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. ii TABLE OF CONTENTS Page Summary…………………………………………………………………………………ix List of figures……………………………………………………………………………xii List of abbreviations……………………………………………………………… ….xv Chapter I Introduction…………………………………………………………………. 1.1 General introduction to fungal development…………………………. 1.1.1 Fungal mating…………………………………… 1.1.2 Morphological switch in Fungi……………………… …… 1.1.3 Fungal asexual development .………………………………… 1.1.4 Fungal pathogenicity…… …………………………… …5 .7 ….11 … 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 iii 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 iv 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 v 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 vi 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 vii 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 viii 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 ix Reitman, M. L., and Weinstein, L. S. (2000). Paternal versus maternal transmission of a stimulatory G-protein alpha subunit knockout produces opposite effects on energy metabolism. J Clin Invest 105, 615-623. Zheng, B., De Vries, L., and Gist Farquhar, M. (1999). Divergence of RGS proteins: evidence for the existence of six mammalian RGS subfamilies. Trends Biochem Sci 24, 411-414. Zhou, X. L., Stumpf, M. A., Hoch, H. C., and Kung, C. (1991). A mechanosensitive channel in whole cells and in membrane patches of the fungus Uromyces. Science 253, 1415-1417. Zhou Yuanxing, Ronald J. Newton and Jean H. Gould. (1997). A simple method for identifying plant/T-DNA junction sequence resulting from Agrobacterium-mediated DNA transformation. Plant Molecr Biol Rep 15, 246-254. Zupan, J., Muth, T. R., Draper, O., and Zambryski, P. (2000). The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J 23, 11-28. 188 Figure Figure Figure Figure Figure Figure Conidium Appressorium Germ tube Figure TMT980 WT TMT1045 TMT1069 TMT1398 Figure Inductive Non-inductive Figure Figure 10 A B C D Figure 11 Non-inductive Figure 12 TMT1398 1h 3h 7h 12h WT Figure 13 A TMT1398 WT 1d 4d _ B WT TMT1398 Figure 14 A WT TMT1398 B WT TMT1398 Figure 15 WT TMT1398 _ [...]... 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 G subunit targets (G s subunit MagA, G i subunit MagB, and G II subunit MagC) in Magnaporthe Characterization of individual G -deletion strains and RGS1-insensitive mutants (magAG187S, magBG183S, and magCG184S)... adenosine monophosphate CM Complete medium d Day DEP Disheveled, Egl -10 and Pleckstrin DEPC Diethyl pyrocarbonate G G protein alpha subunit G G protein beta subunit G 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. .. conidiogenesis……………….… 10 8 Figure 34 G subunit Mgb1 is required for conidiogenesis in Magnaporthe …… 11 0 Figure 35 Rgs1 acts coordinately with MagB to regulate of mycelial hydrophobicity 11 1 Figure 36 Hydrophobin gene expression in wild-type and rgs1∆ 11 3 Figure 37 Rgs1 interacts with MagB………………………………………………… 11 5 Figure 38 Rgs1 physically interacts with MagC……………………………………… .11 6 Figure 39 Soft surfaces... nutrients during mating and virulence, and MAP kinase cascade that senses pheromone during mating, and also regulates haploid fruiting and virulence (Wang and Heitman 19 99) MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea (Xu and Hamer 19 96).The roles of cAMP, PKA and MAP kinase cascade in M grisea pathogenicity will... regulator of G protein signaling, Rgs1 regulates mycelial surface hydrophobicity, asexual reproduction, appressorium development, pathogenicity and thigmotropism in Magnaporthe xi 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–... magCG184S) revealed that Rgs1 directly regulates MagA during appressorium initiation Interestingly, rgs1∆ and magAG187S 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 magBG183S hyperconidiated like rgs1∆, suggesting that Rgs1 regulates MagB during conidiogenesis... Figure 28 Appressorium formation assays with rgs1 G ∆ mutants…………… … 10 0 Figure 29 Conserved switch region I among Magnaporthe G proteins…………… 10 2 Figure 30 Appressorium formation assays in RGS-insensitive G mutants……… 10 3 Figure 31 Rgs1 and MagA regulate intracellular cAMP levels………………… … 10 5 Figure 32 Rgs1 interacts with MagA…………………………………………… … 10 7 Figure 33 Rgs1 acts in concert with MagB during... Figure 16 Easily Wettable phenotype of TMT1398…………………………………….79 Figure 17 Cloning of RGS1 .83 xii 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... conidiogenesis Further characterization of the soaking phenotype of the rgs1∆ and the magBG183S colonies showed that Rgs1 and MagB function together to regulate mycelial x hydrophobicity In biochemical analyses, Rgs1 physically interacted with and individually accelerated the GTPase activity of each of the three G subunits (MagA, MagB and MagC) in Magnaporthe Thus, as a unique and multifunctional regulator. .. 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 . NATIONAL UNIVERSITY OF SINGAPORE 2006 ANALYSIS OF REGULATOR OF G-PROTEIN SIGNALING (RGS) FUNCTION IN GROWTH, DEVELOPMENT AND PATHOGENICITY OF MAGNAPORTHE GRISEA HAO LIU. expressions of candidate hydrophobin genes in rgs1∆ 11 2 4.2 .11 Physical interaction between Rgs1 and MagB………………………… 11 4 4.2 .12 Physical interaction between Rgs1 and MagC…………………… … 11 4 4.3 Discussion………………………………………………………………………. ANALYSIS OF REGULATOR OF G-PROTEIN SIGNALING (RGS) FUNCTION IN GROWTH, DEVELOPMENT AND PATHOGENICITY OF MAGNAPORTHE GRISEA HAO LIU