Studies of these lesion mimic mutants have begun to shed light on the control of PCD and its connections to disease resistance in plants. For example, analyses of Arabidopsis double mutants between the lesion stimulating disease mutant lsd1 (Dietrich et al., 1997) and mutants for two positive regulators for R genes function, EDS1 and PAD4, has indicated that both
EDS1 and PAD4 are required for runaway cell death in the lsd1 mutant (Rusterucci et al.,
2001). It was suggested that EDS1 and PAD4, two signaling genes that mediate some but not all R responses in Arabidopsis, regulate a reactive oxygen intermediates (ROI)/SA- dependent defense signal amplification loop that is modulated by LSD1 (Rusterucci et al.,
2001).
The ubiquitin/proteasome pathway is the major selective protein degradation system in eukaryotes. It is initiated by the formation of a thiol-ester linkage between the
57 ubiquitin molecule and the cysteine residue at the active site of the ubiquitin-activating enzyme (E1) in an ATP-dependent manner. The activated ubiquitin is then transferred to the active site of the ubiquitin-conjugating enzyme (E2). Finally, an ubiquitin-ligase (E3) binds E2 and catalyzes the formation of an isopeptide linkage between the activated ubiquitin and the substrate protein. In the last decade, ubiquitination has emerged as one of the key regulatory mechanisms of apoptosis in mammalian systems (Lee and Peter,
2003). In plants, ubiquitination-mediated protein degradation has been shown to play a significant role in multiple cellular processes such as photomorphogenesis and regulation of hormone signaling (Sullivan et al., 2003). Recent data suggest that ubiquitination may also play an important role in plant defense against pathogens. The identification of two
F-box proteins and several RING-type E3 ubiquitin ligases in the regulation of plant defense as well as the finding of a possible SGT1-mediated link between ubiquitination and R-gene-mediated resistance have suggested a possible role for ubiquitination in plant disease resistance signaling (Devoto et al., 2003). Nevertheless, direct evidence for the involvement of the ubiquitination/proteolysis pathway in signaling and regulating plant
PCD and disease resistance has not been established.
Many lesion mimic mutants have been identified in rice, and some of these mutants display altered early defense signaling or disease resistance(Takahashi et al.,
1999; Yin et al., 2000). Disruption of a heat stress transcription factor was found recently to be responsible for the phenotype of the stress-inducible rice lesion mimic mutant spl7
(Yamanouchi et al., 2002). The rice lesion mimic mutation spotted leaf11 (spl11) was identified from an ethyl methanesulfonate (EMS)-mutagenized indica cultivar IR68 population and was shown to be inherited in a recessive monogenic fashion (Singh et al.,
58 1995). Phenotypic characterization showed that spl11 confers enhanced, non-race- specific resistance to both M. grisea and Xoo, the pathogens that cause rice blast and bacterial blight diseases, respectively (Yin et al., 2000). In addition, correlation between the lesion development on leaves and the activation of several defense-related genes and enhanced resistance of the spl11 mutant to pathogens was also observed. To understand the molecular basis by which Spl11 suppresses cell death and the relationship between the spontaneous cell death and the activation of defenses in spl11, we have isolated the
Spl11 gene by a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection (Leung et al., 2001). The Spl11 gene encodes a novel protein with both a U- box domain and six armadillo (ARM) repeats. A point mutation was identified in spl11 that resulted in a premature stop codon in the SPL11 protein. We also showed that SPL11 possesses an E3 ubiquitin ligase activity in vitro, and the intact SPL11 U-box domain is essential for this activity, suggesting an involvement of ubiquitination in the control of plant PCD and pathogen defense.
MATERIALS AND METHODS
Plant Growth
spl11/spl11 The F2 population derived from the cross between TP309 and Nipponbare used for the mapping analysis was grown in the greenhouse in the winter season 2002-
2003 at Columbus, Ohio, at 28-18°C day and night temperatures. The F3 population of the same cross was grown in the greenhouse in 2003 summer at Columbus, Ohio at 32-
59 26°C day and night temperatures. Genomic DNA was prepared from young leaves of plants around 45-day-old.
Identification of spl11 Allelic IR64 Mutants
A large deletion mutant bank was established at the International Rice Research
Institute (IRRI) from chemical and irradiation-treated IR64 populations as described
(Leung et al., 2001). Among the morphological mutants collected in the bank, more than
30 were lesion mimic mutants (C. Wu and H. Leung, unpublished). Mutants with phenotype similar to that of spl11 were then crossed with IR64 and spl11 or were crossed with each other for genetic analysis. Those mutant lines allelic to spl11 were then subjected to molecular analysis.
DNA Gel Blot Analysis
Genomic DNA from young leaves was extracted and purified according to the method described (Dellaporta et al., 1984) with extraction buffer modification. The extraction buffer included 100 mM pH 8.0 Tris-HCl buffer; 25 mM pH 8.0 EDTA, 2%
(w/v) sorbitol, 0.25% (w/v) hexadecyl trimethyl ammonium bromide (CTAB), 0.25%
(w/v) polyvinyl polyprolidone, 1% N-lauroyl sarcosine, and 1.4 M sodium chloride.
Approximately 2 µg of rice genomic DNA was digested with an appropriate enzyme and fractionated on a 1.0% agarose gel by electrophoresis. For Southern blot analysis, the gel was first soaked in 0.25 M HCl for 10- 20 min, rinsed with distilled water, and then soaked in 0.4 M NaOH for 10 min. The fractionated DNA was then transferred to a
Hybond N+ nylon filter under alkaline conditions (0.4 M NaOH). The prehybridization,
60 hybridization, and washing of the filter were conducted using standard procedures
(Sambrook and Russell, 2001).
Northern Blot Analysis
Total RNA was isolated with the RNAwizTM RNA isolation reagent (Ambion,
Austin, TX) from 3- to 4-week-old rice leaves according to the protocol provided by the manufacturer. Around 10 µg of total RNA from each sample was mixed with an equal volume of northernMax gel loading solution (Ambion, Austin, TX), heated at 50°C for
30 min, then cooled on ice to denature the RNA. The denatured samples were then separated on a 1.4% agarose gel in 1X BPTE electrophoresis buffer (10 mM PIPES, 30 mM Bis-Tris, and 1 mM EDTA) and blotted to Hybond-N+ nylon membrane (Amersham
Bioscience, Piscataway, NJ) with 20X SSC solution. The prehybridization and hybridization were performed using standard procedures (Sambrook and Russell, 2001).
After hybridization, the blot was washed twice in 1X SSC, 0.5% SDS solution at 65°C for 5 min, followed by washing in 0.5X SSC, 0.5% SDS solution at 65°C for 10 min.
RT-PCR Amplification
To detect changes in Spl11 expression in IR64 mutants, 1 µg of total RNA per sample was used to synthesize the first-strand cDNA using the AMV Reverse
Transcriptase system (Promega, Madison, WI). The synthesis was conducted according to the protocol provided by the manufacturer. The 20 µl first-strand cDNA product was diluted to 120 µl final volume with 1X TE buffer. For PCR amplification of the Spl11 cDNA fragment, 1.5 µl of the diluted first strand cDNA was used in a 25 µl reaction 61 volume with the Taq enzyme from New England BioLabs (Beverly, MA). Spl11-specific primers, Uc-3 (5’-GATGCTTGCCTTATTGTCCTCA-3’) and Uc-4 (5’-
ACGGATTGATATGCCTGACGAT-3’) were used for the amplification. The reaction mixture was cycled through the following temperature profiles: 94°C for 210 sec for one cycle, followed by 94°C for 40 sec, 62°C for 40 sec, and 72°C for 60 sec for 22 cycles, and a final incubation at 72°C for 5 min. For amplification of the rice Actin1 gene, primer paire Actin-F (5’-CGTCTGCGATAATGGAACTGG-3’) and Actin-R (5-
CTGCTGGAATGTGCTGAGAGAT-3’) were used.
For amplification of the 5’ end of the Spl11 cDNA, approximately 1.6 µg mRNA was first purified from total RNA using the Oligotex mRNA mini kit (QIAGEN,
Valencia, CA). About 12.5% dimethyl sulfoxide (DMSO) (final concentration) was added to the first-strand cDNA synthesis reaction to break the secondary structures of the
RNA. The oligo(dT) primer was replaced by the Spl11-specific primer Uc-3 ( 5’-
GATGCTTGCCTTATTGTCCTCA-3’) in the synthesis reaction. The reaction was performed at 38.5°C for 1 hr using the AMV Reverse Transcriptase system and was then denatured at 95°C for 5 min. The reverse transcription was followed by PCR that was carried out with primers RACE1 (5’ -CGTCAGGCATATCAATCCGTTCTTT-3’) and
URACE3 (5’-CCCCACTATTTACCATTCTGCCACT-3’) using approximately one- tenth of the reverse transcription products. Two percent DMSO and 0.25 M betaine
(trimethylglycine) were added to the reaction mixture to overcome the difficulty in the amplification of the high GC content region. The reaction mixture was cycled through the following temperature profiles using the ThermalACE Taq enzyme (Invitrogen, Carlsbad,
62 CA): 98°C for 180 sec for one cycle, followed by 98°C for 30 sec, 54°C for 40 sec, and
72°C for 45 sec for 32 cycles, and a final incubation at 72°C for 10 min.
E3 Ubiquitin Ligase Activity Assay
DNA fragments of Spl11 containing sequence for both the U-box domain and the
ARM domain (1749 bp) and mouse E3 ubiquitin ligase gene CBL (GI:38605691) were cloned into the pMAL-c2 vector (New England BioLabs, Beverly, MA) and expressed in
E. coli. The fusion proteins were prepared according to the manufacturer’s instructions.
For the E3 ubiquitin ligase activity assay of the fusion proteins, wheat E1 (GI:136632) and Arabidopsis E2 AtUBC9 (GI: 20136191) were used for the assay. Both wheat E1 and
AtUBC9 were cloned in frame into vector pET28a (Novagen, now part of EMD
Biosciences, Inc., San Diego, CA) and expressed in E. coli strain BL21. Protein from the
E1- or E2-expressing E. coli was used in the E3 ubiquitin ligase assay in which about 50 ng of E1, 50 ng of E2 and 1 µg of E3 were added. The two SPL11 mutants that contain mutation in the U-box domain are prepared using the Quickchange site-directed mutatgenesis kit (Stratagene, La Jolla, CA) according to the protocol provided by the manufacturer. The sequence of the primer pair used for the preparation of the Val290 to
Arg290 mutant is: M1F: 5’-
CTTGAGCTGATGAAGGATCCTAGAATAGTGTCTACAGGGCAGACA-3’
and M1R: 5’-
TGTCTGCCCTGTAGACACTATTCTAGGATCCTTCATCAGCTCAAG-3’. The primers for the preparation of the mutant contains small deletion (ΔC314P315T316) in the U- box are: M2F: 5’-ATAGCATCAGGCCATCATACCACGCAACAGAAGATG-3’ and
63 M2R: 5’-CATCTTCTGTTGCGTGGTATGATGGCCTGATGCTAT-3’. The in vitro E3 ligase assays were performed as described (Xie et al., 2002).
Complementation
The BAC78 that contains the Spl11 gene was first subcloned into the modified transformation-competent BAC vector pTAC8 (Qu et al., 2003) using NotI as the restriction enzyme. The insert of different subclones was determined by pulse-field gel eletrophoresis and PCR amplification using primer pairs specific to each NotI-digested fragment of BAC78. The subclone containing the Spl11 gene, TAC20, was then digested with PacI and separated on a 0.8% agarose gel. The 9.4 kb fragment from the PacI- digested TAC20 that contains the Spl11 gene was recovered from the gel and then digested with XbaI to remove the 1.4 kb PacI-XbaI fragment. A 365 bp nos terminator
DNA was amplified from vector pBI221 (Clontech, Palo Alto, CA) with primers that contain adapter sequences harboring the PacI and HindIII restriction site, respectively.
The 8.0 kb XbaI-PacI genomic DNA fragment containing the entire Spl11 gene and sufficient cis element (a 2.6 kb DNA fragment upstream of the start codon) was then ligated together with the nos terminator DNA fragment into the binary vector pCAMBIA1301 (CAMBIA, Canberra, Australia). This final binary construct (pGW78) was used for the complementation of the spl11 mutation. The pGW78 was mobilized into
Agrobacterium strain LBA4404 (Hoekema et al., 1983) by electroporation and was used
spl11/spl11 to transform spl11 plants (TP309 ) (Qu et al., 2003). The phenotype of the T1 transformants was scored under standard plant growth conditions as described above.
64 Protein sequence alignments and phylogenetic analysis
All the protein sequence alignments were conducted using the program Clustal_X
(Thompson et al., 1997). The aligned sequence data was then inputted into the MEGA2 program (Kumar et al., 2001) to construct the phylogenetic tree.
GenBank accession numbers
The GenBank accession numbers for the mRNA and genomic DNA of the Spl11 gene are AY652589 and AY652590, respectively.
RESULTS
Genetic and Physical Mapping of the Spl11 Locus
Spl11 was previously physically delimited to a 160 kb DNA segment by markers
R1709 and CG16C-7 in the BAC clone BAC78 (Figure 10A) (Zeng et al., 2002). BAC78 was partially sequenced using a shotgun sequencing approach. Assembly of the sequences produced two continuous sequence contigs that cover 95.7% of BAC78
(Figure 10B). To narrow down the Spl11 gene to a smaller region, two new mapping populations were generated by crossing the spl11 mutant with two japonica cultivars,
TP309 and Nipponbare. The spl11 mutation was first backcrossed into TP309.
Homozygous progenies derived from the cross showing the same lesion mimic phenotype as spl11 (designated as TP309spl11/spl11) were then used as the pollen donor to cross with
Nipponbare. Plants from the F2 and F3 generation of the cross were used for subsequent mapping analysis. Seven cleaved amplification polymorphism sequence (CAPS) markers
65 (Weining and Langridge, 1991) were developed from the sequences available in the 160 kb DNA region. Recombination analysis of 297 F2 lesion mimic plants and 1846 F3 individuals indicated that the Spl11 locus was localized within a 27 kb DNA region bracketed by CG47 and CG111 (Figure 10B).
The gene prediction programs GENSCAN (Burge and Karlin, 1997) and Fgenesh
(Solovyev et al., 1995) were then used to identify possible genes in the 27 kb DNA region. Three DNA intervals with high probability of containing a coding region were detected. We designated the putative genes encoded by these DNA intervals G1, G2, and
G3 (Figure 10C). BAC78 was subcloned into a modified transformation-competent bacterial artificial chromosome (TAC) vector using NotI as the cloning restriction enzyme (Qu et al., 2003). The subclone that covers the 27 kb region, named TAC20, was then used as a probe to fingerprint spl11 and IR68 genomic DNA by RFLP analysis
(Figure 10C). Among the 19 restriction enzymes analyzed, TAC20 showed polymorphism between spl11 and IR68 only with restriction enzyme BslI (Figure 10D-1).
Based on these results, we postulated that a mutation is most likely located within the
Spl11 gene, which resulted in the absence of a BslI restriction site normally present in wild type IR68 genomic DNA (Figure 10C). To test this hypothesis, DNA spanning the putative mutated BslI cutting site was then amplified from the TAC20 plasmid DNA and used as a probe to hybridize with the blot previously used in the fingerprinting experiment. The second hybridization gave exactly the same polymorphism pattern between spl11 and IR68 (Figure 10D-2), indicating that a mutation did occur in the vicinity of the BslI restriction site.
66 Sequence alignment between the mutated BslI region and the 27 kb DNA stretch indicated that the putative deleted BslI restriction site (designated as ∆BslI) in spl11 is located in the first predicted exon of G3 shortly downstream of the start codon (Figure
10C). A search for ESTs in the 27 kb DNA sequence using the BLAST2 algorithm and available databases identified rice ESTs matching the 3´ end of G3 only, which suggests that G3 was most probably the candidate Spl11 gene.
Identification of spl11 Alleles in an IR64 Mutant Collection
To facilitate the cloning of the Spl11 gene, we searched for lesion mimic mutants with a similar phenotype to that of the spl11 mutant from an IR64 mutant collection
(Figure 11A). Three mutants were identified from either diepoxybutane (DEB)-treated
(DB2487) or radiation (γ-ray)-treated (GR5612 and GR5717) IR64 populations (Leung et al., 2001). Allelism tests indicated that the mutations occurred in all three IR64 lesion mimic mutants are recessive and allelic to spl11 (Table 3). Moreover, alterations at the
Spl11 locus were detected in two of the three mutants by RFLP analysis (Figure 11B).
PCR analysis was also performed on these mutants using several Spl11-specific primer pairs. A combination of the PCR and Southern analysis data suggested a 2.5 kb genomic
DNA deletion at the 5´ end of the Spl11 locus in mutant GR5612 and a 1.4 kb deletion near the Spl11 start codon in mutant GR5717 (data not shown). However, no visible change at the Spl11 locus was detected in mutant DB2487 when twenty enzymes were used in the RFLP analysis. This could reflect the fact that these enzymes could not detect the small deletion or point mutation in DB2487. Therefore, we evaluated about 2000 F2 plants from each of the two reciprocal crosses between spl11 and DB2487. Phenotypic
67 evaluation indicated that all the F2 plants were lesion mimic phenotype, suggesting that the lesion mimic phenotype in DB2487 was allelic to the spl11 mutation. The segregation ratio in the F2 generation of the cross between IR64 and DB2487 fitted 3:1 (85 wildtype to 23 lesion mimic, χ2 =0.80 P=0.37), further indicating the mutation in mutant DB2487 was controlled by a recessive mutation.
To test whether the mutation at the Spl11 locus affected the transcript accumulation of the gene in these mutants, RT-PCR analysis using Spl11-specific primers was performed. As shown in Figure 2C, the expression of the candidate Spl11 gene in mutant GR5612 was completely disrupted, corroborated by Southern blot analysis data showing deletion of a 2.5 kb fragment (Figure 11B). There was only a trace expression of
Spl11 in mutant GR5717. The expression of Spl11 was reduced in mutant DB2487 compared with that of wild-type IR64. The perfect association between genomic changes at the Spl11 locus and the lesion mimic phenotype of the three IR64 spl11 alleles, along with changes observed in candidate gene expression of the mutants, provided strong evidence that the candidate gene within the G3 fragment encodes Spl11.
Functional Complementation of spl11
To get final confirmation that the candidate gene encoded within the G3 DNA fragment was Spl11, we made a pCAMBIA1301-derived binary plasmid, pGW78, that contains the full-length Spl11 genomic DNA and a 2.55 kb fragment of the upstream sequence to complement the spl11 mutation. To improve the efficiency of transformation, the spl11 mutation was first introgressed into japonica cultivar TP309, the most commonly used cultivar in rice transformation. The seeds produced by the lesion mimic
68 plant TP309spl11/spl11 were then used for the complementation test. Plasmid pGW78 was transferred into the spl11 mutant via the Agrobacterium-mediated transformation system
(Qu et al., 2003). In total, 44 independently-transformed transgenic lines were generated, among which 40 lines were successfully complemented. Southern blot analysis of the transgenic plants revealed that all the plants carry the mutation originally introgressed from the spl11 plant and 0 to 2 copies of the transgene (data not shown). None of the transgenic plants containing the completely integrated transgene showed any development of lesions within 2 months after the regeneration (Figure 12). These results confirmed that the gene encoded within the G3 DNA interval was responsible for the phenotype of the spl11 mutant.
The Spl11 Gene Encodes a U-box/ARM Protein with Homology to Drosophila armadillo
The successful complementation of the spl11 mutation prompted us to obtain the cDNA in the G3 DNA interval. Northern analysis using G3 genomic DNA as a probe revealed an approximately 2.6 kb mRNA in the leaf, stem and root of IR68 and spl11
(Figure 13A). Spl11 shows highest expression in the leaf, while lowest in the root. A
2106 bp cDNA fragment that covers the central region and 3´ end of the G3 gene was identified from a Nipponbare leaf cDNA library. The 2106 bp cDNA sequence completely matches the corresponding predicted exons in G3. A modified RACE amplification method using primers derived from the predicted Spl11 gene sequence was then used to obtain the 5’ Spl11 cDNA. A full-length cDNA sequence of 2518 bp for the
G3 DNA interval was generated when the RACE result and the cDNA clone sequence
69 were combined (Figure 13B). An open reading frame (ORF) of 2085 bp starting at position 81 was detected in this full-length cDNA. The deduced protein of the complete
ORF had 694 amino acids and a molecular weight of 75.3 kD, with a predicted isoelectric point of pH 5.2 (http://us.expasy.org/tools/#primary).
A database search with the deduced SPL11 amino acid sequence showed that the
75 amino acids at positions 272 to 346 of SPL11 share high similarity to the consensus
U-box domain sequence that was first identified in the yeast protein UFD2 (Koegl et al.,
1999) (Figure 13C). The U-box domain contains about 70 amino acids and is conserved among fungi, plants and animals (Aravind and Koonin, 2000). It has been shown that the
U-box domain is indispensable for E3 ubiquitin ligase activity of several U-box proteins
(Jiang et al., 2001). The presence of the U-box domain in the SPL11 protein therefore suggests a probable E3 ubiquitin ligase activity for SPL11.
The database search also showed that the central and C-terminal region of the
SPL11 protein shares similarity to the ARM repeats of ß-catenin, the vertebrate homolog of Drosophila segment polarity protein armadillo (Riggleman et al., 1989) . The ARM repeats are tandemly repeated copies of the armadillo motif, each containing 38-45 amino acid residues (Peifer et al., 1994). Structural characteristics of the ARM motif suggest its involvement in protein-protein interactions, which has been demonstrated in several cases
(Huber et al., 1997). In total, six ARM repeat motifs were detected in SPL11 (Figure 4C).
Alignment of the ARM repeats in SPL11 with ß-catenin repeats 1 to 6 is shown in Figure
4D. Despite the significant variability in sequence among individual motifs, the chemical nature of the residues within each motif is generally conserved. Homologous modeling between the corresponding SPL11 and β-catenin ARM repeat region indicated that their
70 structure matches well (data not shown) (Guex and Peitsch, 1997). This suggests that the
ARM repeats of SPL11, like that of ß-catenin, might be in physical contact with its interactor(s).
Only two plant proteins bearing both U-box and ARM repeat domains similar to those of SPL11 have been functionally characterized so far. One of them, ARC1, was isolated in a yeast two-hybrid screen for S receptor kinase (SRK)-interacting proteins and was shown to possess an E3 ubiquitin ligase activity that positively regulates self- incompatibility of Brassica (Stone et al., 2003). The other one is PHOR1, which is a photoperiod-responsive protein involved in gibberellin signaling (Amador et al., 2001).
In the Arabidopsis genome, over 40 U-box-ARM proteins were identified using sophisticated data-mining approaches (Mudgil et al., 2004). In addition to Arabidopsis and rice U-box/ARM repeat proteins homologous to SPL11, BLAST2 algorithm search of the NCBI database identified several expressed U-box-ARM proteins from other plant species as well. Two of them, ACRE276 and NtPUB4, were isolated from tobacco and were speculated to be involved in Cf9/Avr9 elicited defense and tobacco development signaling, respectively (Durrant et al., 2000; Kim et al., 2003). The parsley CMPG1 responded immediately after pathogen infection and the mangrove bg55 was induced in high salinity stress (Banzai et al., 200; Kirsch et al., 2001). SPL11 is related to these proteins in amino acid sequence and overall structure. The sequence similarity between
SPL11 and these proteins is mostly restricted to the U-box and ARM repeats. The sequence identity between SPL11 and these proteins in the U-box domain ranges from
75% to 47%, with those amino acid residues key to the U-box function highly conserved
(Ohi et al., 2003) (Figure 14A). The overall distribution and position of the ARM repeats
71 in these proteins are shown in Figure 5B. The number of ARM repeats in these proteins is different, varying from 3 to 7. Sequence comparison between the ARM repeats of these proteins and SPL11’s ARM repeats indicated a sequence identity ranging from 19% to
71% in the ARM domain (Figure 14B, and Figure 15). Phylogenetic analysis between
Arabidopsis U-box/ARM repeat proteins (Azevedo et al., 2001) and SPL11 indicated that
SPL11 is evolutionally most close to AtPUB13 (Figure 14C). Compared to other rice U- box/ARM repeat proteins of which full-length cDNAs are available in the public database, SPL11 is most highly related to the protein deduced from the cDNA
AK121978, with overall 57% sequence identity (data not shown). No significant SPL11 homolog was identified in human, animals, and yeast, suggesting SPL11 might be unique to plants.
Molecular Properties of the Spl11 Gene and Spl11 analogs in rice genome
To determine the exact mutation site of the Spl11 gene in the spl11 mutant, the genomic DNA fragments that span the ΔBslI restriction site were identified from both spl11 and IR68. DNA sequencing revealed a unique nucleotide substitution of T for C in the spl11 gene, a substitution that eliminates the BslI restriction site originally present in the wild-type IR68 genome (Figure 10D-3). This point mutation occurs in the first exon of the Spl11 gene, resulting in a premature stop codon in the spl11 transcript.
Southern analysis of 9 rice japonica or indica cultivars indicated that rice genome contains a single copy of the Spl11 gene (data not shown). Nevertheless, a whole genome scale sequence analysis revealed 77 annotated U-box proteins in rice, among which 29 showing a U-box-ARM overall structure (L.-R. Zeng et al., unpublished data). A search
72 for SPL11-like proteins in the rice full-length cDNA database KOME
(http://cdna01.dna.affrc.go.jp/cDNA/) identified 11 U-box-ARM proteins in addition to a partial cDNA of Spl11 (Table 5). The existence of a large number of U-box-ARM proteins in rice genome suggests their probable involvement in a wide range of cellular processes.
SPL11 Possesses E3 Ubiquitin Ligase Activity in vitro and the U-box domain is essential for the E3 ligase activity
As one of the important features of U-box containing proteins is to function as E3 ubiquitin ligases (Hatakeyama et al., 2001), we wanted to determine whether SPL11 also possesses E3 ligase activity. SPL11 (residues 112 to 694) was expressed in Escherichia coli as a fusion with maltose-binding protein (MBP) and was purified by affinity chromatography. Mouse E3 ubiquitin ligase CBL (GI:38605691) was included in the experiment as a positive control. In the presence of wheat E1 and an Arabidopsis E2
AtUBC9, ubiquitination activity was observed for the purified MBP-SPL11 and MBP-
CBL proteins (Lane 5 and 6 from the left, respectively), whereas in the absence of any of the E1, E2, or E3 (lane 1 to 4 from left) no signal was detected (Figure 16A). These results indicated that SPL11 possesses E3 ligase activity.
It has been shown that the U-box is essential for the E3 ligase activity of U-box proteins (Hatakeyama et al., 2001). To test if an intact U-box domain is required for
SPL11 E3 ligase activity as well, two versions of SPL11 bearing mutation in the U-box domain were tested for E3 activity. The first version carried a single mutation that results in a Val290 to Arg290 single amino acid residue change (Figure 14A, denoted by asteroid).
73 This Val is highly conserved among different U-box proteins and a Val to Ile point mutation in the yeast protein Prp19p U-box domain leads to pre-mRNA splicing deficiency in vivo (Ohi and Gould, 2002). The second version carried a three-amino acid residues deletion (ΔC314P315T316) in the U-box domain (Figure 14A, denoted by black arrows). The C314 and P315 also are highly conserved and were reported to be essential for correct folding of the U-box domain to form an appropriate interface interacting with E2 ubiquitin conjugase (Ohi et al., 2003). In vitro ubiquitination analysis indicated that the
E3 ligase activity was completely abolished in both versions of SPL11 (Figure 16B), indicating that an intact U-box domain is required for SPL11’s E3 ligase activity.
Expression pattern of Spl11 in blast-infected rice plants
It is assumed that Spl11 is involved in rice defense signaling based on the enhanced resistance of the spl11 mutant to rice pathogen attack (Yin et al., 2000). To investigate the role of Spl11 in defense against rice pathogens, the expression of Spl11 in rice blast-infected resistant (carrying the Pi2 R gene]) and susceptible (without the Pi2 gene) plants was monitored by northern blot and RT-PCR analyses (Liu et al., 2002).
RNA was isolated from both resistant and susceptible plants 0, 12, 24 and 72 hr after inoculation with isolate PO6-6 avirulent to Pi2. Both northern hybridization and RT-PCR results indicated that Spl11 was induced at 12 and 24 hr after blast inoculation in resistant and susceptible interactions (Figure 17). No difference was detected in the pattern and level of Spl11 expression between the resistant and susceptible plants. Inoculation with
PO6-6 on IR68 plants (resistant reaction) confirmed the induction of Spl11 by rice blast
74 at early infection stages (data not shown). These results suggest that Spl11 is not R gene- dependent and might be involved in the basal defense signaling against rice blast.
DISCUSSION
To expand our understanding of cell death in plant disease resistance, we previously characterized nine rice lesion mimic mutants for their resistance to fungal and bacterial pathogens (Yin et al., 2000). Amongst the nine mutants, spl11 was the only one that showed enhanced non-race specific resistance to both rice blast and bacterial blight diseases, suggesting that the resistance pathway Spl11 involved in does not function in a gene-for-gene fashion. This was supported by the finding that Spl11 was induced in both incompatible and compatible rice-blast interactions. Nevertheless, how this non- differential expression of Spl11 in both resistant and susceptible rice-pathogen interactions could explain the enhanced non-race specific resistance of spl11 remains to be elucidated.
A link between U-box-mediated ubiquitination and cell death has not been clearly established in plants. Our finding that Spl11 encodes a U-box protein endowed with E3 ubquitin ligase activity and the U-box domain is essential for its E3 activity is significant for our understanding of PCD and disease resistance in plants. The ubiquitination related to the spontaneous cell death phenotype of spl11 is analogous to the wide involvement of ubiquitination in the regulation of apoptosis in mammals (Lee and Peter, 2003).
Mechanistically regulation of apoptosis by ubiquitination always occurs via the ubiquitination of key pro- and anti-apoptotic regulators. In animals, a large number of
75 components forming a complicated signaling network involved in the regulation and execution of apoptosis have been identified in the last decade. The HECT domain protein family and RING-finger domain-containing protein family, including the Skp1-
Cdc53/Cullin1-F-box (SCF) multisubunit E3 complexes, have been implicated in targeting these apoptotic regulators for degradation (Wilson et al., 2002; Wing et al.,
2002; Miyazaki et al., 2003). Although it is unclear at present how SPL11-mediated ubiquitination is regulated and how it functionally contributes to PCD and defense activation in the spl11 mutant, the indication of E3 activity for SPL11 suggested an involvement of a new family of E3 ubiquitin ligases in plant PCD and defense.
Non-U-box protein-mediated ubiquitination has been recently shown to be associated with plant disease resistance in several cases. For example, several RING- finger type E3 ubiquitin ligases were induced after elicitor or pathogen treatments
(Durrant et al., 2000; Takai et al., 2002). Recently, the plant SGT1 protein, which interacts with a convergence component of multiple R gene-mediated signaling pathways, RAR1, was found to interact with the SCF ubiquitin ligase complex as well as the COP9 signalosome (Azevedo et al., 2002). Despite all these findings, key questions such as 1) what are the substrates targeted by the ubiquitination in plant defenses and 2) when and where (i.e., at what level) does ubiquitination operate in regulating the defense reactions remain to be addressed. It is possible that one or more substrates targeted by
SPL11 are functionally related in PCD and defense signaling pathways. In animals, the
IAPs (inhibitor of apoptosis proteins) contain at least one baculoviral IAP repeats (BIR) domain at the N-terminal and often a RING domain at the C-terminal. The combination of BIR-mediated binding, and hence inactivation of proteins and RING-mediated
76 proteolysis of proteins, has been shown to be central to the role of IAPs in regulating apoptosis (Lee and Peter, 2003). In this regards, it would be interesting to determine whether the ARM repeat domain in SPL11 functions with the U-box domain in a way similar to that between the BIR domain and the RING domain in animal IAPs. The identification and characterization of the substrates in SPL11-mediated ubiquitination will be essential to answer such questions and to obtain an in-depth understanding of the role of the SPL11-mediated ubiquitination in PCD and defense.
The Spl11 mutant phenotype could be caused by the disruption of negative regulation of PCD and defense activation or simply be a reflection of perturbed cellular homeostasis. Although it is difficult to distinguish these two possibilities with certainty, two lines of evidence support the first explanation. Firstly, most of the genes differentially expressed in spl11 in our microarray hybridization experiment of spl11 are related to cell death and defense (L.-R. Zeng, T. Zhu and G.-L. Wang, unpublished data).
In contrast, few genes involved in other well-defined cellular processes could be identified, suggesting that the spl11 mutant, like the Arabidopsis mutant acd11, is not excessively pleiotropic (Brodersen et al., 2002). Secondly, we have identified several putative spl11 suppressors in the screening for mutants with alleviated spl11 phenotype from a DEB-treated spl11 population (Leung et al., unpublished data). Multiple genes might suppress the lesion formation since there was a wide range of lesion phenotypes in terms of lesion numbers among these mutants. The identification of spl11 suppressors suggests that the spl11 phenotype is genetically programmed.
Only a few U-box proteins can be identified in the genomes of human,
Caenorhabditis elegans and Drosophila melanogaster (Azevedo et al., 2001). By
77 contrast, dozens of U-box proteins were identified in Arabidopsis and rice genome
(Mudgil et al., 2004). Such proteins likely exist widely in other plants as well. The existence of a large number of such proteins in plants suggests that they may play diverse roles in multiple processes. So far, SPL11 represents the first case of a U-box/ARM repeat structure protein related to both PCD and defense. Our results indicated that ubiquitination is involved in PCD and defense in plants. This is consistent with the emerging plant disease defense model suggesting that the signaling components/regulators in the plant PCD and defense pathways need to be inactivated and degraded in a temporally and spatially ordered manner similar to what is observed in animals. This strongly suggests that the ubiquitination pathway could play a significant role in regulating plant PCD and defense as well. Therefore, the cloning and characterization of the Spl11 gene hereby opens a way to dissect the mechanism by which the ubiquitination system contributes to the control of PCD and disease resistance in plants.
78
IR64 GR5717 GR5612 DB2487 spl11 IR64 -b 11c WT/12d 13 WT/14 12WT /13 - GR5717 - 71 LM/71 65 LM/65 33 LM/33 GR5612 - 35 LM/35 56 LM/56 DB2487 - 58 LM/58 a: When crossed to IR64, the lesion mimic mutants were used as the female parent. WT, wild type; LM, lesion mimic b: Corresponding cross was not made. c: The number of F1 plants with corresponding phenotype. d: Total number of F1 plants of the corresponding cross.
Table 4: Allelism tests between rice mutant spl11 and three IR64 lesion mimic mutantsa
79
GI Deduced protein in U-box domain2 ARM domain2 (amino acids) start end start end 37991601 726 2943 367 419 672 32977629 611 228 301 354 558 32981103 604 228 301 354 558 32987300 637 261 335 385 591 32982913 375 2 75 127 249 32982589 796 223 297 542 745 32991766 824 228 302 570 773 32972610 535 103 177 237 400 32984738 405 43 117 176 296 32975325 680 275 347 406 446 37990170 824 13 92 594 635
Table 5. U-box-ARM proteins identified in the rice full-length cDNA database
KOME1. 1. The prediction of the U-box domain and ARM domain is conducted by combination of results from two databases, Pfam
(http://www.sanger.ac.uk/Software/Pfam/) and NCBI (http://www.ncbi.nlm.nih.gov/).
2. The cutoff E-value for prediction of U-box is 1e-10, while for ARM repeat is 1e-05.
3. The position of the corresponding amino acid residue in the protein sequence.
4. A partial Spl11 cDNA AK105835 (GI:32991044) from KOME database is not included in the table
80 Figure 10. Physical delineation of the spl11 mutation in rice. (A) Schematic representation of the BAC contig spanning the Spl11 locus. The overlaps between BAC inserts are displayed to scale as open bars. The dotted vertical lines mark the positions of
DNA markers. The BAC insert containing the Spl11 locus is highlighted in light gray.
Orientation of rice chromosome 12 is indicated in the upper right corner. (B) Fine physical mapping of Spl11 in BAC78. The two cross-hatched gray bars denote the sequenced regions in BAC78. The vertical dotted lines denote the positions of the respective CAPS markers. The number of recombinants/number of segregants tested is indicated for each marker. Arrows above the bars mark the NotI cutting sites of the
BAC78 insert. T7 and Sp6 indicate the orientation of the insert cloned into the BAC vector pBeloBAC11. The position of the subclone TAC20 insert that contains the Spl11 gene is displayed. (C) Prediction of potential coding sequences in the 27 kb region of
TAC20 where the Spl11 gene was physically delimited. The gray bar depicts the sequenced area. The three solid gray lines designated as G1, G2, and G3 indicate the regions with high coding probability. The vertical lines mark the BslI cutting sites. The asterisk denotes the putative mutation site in spl11. Exons predicted in G3 by the programs GENSCAN and Fgenesh using different matrixes are displayed in dark gray.
(D) RFLP fingerprinting of IR68, spl11 and Nipponbare genomic DNA at the Spl11 locus and detection of a point mutation detected in the spl1l gene. Nineteen restriction enzymes were analyzed but only the results of BslI are shown. 1. Genomic DNA was digested with
BslI and then separated on a 1.0% agarose gel. A TAC20 insert digested with HindIII was used as the probe for hybridization. 2. The same blot hybridized with a TAC20 insert in
#1 was used. DNA spanning the putative mutated BslI site was amplified from TAC20
81 and used as the probe. Gray arrows denote the polymorphic bands. M: λ/HindIII DNA marker (New England BioLabs, Beverly, MA). 3. DNA sequence in the vicinity of the spl11 mutation. The C to T point mutation in spl11 is denoted by arrow. This point mutation causes a premature stop codon as marked by underline. The asteroid marks the start codon for the SPL11 protein.
82
83
Figure 11. Analysis of rice IR64 lesion mimic mutants allelic to spl11. (A) Lesion phenotype of spl11 and IR64 background lesion mimic mutants. Picture was taken of leaves from two-month-old plants. (B) Southern blot analysis of the Spl11 locus in wild- type plants and different mutant lines. 1. Genomic DNA was restricted by BslI and then separated on a 1.0% agarose gel. A 2.5 kb genomic DNA fragment at the 5’ end of the
Spl11 gene was used as the probe. 2. Genomic DNA was digested by EcoRI and PstI, respectively. The same probe in #1 was used for the hybridization. Changes detected at the Spl11 locus in the IR64 mutants are described in the text. (C) Transcript analysis of
Spl11 in IR64 lesion mimic mutants by RT-PCR. Spl11- specific primers were used to amplify a 0.84 Kb Spl11 cDNA fragment from total RNA. The rice Actin1-specific primers were used in the RT-PCR to quantify the cDNA template. The experiment was repeated three times.
84
Figure 12. Functional complementation test of rice Spl11 candidate gene. The leaves of two-month-old rice plants are shown. Wild type TP309 and mutant TP2-3
(TP309spl11/spl11) are used as non-transgenic control. Line pGW78-34 and pGW78-161 are
Spl11 transgenic plants: spl11 mutant TP2-3 (TP309spl11/spl11) with the 8.06-kb XbaI-PacI fragment of the wildtype gene. Line pGW78-28 indicated as an example of failure in transformation.
85
Figure 13. Transcript abundance of Spl11 in different rice tissues, gene structure, and deduced amino acid sequence of Spl11. (A) RNA blot analysis of the Spl11 transcript accumulation in different tissue. Total RNAs from 3 week-old leaves, stems, 86 and roots of IR68 were probed with a 0.84 kb cDNA fragment at the 3’ portion of Spl11.
Ethidium bromide staining of rRNA was used as a loading control. L: leaf, S: stem, R: root. (B) Spl11 gene structure. Exons are denoted as black boxes. The number below each exon indicates the length of the exon in basepairs. (C) Deduced amino acid sequence of
Spl11. The N-terminal alanine-rich region is in bold. Amino acids of the coiled-coil domain (145 to 165) are highlighted. The U-box domain (272 to 346) is boxed. Amino acids of the ARM repeat motifs are displayed in italics and boldface. (D) Sequence alignments of the ARM repeat of β-catenin and SPL11. Numbers of the ranges of amino acids composing each repeat are shown on the left. The repeats are structurally similar, with each repeat containing three helices H1, H2, and H3, as indicated. The chemically conserved hydrophobic and polar residues are highlighted in dark and light gray, respectively.
87
Figure 14. Amino acid sequence alignments between rice SPL11 and U-box-ARM proteins from other plant species. (A) Sequence alignments in the highly conserved U- box domain of SPL11 and those of other U-box-ARM proteins. The numbers on the left or right indicate the amino acid residues. Gaps, which were introduced to maximize alignment, are indicated by dashes. The residues conserved among the compared sequences are boxed in black or light gray based on the degree of conservation.
AK121978 from O. sativa (GI:37991601), ARC1 from B. napus (GI:2558938),
ACRE276 from N. tabacum (GI:30013679), AAM91213 from A. thaliana (GI:
22136270), bg55 from B. gymnorrhiza (GI:14149112), PHOR1 from S. tuberosum
(GI:13539578), CMPG1 from P. crispum (GI:14582202), NtPUB4 from N. tabacum
(GI:28974687). Only the one most highly related to SPL11 from rice and Arabidopsis, 88 respectively is included in the alignment due to the large number of U-box-ARM proteins present in rice and Arabidopsis genome. The asteroid and black arrows marked the amino acids that were mutated in the SPL11 E3 ligase activity assay. (B) Schematic representation of SPL11 and other U-box-ARM proteins of plants. The black box indicated the U-box domain and the individual ARM repeat of the ARM domain is indicated by numbered shaded box. The percentage of sequence identity of the ARM repeats from plant U-box-ARM proteins to their most homologous ARM repeats in
SPL11 is indicated. Detail sequence alignment in the ARM domain of SPL11 with those of other U-box-ARM proteins is indicated in Supplemental Figure 1.(C) Phylogenetic relationship between SPL11 and Arabidopsis U-box/ARM repeat proteins. The phylogenies were generated with neighboring joining with 400 bootstrap replicates and were rooted at midpoint. The bootstrap values are shown as percentages. AtPUB8 (locus
At4g21350) was not included in the tree due to no EST, SAGE tag, or cDNA was identified for the corresponding predicted gene.
89 Figure 15. Sequence alignment between the ARM repeats of SPL11 and other plant
U-box-ARM repeat proteins. The numbers on the left or right side indicate the position of the amino acid residues. Gaps, which were introduced to maximize alignment, are indicated by dashes. The ARM repeats of other plant proteins were aligned to their most homologous ARM repeats in SPL11, e.g., the number 2 to 7 ARM repeats of ARC1 were aligned to SPL11 ARM repeat 1 to 6. 90
Figure 16. E3 ubiquitin ligase activity of SPL11.
(A) MBP-SPL11 and MBP-CBL fusion protein were assayed for E3 activity in the presence of E1 (from wheat, GI:136632), E2 (AtUBC9, GI: 20136191), and 32P-labelled ubiquitins. The numbers on the left denote the molecular weight of marker proteins in kilodalton. Mouse E3 ubiquitin ligase CBL (GI:38605691) was used as a positive control.
32P-ubiquitin is indicated by arrow. MBP itself was used as a negative control.
(B) E3 ligase activity of SPL11 and its mutants. CK: MBP; lane 1, WT SPL11; lane 2,
SPL11 (V290R); lane 3, SPL11ΔC314P315T316. 32P-ubiquitin is indicated by arrow. Lower part of figure shows two times of corresponding amount of MBP and MBP fusion proteins used in the E3 activity assay.
91
Figure 17. Expression patterns of Spl11 in rice-blast interaction. Total RNA was isolated from infected leaves at the indicated hours after rice blast inoculation. About 10 µg of total RNA were loaded in each lane in northern blot. 32P labeled-Spl11 cDNA fragment (0.84 kb) was used as the probe in the RNA hybridization. A pair of Spl11- specific primers was used in the RT-PCR analysis. The rRNA gel picture shows the loading quantification of the RNAs in the northern blot analysis. The amplification of the rice Actin1 gene was used as a control for equal amount of total RNAs in the RT-PCR analysis. The numbers denoted the hours after rice blast inoculation.
92 CHAPTER 5
IDENTIFICATION OF SPL11-INTERACTING PROTEINS
ABSTRACT
The discovery that Spl11 encodes an U-box-containing E3 ubiquitin ligase suggested the involvement of ubiquitination system in Spl11-mediated suppression of cell death and/or defense activation. To identify putative substrates of the SPL11-mediated ubiquitination or other components that could putatively be involved in SPL11-mediated suppression of cell death and defense activation, we performed a yeast two-hybrid (Y2H) screening for SPL11-interacting proteins (SPINs) using the ProQuest system (Invitrogen,
CA) in this study. Eight different SPINs were identified when over one million rice cDNA clones were screened using the SPL11 ARM repeat domain as the bait. Two of the
SPINs are pre-mRNA splicing-related proteins, suggesting a connection between alternative splicing and Spl11-mediated cell death and defense signaling. The expression of SPIN1 was induced at 72 hr after M. grisea innoculation.
93 INTRODUCTION
Of the multiple mechanisms employed by eukaryotes to control protein level and activity, the ubiquitin-proteasome pathway is considered to be dominant and its importance in cellular regulation has been increasingly appreciated in the past several years. Substrates targeted by this system include signaling ligands and receptors, transcriptional factors, abnormal proteins and other short-lived proteins (Ciechanover and
Schwartz, 1998). These components participate in basic cellular processes as diverse as regulation of cell cycle and division, cellular response to stress and to extracellular modulators, morphogenesis of neuronal networks, modulation of cell surface receptors, ion channels and the secretory pathway, DNA repair, biogenesis of organelles, regulation of the immune and inflammatory responses and apoptosis. The discovery that Spl11 encodes a U-box-containing E3 ubiquitin ligase suggested the involvement of ubiquitination system in Spl11-mediated suppression of cell death and/or defense activation (see Chapter 4). Some immediate questions followed this discovery include what the substrates of the SPL11-mediated ubiquitination are?; and what components could be putatively involved in the SPL11-mediated suppression of cell death and defense activation? Identification of SPL11-interacting proteins (SPINs) will be essential in seeking answers to these questions.
Physical interactions between proteins are integral to many biological processes, such as signal transduction and transcription. The targeting of substrate proteins for the ubiquitination involves protein-protein interaction as well. The way E3 ubiquitin ligase interacts with the substrate protein during the ubiquitination process depends on the
94 nature of the E3 ligase involved (Vierstra, 2003). Different techniques, from biochemical approaches such as coimmunoprecipitation, glutathione S-transferase (GST) pull-down, and affinity chromatography, to molecular genetic approaches such as the Y2H system have been developed to study protein-protein interactions. The Y2H was originally developed by Fields and Song (1989) to detect interactions between two known proteins or to search for unknown partners (preys) of a given protein (bait). Ever since then, various derivatives of the original system have been developed and the technique has become a routine tool for the study of protein-protein interactions.
We performed Y2H screening for SPINs using the ProQuest system (Invitrogen,
CA) in this study. Eight different SPINs were identified when over one million rice cDNA clones were screened using the SPL11 ARM repeat domain as the bait. Two of the
SPINs are pre-mRNA splicing-related proteins, suggesting a connection between alternative splicing and Spl11-mediated cell death and defense signaling. The expression of SPIN1 was induced at 72 hours after M. grisea inoculation.
MATERIALS AND METHODS
The ProQuest Y2H system
The principle of the ProQuest system in the identification of protein-protein interaction is illustrated in Figure 18. Briefly, yeast strain MaV203 that has three reporter genes (HIS3, URA3 and lacZ) stably integrated in single-copy numbers at different loci in its genome is used in the system. The promoter regions of URA3, HIS3,and lacZ are unrelated (except for the presence of GAL4 binding sites). Two low-copy-number
95 expression vector pDBLEU and pPC86 are used to build bait or prey construct. The interaction of the bait to the prey will lead to the induction of the HIS3 and URA3 reporter genes, which allow two-hybrid-dependent transcription activation to be monitored by cell growth on plates lacking histidine or uracil, respectively. Induction of the lacZ gene results in a blue color when assayed with X-Gal (5-bromo-4-chloro-3- indolyl-β-D-galactopyranoside). Moreover, two-hybrid-dependent induction of URA3 results in conversion of the compound 5-fluoroorotic acid (5FOA) to 5-fluorouracil, which is toxic. Hence, cells containing interacting proteins grow when plated on medium lacking uracil, but growth is inhibited when plated on medium containing 5FOA (Figure
18).
Plasmid constructions
The expression vector pDBLEU from the ProQuest system was used to build the bait constructs. Two baits containing the SPL11 U-box domain and ARM repeat domain were prepared and used in the screenings, respectively. cDNA spanning the SPL11 U-box domain (amino acid number 260-355) was amplified with a primer pair that contains adaptor sequences for restriction enzymes NcoI and SalI. The amplified cDNA was then cloned into the pDBLeu vector using the same two adaptor restriction enzymes. The two primers that were used for the SPL11 U-box domain cloning were: 11Y2Hubox-F: 5’-
ATAAGTCGACCAGGGTATTGGATTCAAATGG-3’; 11Y2Hubox-R: 5’-
TCTTCCATGGTGTTAGGCTGGGTTGAGCG-3’. To build the construct that contains the SPL11 ARM repeat domain (Amino acid number 379-653), cDNA was amplified with a primer pair containing adaptor sequences for restriction enzymes NcoI and NheI at
96 first. The amplified cDNA was then cloned into the pDBLeu vector. The two primers that were used for the SPL11 ARM repeat domain were 11Y2Harm-F: 5’-
ATAAGCTAGCCCAGACACTGAGGAGCAGAG-3’; 11Y2Harm-R: 5’-
TCTACCATGGCTCTTGTTGCTGGACTAGGAA-3’. The insert of both constructs was sequenced and confirmed to be in-frame with the vector sequence and no mutation was introduced in the cloning process.
Construction of the rice cDNA library using prey vector pPC86
Leaves from three-week-old seedlings of rice 75-1-127 plants containing the rice blast resistance gene Pi9 (Liu et al., 2002) were harvested 24 hours after the inoculation of M. grisea isolate PO6-6. Total RNA was isolated using Trizol agent (Invitrogen,
Carlsbad, CA). mRNA was then purified from the total RNA using Absolutely mRNA
Purification Kit (Stratagene, La Jolla, CA). Finally, cDNA library was constructed from mRNA using the cDNA Synthesis Kit (Stratagene, La Jolla, CA). The isolation of total
RNA, purification of poly (A) RNA, and construction of the cDNA library were conducted according to the protocol provided by the manufacturer. Plasmid DNA from pools of all clones in the cDNA library was then purified using the QIAGEN plasmid maxi kit (QIAGEN, Valencia, CA) and cloned into the expression vector pPC86 of the
ProQuest system using the restriction enzymes NotI and SalI. The cloning of the cDNAs into the pPC86 vector was conducted according to the protocol provided by the manufacturer.
97
Screening the rice cDNA prey library
The preparation of competent cell of yeast strain MaV203, determination of the concentration of 3-Amino-1,2,4-Triazole (3AT) required to titrate basal HIS3 expression levels, screening for putative positive clones interacting to one of the two baits, reassessment of the interactions of the putative positive clones identified in the first rounds of screening, and test of self-activation of MaV203 cells containing pDBLeu-X
(X refers to SPL11 U-box domain or SPL11 ARM domain) or pPC86-Y (Y refers to the rice cDNA of putative positive clones identified in the screenings) were performed according to the procedures provided by the manufacturer of the ProQuest system. The concentration of 3AT used in screening for SPL11 interactors was determined to be 25 mM according to the protocol provided by the manufacturer.
After reassessing the interactions of putative positive clones and testing self- activation of them, true positive clones were then determined if they contain the same gene by the following procedures. Plasmid DNA of true positive clones were purified using QIAGEN plasmid mini kit (QIAGEN, Valencia, CA). The plasmid DNA was then digested by NotI and SalI and the digestion product was subjected to electrophoresis on
0.8% agarose gel. The insert of the plasmid DNA was amplified and digested with four different restriction enzymes, AvaII, HinfI, HindIII, and BglII, respectively and the digestion product was separated on 0.8% agarose gel. Those clones that gave same restriction pattern in all five cleavage reactions were considered to contain the same rice cDNA. The primers used for amplification of insert of positive clones are: Y2HPPC86F:
5’-TATAACGCGTTTGGAATCACT-3’; Y2HPPC86R: 5’-
98 GTAAATTTCTGGCAAGGTAGAC-3’. After the grouping of the postive clones, insert of at least one clone from each group was then sequenced.
Northern Blot Analysis
The same procedure as in Chapter 4 (page 60 of this dissertation) was used.
RESULTS
Eight different proteins were identified to interact with SPL11 ARM repeat domain
In total, more than 1.5 million yeast colonies were screened in this study. Among them, around half million were screened using the SPL11 U-box domain as the bait. No positive clone was identified to interact with SPL11 U-box domain. Twenty-nine positive clones were identified when SPL11 ARM repeat domain was used as the bait (Table 6).
Endonuclease restriction pattern analysis classified these positive clones into eight different groups. Eleven clones that displayed strong interaction with SPL11 ARM repeat domain was found to belong to the same group. Sequencing analysis and BLAST2 algorithm search against the NCBI database (http://www.ncbi.nlm.nih.gov/) indicated that these clones contained eight different genes (SPIN1-SPIN8). Except for SPIN6, all proteins encoded by the eight genes have intermediate to strong interactions with SPL11.
It is noteworthy the SPIN1 encodes a K homolog (KH) domain-containing RNA-binding protein and SPIN4 encodes a RNAse P Rpr2/Rpp21 (RPR) domain-containing protein with putative pre-mRNA cleavage function, both are related to pre-mRNA processing.
The eleven clones that displayed strong interaction in yeast cells possess the same rice
99 cDNA, SPIN1. Seven positive clones were found to harbor the same gene SPIN2, which encodes a homolog of plant myosin heavy chain protein that usually acts as the molecular motor for many actin-based motility processes in eukaryotes (Tominaga et al., 2003).
SPIN3 encodes a MOM-like protein that is involves in the maintenance of transcriptional gene silencing (Amedeo et al., 2000). SPIN5 encodes a acetylglutamate kinase-like protein. SPIN6 displayed weak interaction with SPL11 and encodes a Rho-GTPase activating protein. Rho-related GTPases from plants (ROPs) are a class of plant proteins that are closely related to the mammalian Rac family, among which Rac2 is a key regulator of the NADPH oxidase. ROPs trigger H2O2 production and hence the oxidative burst, most likely by activating the NADPH oxidase (Agrawal et al., 2003). Two of the
SPINs, SPIN7 and SPIN8 show no hits to known functional groups in the BLAST2 algorithm search.
SPIN1 is a member of small family in rice genome and is induced be pathogen challenge.
The finding that two SPINs are pre-mRNA processing-related proteins suggested that SPL11-mediated suppression of cell death and defense activation might be connected to pre-mRNA processing. Importantly, eleven of the 29 positive clones encode the same
KH domain protein SPIN1. The KH domain was first identified in the human heterogeneousnuclear ribonucleoprotein (hnRNP) K. It is a domain of around 70 amino acids that is present in a wide variety of quite diverse nucleic acid-binding proteins (Burd and Dreyfuss, 1994). Searching of rice full-length cDNA database KOME
(http://cdna01.dna.affrc.go.jp/cDNA/), TIGR rice gene index database
100 (http://www.tigr.org/) and NCBI database for SPIN1 homologs identified six rice KH proteins homologous to SPIN1 (Figure 19). High homology was detected within the ~120 amnio acids of the C-terminus among the members of the SPIN1 family. This region shows homology to the yeast splicing factor (branch point binding protein) MSL5 (data not shown). SPIN1 is homologous to human Sam68 (Src-associated in mitosis, 68 kDa) which was implicated in human apoptosis (Wong et al., 1992). HEN4 and HUA1 were the only two plant KH-domain containing proteins that have been found to be involved in
Arabidopsis flower development (Cheng et al., 2003). To date, no plant KH-domain containing proteins identified have been implicated in PCD and defense response.
To investigate if the expression of SPIN1 is correlated with cell death occurrence in rice disease resistance, expression of SPIN1 in rice blast-infected resistant (carrying the Pi9 R gene) and susceptible (without the Pi9 gene) plants (Liu et al., 2002) was monitored by northern blot analysis. RNA was isolated from both resistant and susceptible plants 0, 12, 24 and 72 hr after inoculation with isolate PO6-6 avirulent to
Pi9. Results indicated that SPIN1 was induced at 72 hr after blast inoculation in the Pi9 resistant and susceptible interactions (Figure 20).
DISCUSSION
The Y2H has been demonstrated to be a powerful tool to study protein-protein interaction. The interaction between two heterologous, chimeric proteins in a yeast nucleus was detected in this system. The intrinsic limitation of this system, such as the possible misfolding and subsequent instability of the hybrid polypeptides, their
101 inappropriate subcellular localization, the absence of certain post-translational modifications (such as tyrosine phosphorylation or complex glycosylation), and the lack of physiological context (the absence of physiological spatial and temporal regulation of protein interaction), however, could result in a significant proportion of false-negative and false-positive results during the screening (Causier and Davies, 2002). In addition, the possible self-activation of certain bait or prey, which themselves activate reporter gene transcription, could also lead to false-positive results. In the ProQuest system, some precautions have been taken to reduce false-positive results. For example, it uses low- copy-number expression vector pDBLEU and pPC86, the three reporter genes (HIS3,
URA3 and lacZ) to be used in the system are stably integrated in single-copy number at different loci in the yeast genome and the promoter regions of URA3, HIS3, and lacZ are unrelated (except for the presence of GAL4 binding sites). During the screening process, self-activation of the two baits and the identified positive clones and large number of
“positive” clones (which may be a sign of false-positive) were not detected, suggesting some, if not all the SPINs we identified could be true SPL11 partners. Nevertheless, the intrinsic properties of the Y2H could not guarantee zero false-positive result. Therefore, the interactions that were identified in this study need to be further tested in different systems, such as GST pull-down assay or co-immunoprecipitation analsysis. Moreover, other system for detecting in vivo protein-protein interaction, such as the recently developed TAP system (Rohila et al., 2004) may help identify SPL11 partners that were missed in this study.
No positive clones were identified when the SPL11 U-box domain was used as the bait in the screening. At least two factors might have contributed to this result. First,
102 the length of the bait is less than 100 amino acids. It is possible that the bait was incorrectly folded in the yeast cell due to the lack the necessary contextual amino acids for folding. This mis-folding may affect the interaction between the U-box domain with its partner. Second, the interaction of the U-box domain and its partner may be transient that is beyond the capability of detection by the Y2H system. Using a bait containing more amino acids that spans the U-box domain or use a different protein-protein interaction detection system may solve the problem.
The identification of two pre-mRNA processing proteins in this study suggested that SPL11-mediated suppression of cell death and defense activation might be related to pre-mRNA processing. Until now, Prp19p is the only U-box-containing E3 ligase that is associated with RNA splicing complexes (Ohi and Gould, 2002; Patterson, 2002). Prp19p is required to maintain the organization and function of the Ntc units that constitute the general splicesome in the cell. The mechanism underlining the connection between U- box-mediated E3 ligase activity of Prp19p and RNA splicing remains to be elucidated.
Similarly, it is possible that SPL11 may involve in alternative splicing in the cell. SPL11 could be a component of the general alternative splicing machinery in the cell or it could work together with SPIN1 and/or SPIN4 to target specific component(s) of a certain signaling pathway (cell death/defense signaling pathway in this study), as shown in
Arabidopsis flowering transition (Simpson et al., 2003). Confirmation of the interaction between SPL11 and SPIN1 or SPIN4 and functional characterization of them in the context of cell death needs to be explored.
103
Gene NCBI Independent Interacting Homolog/putative function name Accession # Positive Strength Clones SPIN1 AK065405 11 Strong KH domain/Pre-mRNA processing protein SPIN2 AK070162 7 Intermediate Putative myosin heavy chain SPIN3 AK058383 4 Between strong MOM-like protein and intermediate SPIN4 AK099689 2 Between strong Pre-mRNA cleavage complex II and intermediate protein Pcf11, S-locus protein 4-like protein SPIN5 AK071756 2 Between strong Acetylglutamate kinase-like protein and intermediate SPIN6 AK103870 1 Weak Rho-GTPase activating protein SPIN7 - 1 Between strong No hits and Intermediate SPIN8 - 1 Intermediate No hits
Table 6. SPL11-interacting proteins identified in the yeast two-hybrid screening. The SPL11 ARM repeats domain was used as the bait. In total, over 1 X 106 colonies were screened.
104 Figure 18. Basis of the ProQuest Y2H system (from the product manual). Top:
MaV203 cells containing pDBLeu-X and pPC86-Y encode fusion proteins DB-X and
AD-Y, respectively. The interaction of X:Y reconstitutes an active transcription factor that binds to the GAL4 DNA binding sequences present in the promoter regions of the 3 chromosomally-integrated reporter genes and activates transcription. Center: Structure of the promoter regions expressing each of the reporter genes and the expected growth/color results. Bottom: Expected growth or color results when tested for induction of the reporter genes for interacting and non-interacting DB-X and AD-Y.
105 Figure 19. Protein sequence alignments of SPIN1 rice homologs. Six SPIN1 rice homologs were identified from the NCBI (http://www.ncbi.nlm.nih.gov/), KOME
(http://cdna01.dna.affrc.go.jp/cDNA/), and TIGR (http://www.tigr.org/) databases.
Except for BAD13019.1 that have only ESTs identified, full-length cDNA for the members in the SPIN1 family were identified. The under line denotes the putative K homolog (KH) domain.
106 1 40
AK063274 (1) ------AK065093 (1) ------AK073986 (1) ------BAD13019.1 (1) MGGRASAAAAVAAFGRRAARLRSWQIRPVPAWILRAVRFP AK069634 (1) ------SPIN1 (1) ------AK072560 (1) ------
41 80 AK063274 (1) ------MEALT--NAEKCFSPA------RAMSPLPL AK065093 (1) ------MDDRIPPPSPLQYSPSP------VHSSPHPL AK073986 (1) ------MDERIPPPAFFQFLPSG------AHSSPHHQ BAD13019.1 (41) PRFFLWRESDVGFKEFLASDLIPPRRDLPFVVAPLHFDCV AK069634 (1) ------MSGGL--YNHQGFSPA------RTLSPQIR SPIN1 (1) ------MSG----LYSPGFSPA------RNLSPQIR AK072560 (1) ------MDGLH--GTDGCFSPG------RAMSPQVR
81 120 AK063274 (23) VRPPPSPGAAGQYLAELLQEQQKIGPFVQVLPICGRLLNQ AK065093 (26) SSLRYSSSERERYLAELLAERQKLAPFVQVLPFCTRLLNQ AK073986 (26) SPLRSPASERERYLAELLAERQKLAPFMQVLPFCNRLLNQ BAD13019.1 (81) EQVGCSIQRLLRYLAELLAERHKLSPFIPVLPNSVRLLNQ AK069634 (23) SNP----EADSQYLSELLAEHHKLGPFMQVLPICSRLLNQ SPIN1 (21) SNPT---DVDSQYLAELLAEHQKLGPFMQVLPICSKLLSQ AK072560 (23) PPVPPDAASGGQYLAELLQEHQKLGPFMQVLPICSRLLNQ
121 160 AK063274 (63) EIMRMSAIVSHLGVREHDRLPIASPNQMHPLPQVPNFCGN AK065093 (66) EILRASSLPPNHNFVDPERIEHGSPLRLPGLPVNGQ--PM AK073986 (66) EILRASSLPPNPNFVEPERVNHGSPLRLTGHPMNGQ--PM BAD13019.1 (121) EILRVSTLLENASLLNQSGLEHGSP--LTTGGLYSNGAAA AK069634 (59) EIMRVSSMVNDHGFNDFDRRRYRSPSPMSSPIMRPNLHGN SPIN1 (58) EIMRVSSIVHNHGFGDFDRHRFRSPSPMSSPNPRSNRSGN AK072560 (63) EIMRVSGMFRQPGVGDFERSQPASPNQMHPSHIVPNFCGN
161 200 AK063274 (103) GFNPWTGTLPEKNGFPRGT-----MGWEGAAHDP--SYIV AK065093 (104) DLEGWSGMQTENMRVLQAS----SMGWNGPPAITG-TPVV AK073986 (104) DLEGWSGMQTEMG-VLQSP----SMGWNVAPGVAG-SPVV BAD13019.1 (159) DMNGWTSAFQSEG---SP-----AYSWRGGSQGSSSGLIV AK069634 (99) GFGPWNGIHQERLGFPPPPPPGTSMDWQGAPPSHG-SYIV SPIN1 (98) GFSPWNGLHQERLGFP----QGTSMDWQGAPPSPS-SHVV AK072560 (103) AFGPWNGMRPERVSFSQG------PGWQGAPQSPS-SYIV
201 240
107 AK063274 (136) KKIVRLEVPTDAYPHFNFIGRLLGPRGNSLKRVEASTGCR AK065093 (139) KKVVRLDVPVDKYPNYNFVGRLLGPRGNSLKRVEASTQCR AK073986 (138) KKVVRIDVPVDKYPNYNFVGRLLGPRGNSLKRVEATTQCR BAD13019.1 (191) KKTMKVDIPVDKYPTFNFVGRILGPRGNSLKRVEATTDCR AK069634 (138) KKIVRMEVPVDAYPNFNFVGRILGPRGNSLKRVEASTGCR SPIN1 (133) KKILRLDVPVDSYPNFNFVGRILGPRGNSLKRVEASTGCR AK072560 (136) KKILRLEIPTDAYPNFNFIGRLLGPRGNSLKRIEASTGCR
241 280 AK063274 (176) VFIRGKGSIKDPIKEEQLKGRPGYEHLSDPTHILIEAELP AK065093 (179) VYIRGRGSVKDSVKEDKLRDKPGYEHLNEPLHVLVEAEFP AK073986 (178) VYIRGRGSVKDSVKEDKLRDKPGYEHLNDPLHVLVEAEFP BAD13019.1 (231) VLIRGRGSIKDPAREDMMRGKPGYEHLNEPLHILVEAELP AK069634 (178) VFIRGKGSIKDADKEEKLKGKPGYEHLNDPLHILIEAELP SPIN1 (173) VFIRGKGSIKDPGKEDKLRGKPGYEHLSDPLHILIEAEFP AK072560 (176) VFIRGKGSIKDPNKEEQLKGRAGYEHLDDPLHILIEAELP
281 320 AK063274 (216) ADVIDTRLAQAQEILEDLLKPVEESQDFLKRQQLRELAVL AK065093 (219) ADIIDTRLNQAVTILEDLLKPIDESMDYYKKQQLRELAIL AK073986 (218) SDIVDVRLNQAVAILEDLLKPVDESMDYYKKQQLRELAIL BAD13019.1 (271) VEIIDTRLIQARDILEDLLKPVDESQDFFKKQQLRELAML AK069634 (218) ANIIDTRLRQAQEIMDELLKPVDESQDYYKRQQLRELAML SPIN1 (213) ASIIDARLRHAQEVIEELLKPVDESQDFYKRQQLRELAML AK072560 (216) ANVIDARLAKAQEILEELLKPVDESQDYYKRQQLRELALL
321 355 AK063274 (256) NSTYREDSP----HQNGSASPFSNGS-TKLGKQ-- AK065093 (259) NGTLREESPSPHLSP--SVSPFNSTG-MKRAKTGR AK073986 (258) NGTLREESPSPHLSPSPSVSPFNSTG-MKRAKTGR BAD13019.1 (311) NGTLREEGMQ----RSGSASPFHNSLGMKRAKTRG AK069634 (258) NSTLREDSP----HP-GSVSPFSNGG-MKRAKPSH SPIN1 (253) NSTLREDSP----HP-GSVSPFSNGG-MKRAKTGQ AK072560 (256) NSPLREESP----HP-GSASPFSNGG-MKRMKQ--
108
Figure 20. Expression patterns of SPIN1 in rice-blast interaction. Total RNA was isolated from infected leaves at the indicated hours after rice blast inoculation. About 10
µg of total RNA were loaded in each lane in northern blot. 32P labeled-SPIN1 full-length cDNA fragment was used as the probe in the RNA hybridization. The rRNA gel picture shows the loading quantification of the RNAs in the northern blot analysis.
109 CHAPTER 6
GENOME-WIDE IDENTIFICATION OF RICE AND ARABIDOPSIS U-BOX
GENES AND ISOLATION OF A MUTANT FOR THE ARABIDOPSIS SPL11
ORTHOLOG
ABSTRACT
The completion of rice and Arabidopsis whole genome sequencing provided an opportunity to identify members of U-box-containing gene family globally. We identified
77 and 63 U-box proteins from the rice and Arabidopsis genomes using a battery of whole genome analysis algorithms, respectively. Expressed sequence tags (ESTs) or full- length cDNAs were found to be associated with most of the genes we identified. The majority of Arabidopsis and rice U-box proteins share similar domain organization. Some of them, however, possess domain organization unique to either rice or Arabidopsis.
Overall, the U-box orthologs between rice and Arabidopsis share high homology at the amino acid level. We also identify the putative SPL11 ortholog from Arabidopsis
(AtSPL11), which shows high level protein sequence identity to rice SPL11. Disruption of AtSpl11 by T-DNA insertion mutation resulted in cell death and stunted growth
110 phenotype analogous to that of rice spl11 mutant, suggesting that the function of the
Spl11 gene might be conserved between monocots and dicots.
INTRODUCTION
E3 ubiquitin ligase plays a central role in selecting the myriad of appropriate candidate proteins during the ubiquitination process (Ciechanover, 1998). This is manifested in that E3s are the most diverse components in the ubiquitination pathway and over 1300 distinct E3 components are encoded by the Arabidopsis genome (Smalle and
Vierstra, 2004). Until recently, four different families of ubiquitin ligases had been described based on their subunit composition and mechanism of action: HECT, SCF,
APC and RING/U-box (Vierstra, 2003). The HECT (homologous to E6-AP COOH terminus) domain is initially identified in viral E6-associated protein (E6-AP) in the study of human papilloma viruses (Scheffner et al., 1993; Huibregtse et al., 1995). It is comprised of ~350 amino acids and its C-terminus contains a conserved active cysteine residue required for the ligase function. The HECT type ligase is the only known E3 that forms an E3-ubiquitin thiolester intermediate before the final attachment of ubiquitin to the substrate. The SCF type E3 ligase is typically composed of four different polypeptides: SKP1 that serves as an adaptor protein, Cullin1 (CDC53), RING-finger protein RBX1 (or ROC1 and HRT1) that interacts with the cullin and E2 conjugating enzyme and an F-box protein that is responsible for recruiting substrates. The SCF type ligase brings the ubiquitin-loaded E2s and the substrate in proximity to promote the transferring of ubiquitin directly from the E2 to the substrates. Besides Cullin1, recent
111 data indicated Cullin2 and Cullin3 also forms similar groups of E3 ligases, in which an elongin C-SOCS module or a BTB protein replaces the SKP1-F-box protein module in the Cullin1 type SCF E3 ligase, respectively (Stebbins et al., 1999; Kurz et al., 2002;
Pintard et al., 2004). The APC (anaphase promoting complex) type E3s are complexes of over 10 proteins. The complex also contains a cullin protein, though their action mechanism is distinct from that of SCF group E3s. The RING (really interesting new gene) finger motif is characterized by the presence of Zn2+- chelating amino acid residues hallmarked as C3HC4 (C, cysteine; H, histidine) or C3H2C3, which forms two cross- brace arranged free loops. Like SCF E3s, the RING type E3 ligases serve as scaffolds to bring together the activated ubiquitin-E2 and the substrate in promoting the transfer of ubiquitin to the substrate. In contrast to the limited number of HECT type E3s identified, the SCF and the RING type E3 are found in hundreds of proteins (Joazeiro and
Weissman, 2000; Stone et al., 2005).
The U-box is a novel E3 ubiquitin ligase activity-related protein domain that was first identified in the yeast ubiquitination factor UFD2 (Koegl et al., 1999). U-box contains ~75 amino acids and possesses a tertiary structure resembling that of the RING finger (Aravind and Koonin, 2000; Ohi et al., 2003). The major difference between U- box and RING domains is that the U-box lacks the hallmark Zn2+-chelating cysteine and histidine residues of RING-finger. Consequently, the conserved zinc-binding residues supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box (Ohi et al., 2003). U-box proteins are present in yeast, plants, animals, and humans (Koegl et al., 1999; Meacham et al., 2001; Stone et al.,
2003). Nevertheless, only a few U-box proteins have been characterized in detail. U-box
112 proteins with predicted or known biological function include UFD2 (ubiquitin chain assemblying (E4), neuritogenesis), Prp19p (pre-mRNA splicing), and CHIP (co- chaperon, stress response and familial parkinson’s disease) that were originally identified from yeast, animal or human (Ballinger et al., 1999; Koegl et al., 1999; Imai et al., 2002;
Ohi and Gould, 2002; Dai et al., 2003; Okumura et al., 2004) and CMPG1 (defense response), PHOR1 (gibberellin signaling), ARC1 (self-incompatibility in pollination), and SPL11 (cell death) that were identified from plants (Amador et al., 2001; Kirsch et al., 2001; Stone et al., 2003; Zeng et al., 2004).
The distribution of U-box proteins is uneven among organisms of different kingdoms with the larger number of such proteins existing in the plant genomes. Only 3
U-box genes are identified among the 6300 annotated genes in the yeast genome.
Similarly, less than 20 U-box proteins were predicted in the human genome (Patterson,
2002). Analyses of the rice fungal pathogen M. grisea genome identified only two putative U-box proteins (L-R Zeng, unpublished). In contrast, 61 U-box proteins were identified in Arabidopsis when a series of analyses were performed (Azevedo et al.,
2001) (http://www.arabidopsis.org/info/genefamily/pub.html). A large part of them, if not all, demonstrated in vitro E3 ligase activity (Mudgil et al., 2004). In plants, Arabidopsis and rice are two systems that serve as a model plant for the study of basic plant biology and as a model plant for the study of traits of agronomic importance in monocot species, respectively (Arabidopsis-Genome-Initiative, 2000; Goff et al., 2002; Yu et al., 2002).
The completion of their whole genome sequencing provided an opportunity to study biological questions comparatively at the genome level between dicot and monocot plants. In rice, genome-wide analysis of its U-box proteins has not been reported. In this
113 study, we identified 77 U-box genes from the rice genome with high confidence using a battery of extensive whole genome analysis algorithms. Using the same algorithms, we identified 61 U-box proteins from Arabidopsis, which makes the total number of U-box proteins presented in Arabidopsis genome grows to 63 when results from Shirasu’s group was combined (http://www.arabidopsis.org/info/genefamily/pub.html). Expressed sequence tags (ESTs) or full-length cDNAs were found to be associated with nearly eighty percent of the U-box proteins we identified in rice and Arabidopsis. The majority of Arabidopsis and rice U-box proteins share similar domain organizations. Some of them, however, possess domain organization unique to either rice or Arabidopsis.
Overall, the U-box orthologs between rice and Arabidopsis share high homology at amino acid level. We also identify the putative SPL11 ortholog from Arabidopsis
(AtSPL11), which shows high level protein sequence identity to rice SPL11. Disruption of AtSPL11 by T-DNA insertion mutation resulted in cell death and stunted growth phenotype analogous to that of rice spl11 mutant, suggesting that the function of the
Spl11 gene might be conserved between monocots and dicots.
MATERIALS AND METHODS
Identification of U-box containing proteins in the rice genome.
The U-box proteins in the genomes of rice japonica and indica subspecies were identified using a battery of algorithms. The complete results are available as supplementary materials at the following address: http://supfam.org/ubox/ubox.html.
Taxonomically, we place the U-box family as a member of the RING domain
114 superfamily. U-box family shows some distant sequence homology to other member families under the RING domain superfamily. To identify them correctly, the U-box proteins must be distinguished from other members in the same superfamily. A crucial difference between the U-box proteins and other superfamily members is the presence of fewer cysteine/histidine residues. The following seven procedures were used in the identification process: (1) WU-blast of known U-box proteins against the genome, (2) searching PFAM models of non-U-box families within the RING domain superfamily
(Bateman et al., 2004), (3) scoring a SAM hidden Markov models (HMM) built from the
PFAM seed alignment of U-box domains (Sonnhammer et al., 1998), (4) a SAM T99
'family' level model was built for U-box proteins (Karplus and Hu, 2001), (5) scoring an
HMM built from the PFAM 'full' alignment, (6) a SAM T99 'superfamily' level model was built and scored. The final results of the above seven sequential analyses were further examined with respect to the number of aligned cysteine residues and multiple sequence alignments annotated with the structural key residues highlighting the differences between U-box family members and other RING domain superfamily members. Additional evidence such as structural information was consulted in some specific cases.
Phylogenetic analysis.
Rice and Arabidopsis U-box proteins were used as queries to search against the
PFAM or GenBank database and were classified into different groups based on their domain organizations. Rice and Arabidopsis proteins from the same group were then aligned using the clustal_X program (Thompson et al., 1997). The aligned sequence data
115 was then inputted into the MEGA2 program (Kumar et al., 2001) to build the phylogenetic tree.
Growth of Arabidopsis plants.
Arabidopsis plants were first grown in a growth chamber with 8 hr light per day, at 26°C day temperature and 23°C night temperature. Four-week-old plants were then transferred to a growth room with 12 hr light per day, 24°C day and night temperature.
Photos of the mutant and wild-type plants were taken at 8 weeks after germination.
Analysis of the co-segregation between the T-DNA insertion mutation and cell death phenotype in an AtSpl11//Atspl11 segregating population.
Small-scale preparations of Arabidopsis genomic DNA were from ~1 cm long rosette leaves of individual plants as described (Lukowitz et al., 1996). The DNA pellet was resuspended in 50 µL of TE, and 1.5 µL was used in a 25 µL PCR reaction. The reaction mixture was cycled through the following temperature profiles: 94°C for 120 s for 1 cycle, followed by 94°C for 45 s, 62°C for 45 s, and 72°C for 30s for 35 cycles. The
PCR product was then separated on 0.8% agarose gel eletrophoresis. The primers used for the analysis include the primer complementary to the T-DNA sequence used for the
Salk Institute T-DNA insertion lines, b: 5’-TGGTTCACGTAGTGGGCCATCG-3’; and two AtSpl11 gene specific primers: c: 5’-
GAAGGAGGTGTCTGTTAAGTTAGAACAA-3’; a: 5’-
AAACAATAACTGGATCTCTCATCATTTCC-3’ (see Figure 23).
116 RESULTS
Identification of U-box-containing proteins from the rice and Arabidopsis genomes.
To have a comprehensive assessment of the rice U-box proteins, genome sequences of two rice subspecies were explored in our analysis (Goff et al., 2002; Yu et al., 2002). U-box proteins from the two annotated genomes were identified with high confidence using the same battery of algorithms (see Materials and Methods). The two set of identified U-box proteins were then compared and combined. In a comparative analysis of corresponding 2.3 Mb DNA of rice chomosome 4 from japonica and indica subspecies, Feng et al. found that the average occurrence of single nucleotide polymorphism (SNP) in the exons is 379 bp and 304 bp per SNP for japonica and indica rice genome, respectively (Feng et al., 2002; Han and Xue, 2003). This means there is on average ~ 1% polymorphism in protein sequence of the same protein encoded by the two genomes. However, proteins from the same loci of the japonica and indica genome may have lower protein sequence identity due to the outcome of different annotation procedures. Therefore we considered U-box proteins from the two genomes that show over 95% protein sequence identity are likely encoded by the same loci and the corresponding genomic DNA sequences of these proteins were then identified and compared manually. The copy of U-box proteins from the indica genome that are confirmed to be encoded by the corresponding loci of that of the japonica genome were then eliminated from the combined list. Seventy-seven rice U-box proteins were identified after these extensive analyses (Table 7). Using the same algorithms, we identified 61 U-box proteins from the Arabidopsis genome, including 3 that were missed
117 in the previous analyses (Azevedo et al., 2001; Mudgil et al., 2004). Comparison between the U-box proteins we identified and those identified by Shirasu’s group
(http://www.arabidopsis.org/info/genefamily/pub.html) indicated that 59 of them are common, suggesting the effectiveness of the algorithms we used in the survey.
Domain organization the rice and Arabidopsis U-box proteins and expression of their corresponding genes.
The U-box protein sequences were used as queries to search against the PFAM database (Bateman et al., 2004) and against the National Center for Biotechnology
Information (NCBI) protein database using the BLASTP algorithm to identify other domain and motifs present. Besides the U-box domain, there are multiple other protein domains and motifs in these proteins (Table 8). Based on their domain/motif organization, the U-box proteins are grouped into nine sub-classes. The numbering of these sub-classes is mandated so that the previous described classes are incorporated
(Azevedo et al., 2001). In general, E3 ubiquitin ligases have a protein-protein interaction domain to appropriately capture substrates for ubiquitination (Patterson, 2002).
Consistent with the expected function as E3 ubiquitin ligase, most rice and Arabidopsis
U-box proteins contain domains or motifs that are involved in protein-protein interactions. The sub-class that consists the largest number of U-box proteins in rice and
Arabidopsis contains the armadillo (ARM)/HEAT repeats. The ARM repeat is an approximately 40 amino acid long tandemly repeated sequence motif first identified in the Drosophila melanogaster segment polarity gene armadillo involved in signal transduction through wingless (Riggleman et al., 1989). Structural characteristics of the
118 ARM motif suggest its involvement in protein-protein interactions, which has been demonstrated in several cases (Huber et al., 1997). In a few cases, HEAT repeats were detected in proximity to or overlap with the ARM repeats. HEAT repeats derive their name from four diverse eukaryotic proteins in which they were first identified: huntingtin, elongation factor 3, PR65/A subunit of protein phosphatase A, and the TOR
(target of rapamycin) (Andrade and Bork, 1995). ARM and HEAT repeats are grouped into the same class in this study due to their structural similarity (Andrade et al., 2001). It is noteworthy that the ARM/HEAT repeats in these proteins is quite divergent as described (Mudgil et al., 2004). The second largest group U-box proteins in rice and
Arabidopsis show no significant domain or motif hits other than U-box in the PFAM or
NCBI databases. Nevertheless, sequence alignments do detect a conserved domain containing ~ 100 residues that is located closely to the C terminus of these proteins
(Figure 21). In addition to the high percentage of leucine residues described before
(Azevedo et al., 2001), a high percentage of homology and several highly conserved residues were detected. We named this putative domain KEGLERS domain after the four conserved residues and its leucine rich feature. Sequence alignments of rice proteins from the same class gave an extremely similar pattern (data not shown). Since no genes from this class have been functionally characterized, the role of this putative domain remains to be elucidated. The other two major domains/motifs present in plant U-box proteins that are involved in protein-protein interaction are tetratrico peptide repeat (TPR) motif and
WD40 repeats (Das et al., 1998; Li and Roberts, 2001). The two rice or Arabidopsis U- box proteins that contain the WD40 repeats are homologous to the animal Prp19p protein that was shown to be involved in pre-mRNA splicing (Ohi and Gould, 2002). In addition
119 to those involved in protein-protein interactions, domains/motifs involved in signal transduction, such as kinase domain and MIF4G also are detected in rice and/or
Arabidopsis proteins.
The distribution of U-box proteins in various classes is different between rice and
Arabidopsis albeit the overall classification is similar between them (Table 8). One arresting difference is that there is only one TPR-U-box type protein in Arabidopsis while seven are identified in rice genome. The TPR is a structural motif present in a wide range of proteins (Lamb et al., 1995). It mediates protein-protein interactions and the assembly of multiprotein complexes (D'Andrea and Regan, 2003). The only Arabidopsis TPR-U- box protein is homologous to the mammalian carboxyl terminus of Hsc70-interacting protein (CHIP)(AtCHIP) and it is involved in temperature stress tolerance (Yan et al.,
2003). In animals CHIP interacts with the protein chaperons such as Hsp70 and Hsp90 and modules the induction of transcription factor HSF1, which plays a role in tuning the response to stress at multiple levels (Ballinger et al., 1999; Wiederkehr et al., 2002; Dai et al., 2003). In addition to stress response, CHIP also regulates the E3 activity of Parkin that are responsible for familial parkinson’s disease (Imai et al., 2002). No rice TPR-U- box type protein has been functionally characterized yet. The MIF4G domain that was merely associated with two Arabidopsis U-box domain is related to transcription initiation.
Expression of these identified U-box genes was investigated by searching the cDNA and EST databases. Evidence for expression was found for the majority of rice and Arabidopsis U-box genes. Corresponding ESTs or full-length cDNAs or both could be found for 60 out of the 77 rice U-box genes, amongst which 44 have full-length
120 cDNAs (Table 8). For Arabidopsis, 49 out of the 63 U-box genes have ESTs or full- length cDNAs or both deposited in the database (Table 9). The rice and Arabidopsis genes were generally expressed in a variety of tissues, such as root, leaf, flower, and calli etc based on the origin of the ESTs.
Identification of a T-DNA insertion mutant of Arabidopsis Spl11 ortholog (AtSpl11)
The successful cloning of the rice Spl11 gene prompted us to investigate the function of its ortholog in the model dicot plant Arabidopsis. Twenty-nine Arabidopsis
U-box proteins possess U-box-ARM repeat type domain organization that is similar to rice SPL11 (Table 9). Phylogenetic analysis indicated that rice Spl11 is evolutionally closer to Arabidopsis gene at loci At3g54850 (AtPUB14), At3g46510 (AtPUB13), and
At2g28830 (AtPUB12) (Figure 22). Six T-DNA insertion lines of the three Arabidopsis highly homologous genes were obtained from the Arabidopsis Biological Stock Center
(ABRC) (http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm). One mutant line that contains a T-DNA insertion in the third exon of the At3g54850
(AtPUB14) displayed typical cell death and stunt growth phenotypes (Figure 23). Other mutant lines possess T-DNA insertion either in the intron or at 3’ UTR and failed to give visible cell death phenotype. PCR analysis confirmed the co-segregation between the T-
DNA fragment and the cell death phenotype. Whether this T-DNA mutant confers enhanced resistance to pathogens in Arabidopsis is under investigation. Nevertheless, these results suggested that Spl11 may share a conserved biological function in both monocots and dicots.
121 DISCUSSION
Seventy-seven U-box-containing proteins were identified from the rice genome using a battery of algorithms in this study. The actual number could be bigger due to rice genome was not completely sequenced when the survey was performed. Sixty-three U- box proteins were identified from Arabidopsis when data from my study and that from
Shirasu’s lab (http://www.arabidopsis.org/info/genefamily/pub.html) were combined. U- box-containing proteins are supposed to possess E3 ubiquitin ligase activity. Of the 79 rice U-box genes, only Spl11 has been demonstrated to have E3 activity. Thus biochemical assay of the ligase activity of the proteins encoded by these rice genes should be included as part of the study next. Compared to organisms from other kingdoms, plant genomes contain a much larger number of U-box proteins. Why plants require more U-box proteins is unclear at present. One speculation is that U-box proteins may play a role in plants’ responding to various environmental stresses, given the lack of animal-like immune system and the static life of plants (Patterson, 2002). Domain organization analysis indicated that the U-box family could be divided into various groups. A number of protein-protein interaction domains, protein phosphorylation-related domain, and transcription initiation-related domain were found to be associated with the
U-box domain in rice and Arabidopsis genome. U-box proteins with similar domain organization from other organisms have been demonstrated to function in different cellular processes (Amador et al., 2001; Kirsch et al., 2001; Ohi and Gould, 2002; Stone et al., 2003). All these data suggest that the biological functions of U-box proteins might be diverse rather than be limited to stress response signaling. Of the rice and Arabidopsis
122 U-box proteins identified hereby, only a few of them have been functionally characterized. A systemic approach, such as virus-induced gene silencing (VIGS) might be essential to functionally characterize these genes in a large-scale.
A majority of the U-box genes identified in this study were expressed, which was demonstrated by the identification of their ESTs or cDNAs. This suggested the effectiveness of the algorithms we used for the survey. More recent expression data such as serial analysis of gene espression (SAGE) (Velculescu et al., 1995) tags and massively parallel signature sequencing (MPSS) (Brenner et al., 2000) tags were not included in the analysis. These data could be important for confirming the expression of the identified genes. MPSS data on different rice tissues and rice under different biotic or abiotic stresses from our lab indicated that eleven out of the nineteen rice U-box gene that had no
EST or cDNA were identified in the databases were expressed (data not shown). The
MPSS tags for the 11 genes were identified only under certain specific circumstances such as salt-stressed rice plant and/or the expression level of most of them is much lower than the U-box genes with their ESTs and/or cDNA identified. This may explain the failure to identify their ESTs and/or cDNA in the databases.
The identification of the AtSpl11 T-DNA insertion mutant that displayed cell death and stunt growth phenotype suggested that Spl11 might play a conserved role in monocots and dicots. Given the abundance of the characterized genetic resources (e.g. cell death and disease mutants) and other advantages of Arabidopsis as a model system for plant molecular biology, the Atspl11 mutant provided us a promising material to investigate the possible connections between the AtSpl11-mediated cell death signaling and other well-known components such as salicylic acid (SA), jasmonic acid (JA),
123 ethylene, ROS, and NO (nitric oxide) mediated signaling pathways. Due to the fact that counterparts of some Arabidopsis genetic resource (e.g. mutants in the known plant PCD signaling pathways) are not available in rice, results obtained in the characterization of this mutant will be an essential complement to the rice studies.
124
Name Sub- Rice genome Protein ID cDNA EST class locus OsUFD2 I Os03g31400 XP_468783.1 - CB639670, CB640908, (OsPUB1) CB619295, CB619296, AU078410, CB643846, CB643847, CB000450, CA764512, AU078411, CR281540, CF328714, CF328715 OsPUB2 II Os05g39930 AAT01358.1 AK099529 D23908, AU164828, AI096152, C73275 OsPUB3 II Os01g60860 BAB93187.1 AK065307 BI809145, AU070503, CB631190, D48112, AU165954 OsPUB4 II Os02g13960 BAD27662.1 AK062592 CB630469，CB630470， CB643247，CB643248， AU032329，CF325064， CK738435，BQ908529， AU173816 OsPUB5 II Os08g32060 BAC98577.1 - - OsPUB6 II Os09g21120 BAD26307.1 - - OsPUB7 II Os04g28100 CAE02102.2 - CB650396, CB650395 OsPUB8 II Os02g28720 BBAD21861.1 - AU225089, CF310095, BI801229, AA754456, BI807368 OsPUB9 II Os02g49950 BAD16137.1 AK102091 CB672091, CB672092, CB634863, CB634864, AU097467, AU033197 OsPUB10 II Os03g16740 - AK121978 BE040196, CA758916, AK060190 CB636819 OsPUB11 II Os12g38210 AAT94161.1 AK105835 CB680406, BF430451, CF305067, AT003699, AT003719, AA749599, AU173870, AU197209, BI812454 OsPUB12 II Os06g01300 BAD67946.1 AK071080 CB629073, CB629075, AK067611 CB661839, CB661840, CB647707, CB647708, CB663113, CB663114, CB661157, CB661158, CB640944, BM421775, AU070995 OsPUB13 II Os06g51130 BAD61809.1 - - OsPUB14 II Os08g37570 BAD10281.1 AK072890 CA762420 (continued)
Table 7. Rice U-box gene names, their correspondence to sub-class, rice genome locus, rice chromosome, GenBank protein IDs, and accessions for their cDNAs and cognate ESTs.
125 Table 7: continued OsPUB15 II Os08g01900 BAD09539.1 AK106557 AU069544, CB675602, AK102080 CB675603, CB637298, CF304665, C26426, CB631051, AU092305, CA760274, CR281099 OsPUB16 II Os01g66130 BAD82582.1 AK072566 CF326076 OsPUB17 II Os08g32610 BAC24957.1 - -
OsPUB18 II Os09g21740 BAD26106.1 - CB655398, CA756899, CB655399, D49059, D49048, AU070443, AU032876 OsPUB19 II Os06g13090 BAD37863.1 - - OsPUB20 II Os06g13080 BAD37898.1 - - OsPUB21 II Os02g49520 BAD16089.1 AK065834 - AK120552 AK062565 AK068514 AK060172 OsPUB22 II Os06g16410 BAD68030.1 AK110058 CB656843，CB656844， CB638682，CB638681， CB638683，CB654780， CB654781 OsPUB23 II Os12g17900 - AK102429 CB644678, CB644679, BE228945, CA765839 OsPUB24 II Os03g45420 AAP50990.1 AK120547, AU101622, AU164585 AK066133 OsPUB25 II Os12g17880 - AK099846 CB672899，CB672900， AK066847 CF306503，CB640118， CB675872，CB675873， AU056255，CB632057， AU056256，BI809202 OsPUB26 II Os03g10400 - AK102337 CB632246, CB632247, CB629580, CB629581, AU181990, CA757822 OsPUB27 II Os04g41250 CAD41127.2 AK100423 C72885, AU082230 OsPUB28 II Os01g67500 BAD82105.1 AK101205 CB679893, CB679894, AK065658 AU094854, BX899859, C91848 OsPUB29 II Os02g28870 BAD21885.1 AK106789 - OsPUB30 II Os08g04470 BAD03021.1 - AU069933 OsPUB31 III Os01g64570 BAD81908.1 - - OsPUB32 III Os04g58920 CAD41926.1 AK108418 CB641925, CB641926, CB641964, CB641965 OsPUB33 III Os02g33590 BAD28838.1 AK108494 BI808859 OsPUB34 III Os04g34030 CAD40926.3 - - OsPUB35 III Os04g49970 CAE04863.2 AK070506 - AK110959 OsPUB36 III Os02g46500 BAD07682.1 AK110831 - OsPUB37 III Os12g06410 - - - OsPUB38 III Os04g35680 CAD40819.2 AK121647 - (continued) 126 Table 7: continued OsPUB39 III Os06g13870 BAD45713.1 - AU174669 OsPUB40 III Os02g50460 BAD16037.1 - AU100984, AU100985, AU063504, AU172336 OsPUB41 III Os03g13740 - AK109161 BQ907174, C27098, AU166815 OsPUB42 III Os10g03440 AAN04506.1 - - OsPUB43 III Os02g34410 BAD16270.1 AU184395 OsPUB44 III Os05g36360 AAT07620.1 AK121082 CB669445，CB669446， CB650659，CB650660， CB645255，CB645256， CB650137，CB650138， CB649839，CB647557， BM421109， BI807625， BI808006，BQ908725， AI096168 OsPUB45 III Os02g33680 BAD28063.1 - - OsPUB46 III Os04g34140 CAE02914.3 AK121255 AU089778, AU089779, D49041 OsPUB47 IV Os06g37620 BAD61851.1 - CB627135, CB668863, TC257914 OsPUB48 IV Os02g12670 BAD17181.1 - C73105, CB096600, CR280370, CB647037, AU075377 OsPUB49 IV Os10g41220 AAK31267.1 AK099675 AU094827, CB672905 OsPUB50 IV Os03g01160 - - AU222811 OsPUB51 IV Os06g04880 - AK065683 CB634275, CB634276, AK105657 CB663207, CB663208, CB661735, CB661736, CB663242, CB676242 OsPUB52 IV Os09g39620 BAD46225.1 - C73201 OsPUB53 IV Os10g40060 AAK43499.1 AK064032 D48628, CB666071, AK073764 CB666072, BP184834 OsPUB54 IV Os10g40100 AAK43512.1 AK066841 CB674756, BM421191 AK065488 OsPUB55 IV Os02g44610 BAD27770.1 AK109040 OsPUB56 IV Os09g39640 BAD46228.1 AK058797 CB631963, CF316548, CF316549, CA882843 OsPUB57 IV Os03g31070 XP_468750.1 AK069682 CB674493 AK067154 OsPUB58 IV Os03g31040 AAP20862.1 - - OsPUB59 IV Os03g31000 AAP20839.1 - - OsPUB60 IV Os02g44610 BAD28396.1 - - OsPUB61 IV Os10g01060 AAL73560.1 AK102556 CB648412 AK061113 OsPUB62 IV Os02g57700 BAD22918.1 - BQ908820 OsPUB69 V Os08g13780 BAD01285.1 - - OsPUB73 V Os02g47670 BAD08040.1 AK101760 CB659631, CB659632, AK065645 AU056349, CA759692, AK100727 AU056350 OsPUB74 V Os06g28590 BAD35246.1 AK066627 CB633820, CB633821, CA305715 (continued) 127 Table 7: continued OsPUB75 V Os03g13050 AAM19135.1 AK068218 CB636145, CB636146, C91823, CB684588, CB684587, AU094784, BI807109 OsPUB76 V Os04g30470 CAD40573.1 - - OsPUB77 V Os04g49500 CAE04155.2 AK099968 OsPUB71 VI Os01g12930 BAD81324.1 - - OsPUB72 VI Os10g32880 NP_921905.1 AK064778 CB64800, CB64800, CB65460, CB65461, C28936, CB64479, CB64479, CB64050, CB63621, CB63622, CB63994, CB64625, CB64625, CB630643, CB630644, CB633728, CB633729, CA753148, CB684534, CB684533, CB684504, AU063371, AU063355, AU101014, CR288259, AU068591, BI81131, CR288238, AU166674, AU222544, BQ908380, AU092951, CF308939, CF308938 OsPUB63 VII Os06g06490 BAD69206.1 AK063418 CA758128, CF294635, CF294636, CF306389, CF305746, CF303031, CF303481, C73147, CA765281, AU075414 OsPUB64 VII Os06g06470 BAD69204.1 AK068486 CB669618, CB669619, CB666007, CB666008, CB655998, CB655999, CB656035, CB656036, AU068890, CB651921, CB651922, CB662541, CB662542, CB659153, CB659154, CB673778, CB649572, CB672550, CA762292, CA763973, AU068891, BI807157, AU078129, CA999626, C20397, C50849 OsPUB65 VII Os06g06450 BAD69203.1 AK101286 - OsPUB66 VII Os10g40220 AAK43506.1 - - OsPUB67 VII Os10g40490 - AK121815 CA759007, CA759008 OsPUB68 VII Os08g02140 BAD10546.1 AK106523 AU081544 OsPUB70 VIII Os06g06760 BAD67644.1 AK069245 CB684359，CB684360， AK069675 CB661779，BI812595， AK072504 BI811980
128
Sub-class Domain organization Number of proteins (N terminus → C terminus) Rice Arabidopsis I UFD2-like 1 1 II U-box + ARM/HEAT 29 29 III U-box + Leucine rich 16 12 IV Kinase + U-box 16 9 V U-box only 6 7 VI U-box + WD40 2 2 VII TPR + U-box 6 1 VIII TPR + Kinase + U-box 1 0 IX MIF4G + U-box 0 2
Table 8. Domain organization of rice and Arabidopsis U-box proteins
129
Order # Sub- AGI name GenBank cDNA EST accession class Accession AtPUB1(AtU I At5g15400 CAC01739 - AV825909, AV792873, FD2) AV528800, CF773585, AV562161 , CD534503, AV546905, AI996637, AV563966, CD530791 AtPUB10 II At1g71020 AAM98326 AY075626, BX840518, BX840019, AY142062 BX835989, BX841123, BX836705, CB263103, Z25977, Z25945, BE526988, AA650757, AI993133, T44557, AV564335, BP619079, Z33913, AA395481, BP658033, BP659001, BP654945, BP655103, AV814923 AtPUB11 II At1g23030 D86364 BT002518 AV831784, AV817782, AV548528, CK121999, BE528462, AI995305, BP615004, BP565230, BP578961, BP583295, BP564282, BP572485, BP569273, BP571858, BP577487 AtPUB12 II At2g28830 AAC79587 AY035038 AV827460, AV798289, BX839163 AtPUB13 II At3g46510 AAM91213 AY042791, CF651796, AV827706, AY128813 AV799055, BX840044, T22904, R87023, AI998984, BP625917, BP633083, BP592788, AV563817, AI099752, AV546729, BP658394, BP600576 AtPUB14 II At3g54850 AAM20180 AY065279, AV829174, AV804773, AY096530 BE523516, AV565084, AV564015, AV533554, BP601844 AtPUB15 II At5g42340 BAB10475 - - AtPUB16 II At5g01830 AAU45223 - AU237885, AU228988, T76464, AU036576, BP608621, BP620856, AA394623 (continued)
Table 9. Arabidopsis U-box gene names, their correspondence to sub-class, Arabidopsis genome locus, GenBank protein accession, and accessions for their cDNAs and cognate ESTs.
130 Table 9: continued AtPUB17 II At1g29340 AAN13028 AY064045, AV828218, AV800756, AY150512 AV553172, CD529402, H76454, N37242, R65246, AV544711, BP615330, BP654674, BP602831, BP607206, BP607923, AA651040, AI997327 AtPUB18 II At1g10560 AAD39579 AY075683 AV539978
AtPUB19 II At1g60190 AAC24052 CB260551
AtPUB2 II At5g67340 AAU94381 - AU238197, AU229341, CB074615, AV549470, BP617697 AtPUB3 II At3g54790 NP_191039 AK118613 AU239487 , AU230797, AV529193, AV529507, W43546, CF773657, AV523503, AV523751, AV548792, BP659183, AV546722 AtPUB38 II At5g65200 AAU45218 - CD530145, AI996717
AtPUB39 II At3g47820 AAM14040 AY091018 AV829157, AV804692, BP624381, AV542927 AtPUB4 II At2g23140 NP_179895 - BE523996, AV556642
AtPUB40 II At5g40140 NP_198830 - - AtPUB41 II At5g62560 NP_201062 AY128369, AV832065, AV831740, BT000351 AV817622, AV831036, AV814444, BP571263 AtPUB42 II At1g68940 A96714 - -
AtPUB43 II At1g76390 E96791 - AV826575, AV795575, CD530005, BP609660, BP619339, BP616657, AI996212 AtPUB44 II At1g20780 NP_564125 - CB264204, CB259902 AtPUB45 II At1g27910 AAM19837 AY094469, AB015118, AV528505, BT002721 AV829707, AV807489, T21712, AV522949, AV557171, AI998086, BP650331, AA395662 AtPUB46 II At5g18320 NP_197333 - -
AtPUB47 II At5g18330 NP_197334 - -
AtPUB48 II At5g18340 NP_197335 - - AtPUB5 II At4g36550 CAB16838 - - AtPUB6 II At1g24330 NP_173843 AV533367 AtPUB7 II At1g67530 AAP21292 AK117197, AU237998, AU229120, BT006484 BX837657, AV523667 (continued)
131 Table 9: continued AtPUB8 II At4g21350 CAB79134 - - (AtCMPG3) AtPUB9 II At3g07360 AAF02146 AY062107, AV830343, AV811344, AY088961, BE528631, BP562470, AY075636 AV441754, AI997391, CF773458, AV439485, BP586833, BP657201, BP665348, BP606899, BP619036, BP607684, BP671268, AV557454 AtPUB20 III At1g66160 AAL34278 AY087421, AV822591, T76460, (AtCMPG1) AY034913, T75967, T44966, R64779, AY063104 AI996558, BP610587, BP640497, BP609936, BP612992, BP598694, AV815241, BP630807, BP605721 AtPUB21 III At5g37490 NP_198565 AK117575 AU238413, AU229597 (AtCMPG5) AtPUB22 III At3g52450 AAU90053 - AU237485, AU228559, AV538961 AtPUB23 III At2g35930 AAO64764 BT005829 AU235317, AU225985, AV553417, BP608589, BP579884, BP578084, BP570135, BP571174, BP569060, BP570139, BP665829, AV543543 AtPUB24 III At3g11840 NP_566402 AY084238 BP633908, AU228517, CD533052, BP641441, BP665607, BP656610 AtPUB25 III At3g19380 AAM63504 AY058183, AA585864, AV797103, AY086503 AV827046, BX834409, BX837444, BX840032, CB256673, CB255884 CB255873, AV553068, AI997540, BX840996, BX836822, BX837316, T42091, AV544647 AtPUB26 III At1g49780 E96534 AK118346 BX839069, AU239208, AU230489 AtPUB27 III At5g64660 BAB11433 AK118660, CB264680, CK119781, (AtCMPG2) AV556154, AV555153, CD531212, CD529820, AU230853, AU239533, AI996899, AV536351 AtPUB28 III At5g09800 AAR24661 BT010883, CF652568 AtPUB29 III At3g18710 BAB01797 AK117976, AU238816, AU230059 BT005359 AtPUB30 III At3g49810 NP_566927 BT004040, BP562826, AU227323, AY084697 BX836565, BP584286, AV556601, AV548184 (continued)
132 Table 9: continued AtPUB31 III At5g65920 BAB11139 AY062476, CB263044, AV825681, (AtCMPG4) AY093259 AV791948, Z29203, AV793107, Z38035 AtPUB32 IV At3g49060 AAO42772 AY050369, AV825912, AV792889, BT004526 BG459358, AV554146, CD533098, BP570009, BP574080, AV545415, BP567005 AtPUB33 IV At2g45910 NP_182115 BT002512 BP562395, AV813730, AV829993, AV809512, BE524749, W43837 AtPUB34 IV At2g19410 AAM14871 - -
AtPUB35 IV At4g25160 NP_194246 - AA712567
AtPUB50 IV At5g65500 NP_201353 - -
AtPUB51 IV At5g61560 NP_200964 - AU237255, AU228292, BP601360 AtPUB52 IV At5g61550 BAB08999 BT005789 AU236383, AU227293, BP563739, BP580904, BP578835 AtPUB53 IV At5g51270 BAA97390 - -
AtPUB63 IV At5g57035 NP_680448 - AV565864
AtPUB36 V At3g61390.2 NP_191698 AK117154, AU237946, AU229062, BT005367 CD529049, AI998654 AtPUB37 V At2g45920 AAU90054 - R90713, H36412, H36424, AI992468, AV546062 AtPUB54 V At1g01680 AAF78398 - CB074904, BU635944,
AtPUB55 V At1g01660 NP_171672 - - AtPUB56 V At1g01670 BAC42729 AK118101 AU238957l, AU230208
AtPUB62 V At1g15165 - - AtPUB49 VI AT5g67530 AAM13295 AV792481, BP561482, AY062106, AY062516, BP562274, BG459510, AY093296, CB264439, AV441936, AY568528, F14279 , Z29729 , Z29730 BT001006, , Z18013 AB013390
(continued)
133 Table 9: continued AtPUB60 VI At2g33340 NP_850206 - AV823468, AV827950, AV799987, BE037669, AV537027, AI994175, N65335, AV552154, AV547348, AI999388, AV533140, Z24529, AV798870, BP668593, AV543754, BP601443, AV793559, BP616155, BP619325, BP606943, AV800023, BP605481, BP600082, BP612055, BP621270, BP603369, AV803582, AV793800, BP661243, BP612936, BP603822, BP664408, BP645322, AV815530, AV809814, BP651387, BP618762, Z29796, AV802561 AtPUB61 VII At3g07370 AAF02162 AY042807, AV832046, AV818929, (AtCHIP) AY064647 AV525756, CD534547, BP618845, BP602134, BP612488, BP609879, BP602888, BP599847, AV807267 AtPUB57 VIII At1g56030 NP_175999 - -
AtPUB58 VIII At1g56040 NP_176000 - -
134
Figure 21. A putative conserved domain located closely to the C terminus of
Arabidopsis U-box-Leucine-rich type U-box proteins. In the ~ 100 amino acid residues (denoted by empty box) located at the C-terminus half of these proteins, a high percentage of leucine residues was presented and four highly conserved residues were detected. We named this putative domain KEGLERS domain after the four conserved residues and its leucine rich feature. No biological function has been assigned to this domain.
135 Figure 22. Phylogenetic relationship between rice and Arabidopsis U-box/ARM repeat proteins. The phylogenies were generated with neighboring joining with 400 bootstrap replicates and were rooted at midpoint. The bootstrap values are shown as percentages. The sold arrow marks the position of rice SPL11 (OsPUB12). Three empty arrows denote the Arabidopsis U-box/ARM type genes evolutionally closest to rice Spl11.
136 137
Figure 23. Atspl11 displays cell death and stunt growth phenotype. (A). Gene structure of Atspl11. Exons are denoted as black boxes. The number below each exon indicates the length of the exon in basepairs. The empty arrow a, b, and c mark the relative position of primers used for the identification of Atspl11. Vertical arrow denotes the location at which the T-DNA was inserted. (B). Phenotype of the wild-type and the mutant. Photos were taken 8 weeks after germination. The wild-type and mutant plants were grown in the same condition. Top panel: whole plants grown in the soil. Lower panel: close look of the leaves of wild-type and mutant plants
138 CHAPTER 7
A NOVEL MECHANISM UNDERLYING PCD IN PLANT DISEASE
RESISTANCE
Amongst the various types of plant PCD, HR is intimately associated with plant disease resistance (Heath, 2000) and has received considerable attention in recent years.
The complexity of HR cell death signaling is manifested in the identification of a variety of physiological processes correlated with the occurrence of HR, such as ion fluxes, generation of ROS (Laloi et al., 2004) and nitric oxide (NO) (Delledonne et al., 1998;
Delledonne et al., 2001), expression of defense-related genes, production of anti-microbal molecules, and formation of cell wall apposition. Though a common framework in signaling and execution of plant PCD has been proposed (see chapter 1), the distinct feature of HR in its consistent association with the induction of local and systemic defense responses suggests that certain molecular events might be uniquely involved in this disease resistance-related PCD.
In this study, a rice lesion mimic mutant called spl11 was used to investigate PCD in plant defense. Several characteristics make the spl11 mutant a good subject to study
PCD in plant defense signaling (Yin et al., 2000). Firstly, it displayed constitutive cell death (lesion mimic) formation without abiotic and biotic challenges. Secondly, it
139 displayed enhanced resistance to rice fungal and bacterial pathogens infections. Thirdly, the northern blot analysis indicated that the induction of several defense-related genes in spl11 is intimately correlated with the cell death formation. The induction of defense signaling along with the activation of cell death in spl11 was corroborated by data from genome-wide gene expression profiling analysis of the spl11 mutant in this study
(Chapter 2). Of the 133 genes that were over 3.5-fold up-regulated at three different leaf lesion development stages in spl11, more than 50 percent are defense-related or oxidative stress/cell death-related. It is noteworthy that with more cell death (lesion mimic) developed on the leaf (fully expanded leaf and old leaf), more defense-related genes were induced (Table 1). Though the details of the signaling cascade of Spl11-mediated cell death/defense signaling are largely unknown at present, the involvement of ROS production in the process is beyond dispute, as demonstrated by the identification of a variety of oxidate stress-related genes being induced in the mutant. The failure to identify well-defined groups of target genes for other hormonal or cellular processes in the microarray analysis indicated that the Spl11 gene might be a component in the plant PCD pathway.
The molecular cloning of the Spl11 gene and characterization of the protein as an
E3 ubiquitin ligase are the central part of this doctorate research. Spl11 is the first case in plants that a component in the ubiquitination pathway is related to both cell death and defense activation. The wide involvement of ubiquitination in the regulation of apoptosis in mammals has been well documented (Lee and Peter, 2003). In animals, a large number of components, especially those apoptosis regulators have been identified in the past three decades. Various E3s have been implicated in targeting some key apoptosis
140 regulators for modification or degradation (Wilson et al., 2002; Wing et al., 2002;
Miyazaki et al., 2003). The lack of homologs of most core animal apoptosis signaling components in plant genomes suggested that the mechanism underlying PCD in plants might be, at least in some aspects, distinct from the apoptosis in animals (see Chapter 1).
Nevertheless, the existence of plant proteases that are structurally non-orthologous but functionally equivalent to caspases from animals (Solomon et al., 1999; Zhao et al., 1999) implied that the framework of the molecular signaling underlying plant and animal PCD might be similar. Therefore, it is possible that the substrate of SPL11 is a key regulator of plant PCD. In the ubiquitination system, the members of the E3 families help choose which proteins should be ubiquitinated. In Arabidopsis, it has been proposed that most
Arabidopsis substrates have their own ubiquitination cascades, which could in turn perceive unique degradation signals as a way to confer precise specificity (Vierstra,
2003). In this sense, the SPL11-mediated ubiquitination might specifically target its substrate(s) only when it recognizes the cellular signal to suppress the cell death/defense process. Given all these considerations, identification of the substrate(s) for SPL11- mediated ubquitination will be crucial to the future study on this project.
Identification of substrates for the ubiquitination system has met extraordinary challenges probably due to the heterogeneity, short half-life and low abundance of these proteins in the cell. To date, only a few substrates of E3 ligases (i.e. phyA, HY5/HYH,
AUX/IAA, NAC1, E2F and ABI5) have been identified in plants (Vierstra, 2003). The identification of eight different genes that interacted with SPL11 in the Y2H system could be the first step toward the isolation of SPL11 substrate(s) (Chapter 5). Importantly, two of eight genes encode proteins that are related to pre-mRNA processing. SPIN1
141 encodes a protein containing a KH domain that involves in RNA binding or pre-mRNA processing. SPIN4 encodes a RPR domain containing protein with putative pre-mRNA cleavage function. The ubquitination system has been demonstrated to be involved in chromatin structure modification and DNA repair-related transcriptional regulation
(Jason et al., 2002; Kleiman et al., 2005). In apoptosis, a number of PCD regulatory genes are expressed as functionally distinct or even antagonistic isoforms as a result of alternative splicing (Shaham and Horvitz, 1996; Jiang and Wu, 1999). However, the involvement of ubiquitination in pre-mRNA splicing regulation has not been reported.
Until now, Prp19p is the only U-box-containing E3 ligase that is associated with RNA splicing complexes (Ohi and Gould, 2002; Patterson, 2002). Mutation of conserved amino acids in the U-box domain abolished the interaction of Prp19p with other components in the complex and the alternative splicing activity of Prp19 (Ohi and Gould,
2002). However, what role the U-box-mediated E3 ligase activity of Prp19p plays in
RNA splicing remains to be answered. Preliminary results from my GST pull-down assay indicated that SPIN1 interacted with SPL11 ARM repeat domain and full-length SPL11
(data not shown) in vitro, indicating that SPIN1 is unlikely a false-positive interactor.
Like Prp19p, SPL11 could be a component of the general splicesome in the cell or it may act together with SPIN1 and/or SPIN4 to alternatively splice specific cell death regulator in plant cell, like what is observed in some apoptosis regulators (Minn et al., 1996;
Shaham and Horvitz, 1996). Alternatively, SPL11 may target SPIN1 or SPIN4 for ubiquitination to affect their function in alternative splicing. Functional demonstration of
SPIN1 and SPIN4 as pre-mRNA processing protein and identification of the pre-
142 mRNA(s) that bind to SPIN1 and SPIN4 will provide essential information to reveal the nature of connection between SPL11 and pre-mRNA processing.
The presence of a large number of U-box proteins in the rice and Arabidopsis genomes and the diversity in their domain organization suggested they could play multiple roles in the cell (Chapter 6). It was estimated that 10% of total eukaryotic proteins are targeted by the ubiquitination system (Ciechanover et al., 1984).
Accordingly, based on the predicted number of rice genes (Goff et al., 2002; Yu et al.,
2002), there will be as many as over 4000 substrates in the rice genome are regulated by the ubiquitination system. Based on the information from the Arabidopsis genome, it is likely that most rice U-box proteins may target different substrate. In cells, the fates of ubquitin-tagged substrates vary depending on the nature of ubiquitin linkage. While proteins under poly-ubiquitination are mostly targeted to the proteasome for degradation, proteins with a single (mono-ubiquitination) or a few (oligo-ubiquitination) ubquitins have been implicated in protein relocation, transcription and cell size control, DNA repair, and receptor internalization (Lee and Peter, 2003). In addition, the choices of different lysine residue of the ubiquitin molecule for the extension of the ubquitin chain also influence the fate of the ubiquitinated protein (Marx, 2002). It is likely that the substrate of some U-box proteins may not be targeted for degradation. In our in vitro analysis, SPL11 had only a few ubiquitin attached to itself (Figure 16). Whether it has same activity in vivo and the relevance of this oligo-ubiquitination to its biological role will be an interesting question to study.
Doubtlessly, much work requires to be done before we can draw a clear flow chart of the molecular events underpinning Spl11-mediated cell death and defense
143 signaling. Nevertheless, data presented in this dissertation do point to a novel mechanism that involves both ubiquitination and pre-mRNA processing in regulating rice cell death and defense signaling. The identification of a T-DNA insertion mutant of AtSpl11 displaying cell death and stunt growth phenotypes suggested that this mechanism might be conserved between dicot and monocot plants. Given this universality, it would be intriguing to find out in future studies how two basic cellular mechanisms, ubiquitination and alternative pre-mRNA processing, work together in modulating plant cell death and defense.
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