Analysis of regulator of g protein signaling (RGS) function in growth, development and pathogenicity of magnaporthe grisea 1

ANALYSIS OF REGULATOR OF G-PROTEIN SIGNALING RGS FUNCTION IN GROWTH, DEVELOPMENT AND PATHOGENICITY OF MAGNAPORTHE GRISEA HAO LIU NATIONAL UNIVERSITY OF SINGAPORE 2006... ANALYSIS OF R

ANALYSIS OF REGULATOR OF G-PROTEIN SIGNALING (RGS) FUNCTION IN GROWTH, DEVELOPMENT AND

PATHOGENICITY OF MAGNAPORTHE GRISEA

HAO LIU

NATIONAL UNIVERSITY OF SINGAPORE

2006

ANALYSIS OF REGULATOR OF G-PROTEIN SIGNALING (RGS) FUNCTION IN GROWTH, DEVELOPMENT AND

PATHOGENICITY OF MAGNAPORTHE GRISEA

HAO LIU

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY

NATIONAL UNIVERSITY OF SINGAPORE

ACKNOWLEDGEMENTS

I would like to express my heartfelt thanks to my thesis supervisor Dr Naweed Naqvi for

giving me the opportunity to work on M grisea and for his constant support and guidance

through out the course of this work I thank all the members of my thesis committee: Prof William Chia, A/Prof Mohan Balasubramanian and A/Prof Yue Wang for helpful suggestions Special thanks to Dr Hongyan Wang, who introduced IMA to me I also thank Dr Fengwei Yu for his fruitful discussion and suggestions

Many thanks to Angayarkanni Suresh for excellent technical assistance, and to all members of the Fungal Genomics Group for suggestions and discussions I also extend

my thanks to all the members of the Cell Biology Forum for constructive criticism I thank TLL administrative and support staff for all the help Financial support from the Temasek Life Sciences Laboratory is duly acknowledged

TABLE OF CONTENTS

Page

Summary………ix

List of figures………xii

List of abbreviations……… ….xv

Chapter I Introduction……… 1

1.1 General introduction to fungal development……… 1

1.1.1 Fungal mating……… …5

1.1.2 Morphological switch in Fungi……… …… .7

1.1.3 Fungal asexual development ……… ….11

1.1.4 Fungal pathogenicity…… ……… … 14

1.2 General introduction of G-protein-mediated signaling cascade …… 17

1.2.1 Heterometric G proteins……… 18

1.2.2 Model organisms to study G proteins……… 21

1.2.2.1 G proteins in plants……… 21

1.2.2.2 G proteins in yeast……… 22

1.2.2.3 G proteins in mammals……… 23

1.2.3 Desensitization of G protein Signaling……… …… 24

1.2.3.1 The discovery of Regulator of G-protein signaling (RGS)………24

1.2.3.2 The mechanism of RGS function……… …… 26

1.3 G proteins in fungal pathogens………28

1.3.1 G proteins in Aspergillus……… 28

1.3.2 G proteins in Candida albicans………29

1.3.3 G proteins in Ustilago maydis……….…… 29

1.3.4 G proteins in Cryphonectria parasitica………30

1.4 Magnaporthe grisea, the rice blast pathogen……….……… 31

1.4.1 General introduction to Magnaporthe……… …31

1.4.2 Magnaporthe conidiation … ……33

1.4.3 Morphology of appressorium ……… 33

1.4.4 Signal perception and transduction for appressorium development…….…35

1.4.4.1 Surface signal perception……… 35

1.4.4.2 Intracellular signal transduction……….37

1.4.5 G proteins in Magnaporthe……… 38

1.5 Aims and objectives of this thesis………39

1.6 The significance of this study……… 40

Chapter II Materials and Methods………42

2.1 Strains, Growth, Infection Assays and Reagents……….42

2.1.1 Magnaporthe grisea strains and growth conditions……….………….42

2.1.2 E coli strains and growth conditions……… 42

2.1.3 Agrobacterium tumefaciens strains and growth conditions……… …43

2.1.4 Appressorium formation assay with Manaporthe conidia………43

2.1.5 Barley related methods and infection with Magnaporthe conidia……… 44

2.1.6 Rice related methods and infection with Magnaporthe conidia………… 44

2.2 Molecular Methods……… 44

2.2.1 DNA techniques………44

2.2.1.1 PCR amplification……… 44

2.2.1.2 Agarose gel electrophoresis and gel purification of nucleic acid fragments……….…… 45

2.2.1.3 Recombinant DNA techniques……… 45

2.2.1.4 Genomic DNA extraction from Magnaporthe……….…… 46

2.2.1.5 Southern Blot……….47

2.2.1.6 Ligation-mediated PCR ……… 49

2.2.1.7 Transformation of E coli by heat shock method………… …….49

2.2.1.8 Transformation of Agrobacterium by electroporation methods…50 2.2.2 RNA techniques………50

2.2.3 T-DNA random insertion, gene targeting and genetic complementation….51 2.2.3.1 Gene disruption strategy……… 51

2.2.3.2 Targeted gene replacement………52

2.2.3.3 Genetic complementation of rgs1∆……… 52

2.3.3.4 RGS1 overexpression……….……53

2.2.3.5 Site-directed mutation in MAGA, MAGB and MAGC……… …53

2.3 Protein and immunology related methods……… 54

2.3.1 Total protein lysates from Magnaporthe (Denatured and Native)……… 54

2.3.2 Expression and purification of fusion protein in E coli……… 54

2.3.3 Rgs1 antiserum and its specificity……… 55

2.3.4 Protein electrophoresis, immunoblots and reprobing protocals………… 56

2.3.5 Rgs1 and Gα protein interaction in vitro……… 57

2.3.6 Endogenous Rgs1 interacts with recombinant Gα proteins……….58

2.4 cAMP extraction and analysis……….58

2.4.1 Extraction of cAMP from Magnaporthe mycelium and germ tubes… ….59

2.4.2 Analysis of cAMP with cAMP Biotrak Enzymeimmunoassay System… 59

2.5 Hardness Assay………60

2.6 Light Microscopy……….60

Chapter III Identification and Characterization of RGS1 deficient mutant in M grisea 62

3.1 Introduction……… 62

3.2 Results……… 63

3.2.1 Agrobacterium T-DNA mediated insertional mutagenesis in Magnaporthe……… 63

3.2.2 Identification of the disrupted locus……… 64

3.2.3 Characterization of TMT1398 mutant……… 71

3.2.4 Cloning of RGS1 in Magnaporthe……… …… 76

3.2.5 Creation and Characterization of rgs1∆ mutant………81

3.2.6 Genetic complementation of rgs1∆ 81

3.2.7 Excessive Rgs1 reduces conidiation but not appressorium development….82 3.3 Discussion………82

Chapter IV Mechanism of Rgs1 function……… 94

4.1 Introduction……… 94

4.2 Results……… 95

4.2.1 RGS-domain containing proteins in Magnaporthe……… 95

4.2.2 Appressorium formation in Gα deletion mutants……….96

4.2.3 Appressorium formation in rgs1∆ Gα ∆ mutants……….98

4.2.4 Appressorium formation in RGS-insensitive Gα mutants………98

4.2.5 Rgs1 dependent regulation of cyclic AMP level……… 104

4.2.6 Rgs1 physically interacts with MagA……….104

4.2.7 Gαi subunit MagB is critical for conidiogenesis……… …….106

4.2.8 Mgb1, the Gβ subunit, is required for conidiogenesis in Magnaporthe………109

4.2.9 Water soaking phenotype of Gα mutant colonies……… 109

4.2.10 The expressions of candidate hydrophobin genes in rgs1∆ 112

4.2.11 Physical interaction between Rgs1 and MagB……… 114

4.2.12 Physical interaction between Rgs1 and MagC……… ……114

4.3 Discussion……… 117

4.3.1 Rgs1 regulates MagA for appressorium development……… 117

4.3.2 Rgs1 regulates MagB for conidiogenesis and surface hydrophobicity… 118

Chapter V Thigmotropic signaling in M grisea pathogenesis……… 121

5.1 Introduction………121

5.2 Results………121

5.2.1 Surface hardness stimulus is essential for appressorium differentiation in Magnaporthe………121

5.2.2 Timing of the thigmotropic signal sensing……….125

5.2.3 Relationship between thigmotropism and cAMP levels……….127

5.2.4 Mechanosensitive channels in Magnaporthe……… ……131

5.3 Discussion……… 134

Chapter VI General Discussion………137

Appendix 1……… ………144

References……… 149

SUMMARY

The Magnaporthe-rice interaction is a major model for understanding plant disease,

largely because of its economic importance, and also due to the molecular genetic

tractability of the blast fungus Rice-blast disease caused by Magnaporthe is initiated

when conidia germinate upon attachment to the host surface In response to surface and environmental cues, the resultant germ tubes undergo infection-specific differentiation to form the dome-shaped appressoria, which are employed to forcibly penetrate host cuticle

Signaling between Magnaporthe and rice is therefore predicted to be critical for initiating

the pathogenesis cycle However, the molecular mechanisms underlying this parasite-host communication are not fully understood

This study initially describes an Agrobacterium Transferred-DNA mediated insertional mutagenesis in Magnaporthe, aimed at identifying genes required for fungal

pathogenicity This led to the identification of an insertional mutant TMT1398, in which

a gene (RGS1) encoding an RGS-domain containing protein was disrupted TMT1398 and an rgs1∆ strain showed pleiotropic defects such as soaking phenotype,

hyperconidiation, appressoria formation on non-inductive surfaces and reduced pathogenicity Through genetic complementation it was ascertained that the defects

displayed by TMT1398 and rgs1∆ were due to the loss of Rgs1 function

In Magnaporthe, appressorium formation has been shown to be induced by surface hydrophobicity Unlike the wild-type, TMT1398 and rgs1∆ did not depend on surface

hydrophobicity signals for appressorium development and could form appressoria on hydrophilic or hydrophobic surfaces with equal efficiency However, neither the wild-

type nor the rgs1∆ could form appressoria on soft surfaces, regardless of the

hydrophobicity, suggesting that surface hardness signal could be crucial for appressorium development A major highlight of the study presented here is the identification of such a thigmotropic response, which was found to be essential for initiating appressorium

formation in Magnaporthe The critical surface hardness sufficient to induce

appressorium formation was estimated through biophysical analysis of host leaf surface

as well as the inductive artificial membranes

Regulators of G-protein signaling (RGS) accelerate the intrinsic GTPase activity of the constituent Gα subunits and thus negatively regulate the heterotrimeric G-protein signaling cascades The mechanism of Rgs1 function was therefore investigated and elucidated by analyzing the function(s) of its potential Gα subunit targets (Gαs subunit MagA, Gαi subunit MagB, and GαII subunit MagC) in Magnaporthe Characterization of

individual Gα-deletion strains and RGS1-insensitive mutants (magAG187S

, magB G183S, and

magC G184S

) revealed that Rgs1 directly regulates MagA during appressorium initiation

Interestingly, rgs1∆ and magA G187S

accumulated higher levels of cAMP compared to the wild-type further suggesting that cAMP-mediated downstream signaling is important for

appressorium formation The magB∆ failed to conidiate, whereas magB G183S

hyperconidiated like rgs1∆, suggesting that Rgs1 regulates MagB during conidiogenesis Further characterization of the soaking phenotype of the rgs1∆ and the magB G183S

colonies showed that Rgs1 and MagB function together to regulate mycelial

hydrophobicity In biochemical analyses, Rgs1 physically interacted with and individually accelerated the GTPase activity of each of the three Gα subunits (MagA,

MagB and MagC) in Magnaporthe Thus, as a unique and multifunctional regulator of G

protein signaling, Rgs1 regulates mycelial surface hydrophobicity, asexual reproduction,

appressorium development, pathogenicity and thigmotropism in Magnaporthe

LIST OF FIGURES

Figure……… Page

Figure 1 Morphism in fungi……… 3

Figure 2 Conidiogenesis in fungi …… ……… 13

Figure 3 Schematic representation of canonical G protein signaling……… 21

Figure 4 The RGS4– Giα1 Complex……….………28

Figure 5 Two life cycles in Magnaporthe……….33

Figure 6 Morphology of appressorium……….35

Figure 7 Infection assay on barley leaves……….65

Figure 8 Appresorium formation assay……….66

Figure 9 Ligation-mediated PCR for identifying flanking sequences for Agrobacterium T-DNA insertions in Magnaporthe ……….………67

Figure 10 Identification of the T-DNA insertion sites……… ……… … 70

Figure 11 Appressorium formation defect in TMT1398… ……… 72

Figure 12 Accelerated appressorium formation in TMT1398……… 74

Figure 13 Defect in conidiogenesis of TMT1398……….75

Figure 14 Defect in pathogenicity of TMT1398……… 77

Figure 15 Penetration peg formation of TMT1398……… 78

Figure 16 Easily Wettable phenotype of TMT1398……….79

Figure 17 Cloning of RGS1 83

Figure 18 Alignment of Rgs1 with fungal orthologs………84

Figure 19 Analysis of RGS domain of Rgs1 with multiple sequence alignments………85

Figure 20 Creation of RGS1 deletion mutant………86

Figure 21 Rgs1 negatively regulates conidiation……… 87

Figure 22 Rgs1 negatively regulates appressorium formation on non-inductive / hydrophilic surfaces……… 88

Figure 23 RGS1 is required for full pathogenicity in Maganporthe……….89

Figure 24 RGS1 is involved in surface hydrophobicity……… 90

Figure 25 RGS1 overexpression in Magnaporthe……….91

Figure 26 RGS proteins in Magnaporthe……….…….97

Figure 27 Appressorium formation in Gα deletion mutants……… 99

Figure 28 Appressorium formation assays with rgs1∆Gα∆ mutants……… … 100

Figure 29 Conserved switch region I among Magnaporthe Gα proteins……… 102

Figure 30 Appressorium formation assays in RGS-insensitive Gα mutants……… …103

Figure 31 Rgs1 and MagA regulate intracellular cAMP levels……… ……105

Figure 32 Rgs1 interacts with MagA……… … 107

Figure 33 Rgs1 acts in concert with MagB during conidiogenesis……….… 108

Figure 34 Gβ subunit Mgb1 is required for conidiogenesis in Magnaporthe…………110

Figure 35 Rgs1 acts coordinately with MagB to regulate of mycelial hydrophobicity 111

Figure 36 Hydrophobin gene expression in wild-type and rgs1∆ 113

Figure 37 Rgs1 interacts with MagB……… 115

Figure 38 Rgs1 physically interacts with MagC……….116

Figure 39 Soft surfaces do not induce appressorium formation……….123

Figure 40 Contact with hard surface induce appressorium development……… 124

Figure 41 The increased induction ability of appressorium formation on dried agar.…126

Figure 42 Surface-hardness signal is perceived and integrated within two hours of

conidia germination….………128

Figure 43 Inhibition of appressorium formation with Gadolinium……….129

Figure 44 Hardness signaling and intracellular cAMP……… 130

Figure 45 Mechanosensitive channels are not required for appressorium formation….133

Figure 46 Growth characteristics of mechanosensitive channel mutants under stress

conditions……….135

LIST OF ABBREVIATIONS

aa Amino acid

ABA Abscisic acid

BAC Bacterial artificial chromosome

cAMP Cyclic adenosine monophosphate

CM Complete medium

d Day

DEP Disheveled, Egl-10 and Pleckstrin

DEPC Diethyl pyrocarbonate

Gα G protein alpha subunit

Gβ G protein beta subunit

Gγ G protein gamma subunit

GAP Guanosine triphosphatase activating proteins GDP Guanosine diphosphate

GEF Guanine-nucleotide exchange factors

GIRK G protein-coupled inwardly rectifying potassium

G protein Guanine nucleotide binding protein

h Hour

HPH Hygromycin phosphotransferase

LB Left board of T-DNA

MAPK Mitogen-activated protein kinase

MBP Maltose binding protein

m Minute

MscL Mechanosensitive channels large current

MscS Mechanosensitive channels small current ORF Open reading frame

PA Prune agar

PAGE Polyacrylamide gel electropheresis

PCR Polymerase chain reaction

RB Right board of T-DNA

RGS Regulator of G-protein signaling

RPM Revolution per minute

RT Room temperature

RT-PCR Reverse transcription polymerase chain reaction

s Second

SDS Sodium dodecyl sulphate

T-DNA Transfer DNA

Tris Tris(hydroxymethyl)aminomethane

WT Wild-type

Chapter I INTRODUCTION

1.1 General introduction to fungal growth and development

The eukaryotic kingdom of fungi encompasses a tremendously diverse and enormously versatile range of organisms, including yeasts, molds, or a combination of both forms Fungi have adapted

to different modes of growth and development in evolution to spread in the environment, or to survive unfavorable conditions Yeasts are true fungi whose usual and dominant growth form is unicellular (Scherr and Weaver 1953) Budding or fission is the favorite mechanism for yeast reproduction (Chant and Pringle 1991; Chang and Nurse 1996) Budding is asexual reproduction

of a new organism, or daughter cell, by polarized protrusion of part of another organism, or mother cell (Chant and Pringle 1991) A bud can first develop on different part of the mother cell The nucleus of the mother cell splits into two daughter nuclei, one of which migrates into the bud cell (Chant and Pringle 1991; Chang and Nurse 1996; Chant 1999) The bud continues to grow until it separates from mother cell, forming a new cell, which is genetically identical to the mother cell In contrast, growth by fission is a form of symmetric yeast reproduction As seen in

Schizosaccharomyces pombe, the cell enters mitosis, followed by elongation of the cell and

formation of a septum that divides the cell into halves, separating the two nuclei into two cells of the same size (Feierbach and Chang 2001) Molds, in contrast to unicellular yeasts, occur in long filaments known as hyphae consisting of one of more cells surrounded by a tubular cell wall, which grow by hyphal apical extension (Gow 1994) Apical growth in filamentous fungi is a polarized cellular mitotic growth in which nearly all cell growth occurs only at the hyphal tip, followed by fission of cells through the formation of incomplete septa (Horio and Oakley 2005) Hyphal tip growth is characterized by the initial establishment of one growth site, which is

followed by its continuous maintenance (Steinberg 2007) Hyphal tip growth, branching, and hyphal fusion results in the formation of a complex tri-dimensional hyphal network in fungi (Gow 1994)

The unicellular growth form is the preferred mode of reproduction of yeasts However some yeasts can grow in a variety of morphological forms, ranging from budding cell (unicellular yeast) to pseudohyphae (chains of elongated cells with visible constrictions at the sites of septa) and true hyphae (linear filaments without visible constrictions at the septa) (Figure 1) (Kron and Gow 1995; Liu 2001; Berman 2006) This kind of reversible growth type switch and transition in fungi is designated as dimorphism (Bolker 2001) The yeast to pseudohyphal or hyphal switch is triggered by environmental signals, such as temperature or pH or nutritional starvation, which usually imposes the stress response in fungi (Brown and Gow 1999) The molecular mechanism involved in this morphogenesis has been well investigated and revealed to be well conserved in

yeast and fungi The morphogenetic switch in Saccharomyces cerevisiae requires the cooperation

of two different intracellular signaling pathways, a MAP kinase cascade and a cAMP-dependent signal network (Gancedo 2001)

1.1.1 Fungal mating

The potential benefits of fungal sex are to purge the genome of deleterious mutations, to generate

diversity, or both (Heitman 2006) Saccharomyces cerevisiae is a model organism used to study fungal mating S cerevisiae is a single celled eukaryote reproducing by mitosis, with daughter cells budding off of mother cells S cerevisiae can survive and grow in two forms, diploid and

haploid The haploid cells undergo a simple life cycle of mitosis and growth, and under

Figure 1 Vegetative morphology of fungal cells

(A) Yeast cells can form both (B) pseudohyphae and (C) true hyphae Switching between the pseudohyphal and hyphal morphologies is less frequent

(Adapted from Berman J Morphogenesis and cell cycle progression in Candida albicans Curr

Opin Microbiol 2006 Dec; 9(6):595-601)

conditions of high stress will generally simply die (Hirschberg and Simchen 1977) The diploid cells undergo a simple life cycle of mitosis and growth as well, but under conditions of stress can undergo sporulation, entering meiosis and producing a variety of haploid spores (Hirschberg and Simchen 1977; Schild and Byers 1980) Thus, diploid cells can undergo both mitosis and

meiosis, but haploid cells can only undergo mitosis S cerevisiae has both the asexual and sexual

life phases Haploid cells are one of two mating types (a or α), and are capable of responding to the mating pheromone, a short peptide produced by other haploid cells of the opposite mating type and mate with them to produce stable diploid cells (Dohlman 1993) The mating of yeast only occurs between haploids, which can be either the a or α mating type and thus display simple sexual differentiation (Dohlman 1993)

S cerevisiae mating type is determined by a single locus, MAT, which in turn governs the sexual

behavior of both haploid and diploid cells (Nasmyth 1982; Harashima et al 1989) Two alleles

of MAT (MATa in a cells and MAT α in α cells, respectively) govern transcriptional repression

and activation of mating type specific genes (Nasmyth 1982; Haber 1992; Bardwell et al 1994;

Haber 1998) In brief, the MATa allele of MAT encodes a pair of genes called a1 and a2, which in

haploids direct the transcription of the a-specific transcriptional program, such as producing the mating pheromone a-factor and expressing α-factor cell surface receptor STE2 (Nasmyth 1982)

Moreover, MATa allele also represses the transcription of the α-specific transcriptional program

by inhibiting the producing the mating pheromone α-factor and a-factor cell surface receptor

STE3 (Nasmyth 1982) This defines an “a” cell The MAT α alleles of MAT encodes the α1 and

α2 genes, which in haploids direct the transcription of the α-specific program (such as producing

α-factor, expressing STE3, and repressing STE2), which causes the cell to be an α-cells a-cells

respond to α-factor and α-cells respond to a-factor, respectively, by growing a projection for cell conjugation (Nasmyth 1982) The response of haploid cells only to the mating pheromone of the opposite mating type allows mating between a and α cells, but not between cells of the same mating type (Hirsch and Cross 1992; Elion 2000) Diploid cells do not produce or respond to either mating pheromone and do not mate (Hirsch and Cross 1992; Elion 2000)

Cellular responses to mating pheromone ultimately elicit important changes including (1): cytoskeletal structural reorganization leading to polarized cell growth; (2) induction of gene transcription; (3) arrest of cell cycle progression in G1 phase; (4) changes in nuclear architecture (Kurjan 1993; Elion 2000; Dohlman 2002) All these cellular changes are critical to the successful progression of mating Briefly, polarized cell growth is required to establish the site for cell fusion New gene transcription is required to produce, for example, proteins that mediate cell adhesion and cell fusion Growth arrest is required to synchronize the cell cycles of the two opposite mating partner Nuclear changes are required in preparation for nuclear fusion and the completion of zygote formation (Kurjan 1993; Elion 2000; Dohlman 2002)

The molecular mechanism involved in mating has been well investigated The pheromone (a or

α factor) signal is perceived and relayed to the intracellular heterotrimeric G protein by the cell surface receptor (Ste3 or Ste2, respectively) to initiate the mating program (Dohlman 1993) The

role of G protein in S cerevisiae mating will be discussed in chapter 1.2.2.2 in detail Study of S

cerevisiae mating leaded to the identification of the first mitogen-activated protein kinase

(MAPK) signaling cascade, a three-tired signal transduction module known as MAPK cascade is conserved ubiquitously throughout the eukaryotic kingdom (Posas et al 1998) A canonical

MAPK pathway is composed of MAP kinase, MAP kinase kinase (MAPKK) and MAP kinase kinase kinase (MAPKKK) G-protein mediated mating signaling activates Ste11, a MAPKKK in

S cerevisiae Ste11, in turn, phosphorylates and activates Ste7, a MAPKK, which likewise,

phosphorylates and activates Fus3, a MAPK (Gustin et al 1998) Fus3 is a Ser/Thr-specific protein kinase which has multiple targets in both the cytosol and the nucleus The phosphorylation of Fus3 leads to activation of transcription and other mating specific events in nucleus (Dohlman 2002)

1.1.2 Morphological switch in fungi

Fungal dimorphism is the ability to produce either separated yeast cells or filamentous forms (Sanchez-Martinez and Perez-Martin 2001) This morphological switch represents an important

developmental stage of some fungal pathogens, such as Candida albicans (Ernst 2000) C

albicans (sometimes referred to as monilia) is the major fungal pathogen that colonizes medical

implants and cause device-associated infections with exceptionally high mortality (Douglas

2002; Filler 2006; Pfaller and Diekema 2007) C albicans is a diploid asexual fungus that is

normally present on the skin and in mucous membranes such as the vagina, mouth, or rectum (Romani et al 2003) The fungus can travel through the blood stream and affect the throat,

intestines, and heart valves (Whiteway and Oberholzer 2004) C albicans is also a causal agent

of opportunistic oral and vaginal infections in humans (Loh and Sivalingam 2003) Under normal

circumstances, C albicans lives in 80% of human population with no harmful effects, although

overgrowth results in Candidiasis (Chapman 2003) However, in immunocompromised patients

(e.g AIDS, cancer chemotherapy, organ or bone marrow transplantation) C albicans infection

has emerged as important causes of morbidity (Altamura et al 2001) In addition,

hospital-related infection with C albicans in patients not previously considered at risk (e.g patients on an

intensive care unit) has become a cause of major health concern (Lim and Stern 1986; Mukherjee

et al 2005)

C albicans can grow in a variety of morphological forms, ranging from budding yeast to

pseudohyphae and hyphae (Mitchell 1998) To infect host tissue, the usual unicellular yeast-like

form of C albicans reacts to environmental cues, such as serum and increasing temperature, and

switches into an invasive multicellular filamentous form (Mitchell 1998) This transition is important for the development of pathogenicity because the mutant strain ‘locked’ in the yeast form is avirulent (Ernst 2000) This switching between two cell-types is known as dimorphism

Molecular study has revealed the underlying mechanism of C albicans dimorphism (Ernst

2000) Through differential expression screen, some filamentation response genes are found to

be expressed at higher levels in filamentous cells than in yeast cells (Liu 2001) Two parallel pathways involved in regulation of filamentation have been identified (Ernst 2000) The first is

C albicans MAPK cascade, including the upstream kinases Cst20p and Hst7p and the

downstream transcription factor Cph1p (Leberer et al 1996) Mutants lacking any of these components are non-filamentous under some inducing conditions, but produce filaments in the presence of serum (Navarro-Garcia et al 1998) The simple inference from this finding is that an alternate pathway must act in parallel with the MAPK components to govern filamentation Each pathway may respond to a subset of inducing signals, or that may be a graded response in which both pathways are required for weak signals but a single pathway is sufficient for strong signals

The identification of Efg1-deficient mutant (Efg1 is homologue to S cerevisiae Phd1, a

transcriptional regulatory protein involved in regulation of pseudohyphal growth) confirmed this

hypothesis and revealed the second pathway in C albicans dimorphism An Efg1-deficient

mutant produces filaments with aberrant morphology, suggesting Efg1p has some role in

filamentation (Stoldt et al 1997) Moreover, an Efg1 - Cph - double mutant produces no filaments, even in the presence of serum (Lo et al 1997)

In addition to the bud-filament transition, C albicans is capable of undergoing a different type of

morphological change (superficially resembling dimorphism) that has been termed ‘phenotypic switching’ (Slutsky et al 1987) This switching is most easily observed in the morphology of colonies A single cell can divide, and in the absence of environmental signals give rise to several distinct types of colonies (Bergen et al 1990) This switching occurs spontaneously at frequencies well above those produced by point mutation and has been reported to be reversible

In addition, cells isolated from each type of colony usually produce the same type of colony on replating, indicating that a variant colony morphology, once formed, is heritable One of the classically studied strains that undergoes phenotypic switching is WO-1, which consists of two phases, one that grows as smooth white colonies and the other that grows as flat gray colonies (Slutsky et al 1987; Soll et al 1994) The other strain known to undergo switching is 3153A, which produces at least seven different colony morphologies (Soll and Kraft 1988) The

molecular basis of phenotypic switching in C albicans is not well understood While several

gene that are expressed differently in different colony morphologies have been identified, and

several possible mechanisms are suggested In the 3153A strain, a gene called SIR2 (for Silent

Information Regulator) has been found that seems to be important for phenotypic switching,

SIR2 was originally identified in S cerevisiae where it is involved in chromosomal silencing, a

form of the genome is reversibly inactivated by changes in chromatin structures (Pillus and Rine

1989; Perez-Martin et al 1999) Efg1p recently has been suggested to help regulate phenotypic switching in WO-1 Efg1p is expressed only in the white and not in the gray cell type, and overexpression of Efg1p in the gray form causes a rapid conversion to the white form (Sonneborn et al 1999)

C albicans generally exhibit two distinct modes of behavior The first is the familiar free

floating or planktonic form in which single cells float or swim independently in some liquid medium The second is as a biofilm, an attached state in which cells are closely packed and firmly attached to each other and usually to a solid surface Biofilm is defined as structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric

material (Chandra et al 2001) Biofilm is a protected niche for C albicans, where it is safe from

antibiotic treatment and can create a source of persistent infection (Chandra 2001; Kumamoto

2002) C albicans infection, the most common hospital-acquired infection, involves the

formation of biofilm on implanted devices such as catheters or prosthetic heart valves (LaFleur et

al 2006) Non-device-related infection with C albicans can involve bioflim, too Candida

endocarditis, for example, can result from the formation of biofilm on damaged vascular endothelium of native valves in patients with pre-existing cardiac disease (Donlan 2001) Quorum sensing or population dependent gene expression, is a well-known cell signaling

mechanism in bacteria, is now also thought to be significant in C albicans biofilm formation It

has been shown that farnesol acts as a quorum-sensing molecule that inhibits biofilm formation

in C albicans (Hornby et al 2001) It has also been demonstrated that morphogenesis plays a pivotal role in C albicans biofilm development It was found that EFG1-deficient mutant and double mutant EFG1 - CPH1 - , both are defect in filamentation, formed rather poor biofilm lacking

in three dimensional structure and composed mainly of sparse monolayers of elongated cells (Lo

et al 1997)

1.1.3 Fungal asexual development

Fugal asexual development (conidiation) is an important aspect of fungal growth Aspergillus

nidulans has been used as a model organism to study fungal asexual development Aspergillus is

a filamentous, cosmopolitan and ubiquitous fungus found in nature It is commonly isolated from

soil, plant debris, and indoor air environment Aspergillus species are highly aerobic and are

found in almost all oxygen-rich environments, where they commonly grow as molds on the surface of a substrate, as a result of the high oxygen tension (Kreiner et al 2003) Commonly,

Aspergillus grows on carbon-rich substrates such as monosaccharides and polysaccharides In

addition to growth on carbon sources, many species of Aspergillus demonstrate oligotrophy

where they are capable of growing in nutrient-depletion environments, or environments in which

there is a complete lack of key nutrients For example, Aspergillus can be found growing on

damp walls as a major compound of mildew

Aspergillus is a genus of around 200 molds found through much of nature world Aspergillus are

important microorganisms, including medically, agriculturally and industrially important species (Timberlake and Marshall 1988) Some species are pathogens causing infection in humans, other animals and plants, while others are important in commercial microbial fermentations For

example, Aspergillosis is a large spectrum of diseases caused by pathogenic Aspergillus, such as

Aspergillus fumigatus, Aspergillus terreus and Aspergillus flavus These fungi are opportunistic

pathogens (Denning 1998; Galagan et al 2005) The clinical manifestation and severity of the

disease depends upon the immunologic state of the patient Aspergillus flavus is also an agricultural pathogen causing disease on many grain crops, especially maize Aspergillus oryzae

is used in Chinese and Japanese cuisine which ferments soybeans to produce soy sauce and miso

Aspergillus niger is most well-known for its application as the major source of citric acid; this

organism accounts for over 99% of global citric acid production (Papagianni 2007) Aspergillus

niger is also commonly used for the production of native and foreign enzymes, including glucose

oxidase and hen egg white lysozyme Recently, the genomes of three well studied Aspergillus species (Aspergillus nidulans, Aspergillus fumigatus and Aspergillus oryzae) were sequenced

and comparative analysis was performed (Galagan et al 2005; Machida et al 2005; Nierman et

al 2005) The completion of genome sequencing and a well characterized genetic system make

Aspergillus a model system to study biological process (Galagan et al 2005)

Conidiation is a biological process in which filamentous fungi asexually form spores (conidia or

conidiospores) Aspergillus nidulans has been used as a model organism to study multiple aspects of fungal development, especially asexual development Conidiation in A nidulans

involves many common developmental themes including spatial and temporal regulation of gene expression, specialized cellular differentiation, and intercellular communication (Timberlake and

Marshall 1988) Conidiation in A nidulans is a continual sequence from vegetative growth to asexual development A nidulans asexual reproductive cycle can be divided into at least three

different stages, beginning with a growth phase that is required for cells to acquire the ability to respond to induction signals, proceeding through initiation of the developmental pathways, and culminating with execution of the developmentally regulated events leading to conidiation (Adams et al 1998)

Spore formation is a common mechanism among most fungi to reproduce, to spread in the environment, or to survive unfavorable conditions The process of fungal conidiation involvesmany common developmental themes including temporal and spatial regulation of gene expression, cell specialization, and morphogenesis (Adams et al 1998) Conidia are asexual spores of fungi generated through the cellular process of mitosis (Adam 1994) For example, after a certain period of vegetativegrowth, under appropriate conditions such as constant light

illumination, Aspergillus nidulans hyphal cells stop normal growth and begin conidiation by forming conidiophoresthat bear multiple chains of conidia (Figure 2) (Mooney and Yager 1990; Adams et al 1998; Yu et al 2006) According to the different morphologies of conidiophores,

fungal conidium development can be largely classified into two fundamental types: blastic

conidiogenesis, where the spore is already evident before it separates from the conidiogenic

hyphae which is giving rise to it, and thallic conidiogenesis, where first a cross-wall appears and

then the thus created cell develops into a spore (Sigler 1989)

A nidulans conidiation is a precisely timed and genetically programmed event responding to

intertal and external cues The study of asexual development in A nidulans has provided important information on the mechanisms controlling such growth and development A nidulans

reproduces multicellular organs termed conidiophores (specialized fungal hyphae) bearing thousands of mitotically derived asexual conidia (Adams et al 1998) A key and essential step

for conidiophore development is activation of the BRLA gene encoding a C2H2 zinc-finger

transcription factor, which induces expression of other genes required for asexual development

BRLA deficient mutants form structures that resemble conidiophore stalks, except that they grow

indeterminately and fail to produce the other specialized cell types needed for sporulation,

Figure 2 Morphological changes during conidiophore formation

Shown are scanning electron micrographs of the stages of conidiation (A) Early conidiophore stalk (B) Vesicle formation from the tip of the stalk (C) Developing metulae (D) Developing phialides (E) Mature conidiophores bearing chains of conidia

(Adapted from Adams, T H et al 1998 Asexual sporulation in Aspergillus nidulans Microbiol

Mol Biol Rev 62(1): 35-54.)

suggesting that activation of BRLA expression early in conidiophore development represents a

major and essential control point for initiating conidiation (Adams et al 1988) Six genes

required for proper activation of BRLA expression were identified by investigating a large number of fluffy mutants with severe defects in BRLA expression (Adams et al 1998) The corresponding mutants were designated as FLB (fluffy low –BRLA expression) mutants (Adams

et al 1998) Most of the FLB gene products are G-protein cascade elements, demonstrating that

heterotrimeric G-protein mediated pathway plays an essential role in A nidulans conidiation (Adams and Yu 1998) The role of G-proteins in A nidulans conidiation will be discussed in

more detail in chapter 1.3.1

Cyclic-AMP dependent PKA signaling was also found to play important roles in regulation of A

nidulans vegetative growth and conidiation (Yu et al 2006) In A nidulans, PkaA (primary

PKA) and PkaB (secondary PKA) represent the sole PKA catalytic subunits and play

overlapping roles in diverse biological process It was found that the absence of PKAA function

resulted in restricted vegetative growth coupled with hyperactive conidiation and suppressed the

fluffy autolytic phenotype caused by FLB mutation (Yu et al 2006) In addition, overexpression

of PKAA led to elevated hyphal proliferation and reduced sporulation Based on these

observations, it has been proposed that cAMP and PKA signaling cascades play major roles in activation of vegetative growth and in repression of conidiation (Shimizu et al 2003; Yu et al 2006)

1.1.4 Fungal pathogenicity

In addition to bacteria, virus, and parasites, fungi are emerging as an increasingly common threat

to human health and world-wide crop production For example, over the last two decades, the

opportunistic fungal pathogens Candida albicans and Cryptococcus neoformans have emerged

as the leading causes as of fungal meningitis and infect immunocompromised individuals (Oseiwacz 2002) Fungal pathogens can be divided into two general classes: mammalian (human) pathogens and plant pathogens The most common fungi in the former class include

Candida, Aspergillus and Cryptococcus, which are opportunistic pathogens infecting

immunocompromised hosts (van Burik and Magee 2001) The fungal plant pathogens themselves

fall into two major groups, the biotrophs which feed from living plant tissue, such as Erysiphe

necator, which causes powdery mildew of grapes and Ustilago maydis causing smut disease in

maize, and the necrotrophs which kill plant cells and then live off the nutrients released,

exampled by and Magnaporthe grisea that causes rice blast disease (Talbot 2003; Belhadj et al

2006; Kamper et al 2006)

Due to the complex nature of the host-fungus interaction, both human pathogenic fungi and phytopathogens have developed different mechanisms to invade the hosts The infection-related morphogenesis is important for fungal pathogenicity Most of these human opportunistic fungi can exist in either non-pathogenic yeast form or pathogenic hyphae form (Vaughn and Weinberg

1978; Mitchell 1998) The ability to switch from the yeast form to the hyphal form in C albicans

is crucial to the process of pathogenesis, because mutants that are unable to make the switch are typically less effective in causing human disease (Whiteway and Oberholzer 2004; Ernst 2000)

C albicans switches from yeast to mycelial form in response to specific environmental stimuli,

such as serum, pH and glucose (Mitchell 1998) Disruption of the genes governing filamentation

also disrupts C albicans pathogenicity (Ernst 2000) Fungal plant pathogens have also evolved

diverse mechanisms for penetrating into host plant tissue, ranging from entry through natural plant openings to various mechanisms of direct penetration through the outer surface with different structures including hyphae and germ tubes, encysted zoospores and elaborate piercing

structures (Howard and Valent 1996) Ustilago maydis, the model plant pathogen, also employs hyphae to invade its host (Kronstad and Kaiser 2000) Interestingly, U maydis displays a

dimorphic switch between budding growth of haploid cells and filamentous growth of the

dikaryon (Klosterman et al 2007) When grown in the laboratory on very simple media, U

maydis behaves like budding yeast S cerevisiae, forming single cells called sporidia These cells

multiply by budding off daughter cells U maydis sporidia are not infectious and are pathogenic However, when two compatible U maydis sporidia meet on the surface of the plant,

non-they will switch to a different mode of growth (Casselton and Olesnicky, 1998) First, non-they send out conjugation tubes to find each other, after which they fuse and make a hyphae to enter the maize plant Hyphae growing in the plant are dikaryotic possessing two haploid nuclei per

hyphal compartment (Banuett, 1995) In contrast to sporidia, the dikaryotic phase of U maydis

requires infection of the plant in order to grow and differentiate and cannot be maintained in the laboratory Proliferation of the fungus inside the plant leads to disease symptoms as chlorosis, anthocyanin formation and the appearance of tumors harboring the developing teliospores Thus,

the dikaryon phase represents the pathogenic stage of U maydis

In contrast to U maydis, many other phytopathogenic fungi, such as M grisea and

Colletotrichum gloeosporioides (banana pathogen) require germination and differentiation of

germ tube into an infection structure called appressorium in order to penetrate the plants

(Flaishman and Kolattukudy 1994; Talbot 2003) Some fungi are thought to produce appressoria

in response to specific physical signals and topography of leaf surface (Flaishman and Kolattukudy 1994) In other fungi, the appressoria formation has been suggested to involve specific chemical signals from the plant (Flaishman and Kolattukudy 1994) Appressoria mechanically breach host surface by employing high turgor pressure (Howard and Valent 1996) Upon entering into the plant, the fungal invasive hyphae grow aggressively in the host resulting

in the disease lesion on colonized plant surfaces

Though diverse mechanisms are employed by different pathogens to invade their hosts (human and plants), the same pathways are found to govern fungal pathogenicity Both cAMP-PKA pathway and MAP kinase cascade are suggested to govern fungal pathogenicity (Xu 2000; Lengeler et al 2000) The two isoforms of the cAMP-dependent protein kinase catalytic subunit are involved in the control of dimorphism and infection ability in the human opportunistic

pathogen C albicans (Cloutier et al 2003) Mitogen-activated protein kinase-defective C

albicans is avirulent due to the defect in yeast to hyphal transition (Guhad et al 1998)

Differentiation and virulence of C neoformans are coordinately regulated by cAMP-PKA

cascade, which is involved in sensing nutrients during mating and virulence, and MAP kinase cascade that senses pheromone during mating, and also regulates haploid fruiting and virulence (Wang and Heitman 1999) MAP kinase and cAMP signaling regulate infection structure

formation and pathogenic growth in the rice blast fungus Magnaporthe grisea (Xu and Hamer 1996).The roles of cAMP, PKA and MAP kinase cascade in M grisea pathogenicity will be

discussed in detail in chapter 1.4.4.2 Characterization of constitutively filamentous mutants

resulted in identification of the UAC1 adenylyl cyclase in U maydis (Durrenberger et al 1998;

Kruger et al 1998) Subsequently, the UBC1 PKA regulatory subunit gene (UBC: Ustilago bypass of cyclase) was identified by its ability to restore filamentous growth in a uac1 suppressor

mutant (Durrenberger and Kronstad 1999) The identification of several components of the MAP

kinase cascade as suppressors of uac1 mutant indicates that there is crosstalk between cAMP and MAP kinase signaling pathways in U maydis pathogenicity (Lee et al 2003) Coordinated regulation of mating and pathogenicity in U maydis by the cAMP and MAP kinase signaling

pathways is mediated by Prf1, an HMG-box domain transcriptional regulator and a common target of both pathways (Kaffarnik et al 2003) Prf1 binds sequence specifically to pheromone

response elements present in the a and b loci (Hartmann et al 1999) Disruption of PRF1 in

pathogenic haploid strains results in a complete loss of infection ability (Hartmann et al 1999)

1.2 General introduction to cell signaling

“Signal transduction” describes how individual cells receive, process, and ultimately transmit

information derived from external "signals," such as hormones, drugs, or even light (Rodbell

1995) Since the first use of the term “signal transduction” in molecular biology by Rodbell and Hechter in 1969, the concept of biological information system composed of discriminators, transducers, and amplifiers was outlined and a new field to investigate the mechanism of intracellular communication was initiated (Rodbell 1995) The discriminator, or cell receptor, receives information from outside the cell; a cell transducer processes this information across the cell membrane; and the amplifier intensifies these signals to initiate reactions within the cell and /or to transmit information to other cells

All eukaryotic cells have the capacity to respond to chemical and sensory stimuli in their environment Heterotrimeric (αβγ) G protein mediated signaling is one of the most important mechanisms by which eukaryotic cells sense extracellular stimuli and convert them into intracellular signals The evolutionary conservation of G protein signaling mechanisms in eukaryotes has been a remarkable finding from research done in the last few decades (Dowell and Brown 2002) Such signaling is often used to regulate transcription activators or repressors

to control cell function and development (Hoffman 2005)

1.2.1 Heterometric G proteins

Heterotrimeric guanine nucleotide binding proteins (G proteins) are composed of non-identical alpha, beta and gamma subunits, which mediate signaling from a superfamily of heptahelical receptors (G protein coupled receptors, GPCRs) to a smaller number of effectors that include adenylyl cyclase, phospholipase C and various ion channels (Malbon 2005) These molecules received their names from their typical three-subunit composition and the presence of a Ras-like domain in the G alpha subunit, which can tightly bind the guanine nucleotides GDP or GTP (Hamm 1998) To date, sixteen distinct mammalian G protein alpha subunits have been identified and divided into four families based on their sequence similarity and biological function: Gαs, Gαi, Gαq, and Gα12 Functional analyses revealed that Gαs proteins are involved

in stimulation of adenylyl cyclase; Gαi proteins are coupled to inhibition of adenylyl cyclase as well as to activation of G protein-coupled inwardly rectifying potassium channels (GIRK); Gαq proteins are coupled to the activation of phospholipases Cβ, and Gα12 proteins activate Rho guanine-nucleotide exchange factors (GEFs) (Pierce et al 2002) Heterotrimeric G proteins can act as molecular switches for signal transduction: when Gα is bound to GDP, docking sites for

the downstream effectors are blocked by the Gβ and Gγ subunits When GDP is exchanged for GTP, however, three so-called switch regions change their conformation so that the Gβ and Gγ subunits dissociate and thus allow downstream effectors to bind the active Gα molecule (Hampoelz and Knoblich 2004) Binding of the ligands, such as growth factors, vasoactive polypeptides, chemoattractants, neurotransmitters, hormones, phospholipids, odorants and taste ligands on the GPCRs provokes rapid conformational changes in the transmembrane alpha helices, which results in the exchange of GDP with GTP at the Gα subunit and dissociation of the heterotrimer (Bourne 1997) Either the GTP bound Gα or the released Gβγ, or both, are then free to activate downstream effectors (Dohlman 2002) (Figure 3) Typically, these effectors produce second messengers or other biochemical changes that lead to stimulation of a downstream protein kinase or a protein kinase cascade The resultant changes in protein phosphorylation can affect metabolism, ion flux, gene expression, cell morphology, cell movement, cellular differentiation and organismal development (Pierce et al 2002) Hydrolysis

of the GTP by the Gα intrinsic GTPase activity, which can be accelerated by regulator of G protein signaling (RGS), leads to the replacement of GTP with GDP and reassociation of

Gα with Gβγ heterodimer, leading to the termination of G protein-mediated signaling (Figure 3)

1.2.2 Model organisms to study G proteins

1.2.2.1 G proteins in plants

Heterotrimeric G proteins have been implicated in a wide range of plant processes including response to hormones, drought, pathogens and in developmental events such as lateral root formation, hypocotyl elongation, hook opening, leaf expansion and silique development (Perfus-Barbeoch et al 2004) Results and concepts emerging from the phenotypic analyses of G protein

Figure 3 Schematic representation of canonical G protein signaling

Gα-GDP and Gβγ heterodimer form the inactive G protein complex attached to the cytoplasmic loops of seven-transmembrane receptors (7TM) Binding of ligand to 7TM cell-surface receptors stimulates signal onset by acting as guanine nucleotide exchange factors (GEFs) for

Gα subunits, facilitating GDP release, subsequent binding of GTP, and release of the Gβγ dimer Either or both the GTP-bound Gα and liberated Gβγ moieties are then able to modulate the activity of downstream effectors Regulator of G-protein signaling (RGS) proteins stimulate signal termination by acting as GTPase-accelerating proteins (GAPs) for Gα, dramatically enhancing their intrinsic rate of GTP hydrolysis PM: Plasma Membrane

(Adapted from Siderovski and Willard, 2005, Int J Bio Sci., 1: 51-66)

signaling mutants in Arabidopsis and rice have added to a better understanding of G protein

signaling functions in plants The genomes of diploid plant species encode single canonical G

alpha subunit and G beta subunit proteins: Gpa1 and Agb1 in Arabidopsis, and Rga1 and Rgb1

in rice (Ma 1994; Assmann 2002) Two Arabidopsis proteins, Agg1 and Agg2, are likely Gγ

subunits on the basis of their ability to interact with plant Gβ subunits, and the corresponding rice orthologs are Rgg1 and Rgg2 (Mason and Botella 2000; Kato et al 2004) G proteins

regulate ion channels and abscisic acid (ABA) signaling in Arabidopsis guard cells The gpa1∆

mutant lacks both ABA inhibition of guard cell inward K+ channels and pH-independent ABA

activation of anion channels Moreover, stomatal opening in gpa1∆ plants is insensitive to inhibition by ABA, and the rate of water loss from gpa1∆ plants is greater than that from the

wild-type plants, suggesting that manipulation of G protein status in guard cells may provide a

mechanism for controlling plant water balance (Wang et al 2001) Arabidopsis gpa1∆ shows

decreased cell division in hypocotyl and in leaves, and exhibits an altered leaf shape (Ullah et al

2001) Further characterization of gpa1∆ plants revealed that the cell proliferation in Arabidopsis

is regulated by heterotrimeric G protein and this regulation is cell type specific (Chen et al

2003) To date, there are no reports as yet on biotic stress signaling in Arabidopsis G protein

mutants, but some defense signaling pathways in rice appear to rely on Rga1 Upon infection

with a virulent strain of bacterial blight, symptom development in d1 mutants is more severe than

that in wild-type plants (Perfus-Barbeoch et al 2004)

1.2.2.2 G proteins in yeast

G alpha Gpa1 mediated pheromone sensing pathway and Gpa2 mediated glucose signaling

pathway have been identified in S cerevisiae (Versele et al 2001) GPA1 and GPA2 genes were

identified using probes based on the sequence of mammalian Gα proteins (Nakafuku et al 1987) To date, Gpa1 and Gpa2 are the only two identified Gα proteins in S cerevisiae, and Ste4 and Ste18 are the only Gβ and Gγ subunits, respectively (Whiteway et al 1989; Versele et al 2001) The Gpa1 mediated mating signal transduction pathway is arguably the best understood multicomponent signaling system in any eukaryotic organism (Dohlman 2002) All the key gene products responsible for propagating the signal from the cell surface through the cytosol and into the nucleus have been identified and their biochemical properties characterized (Dohlman and Thorner 2001) In brief, pheromone sensing depends on Ste2 and Ste3 receptors (GPCRs) that respectively bind α- and a-factor, and transmit the signal to the heterotrimeric G proteins In either mating type, the pheromone receptor is coupled to the same heterotrimeric G protein, consisting of a Gα subunit (Gpa1) and a Gβγ heterodimer (Ste4-Ste18) (Bender and Sprague 1986) Binding of pheromone to the receptor triggers the dissociation of Gβγ from Gα, following which the heterodimer proceeds to activate a mitogen-activated protein kinase (MAPK) cascade, resulting in new gene transcription, cell cycle arrest, cytoskeletal structure change and eventually cell fusion to form the a/α diploid (Wittenberg and Reed 1996) The relatively recent discovery

of the role of G protein Gpa2 in glucose sensing serves as another model for G protein function Yeast cells prefer glucose as a carbon source due to its rapid fermentation to ethanol, which inhibits the growth of competing micro-organisms (Versele et al 2001) Glucose triggers the switch to fermentative life style by activating the G protein (Gpa2) mediated signal transduction system to activate the cAMP synthesis, thereby activating cAMP-dependent protein kinase (PKA), which controls a broad range of targets (Kraakman et al 1999)

1.2.2.3 G proteins in mammals