Evaluation of the role of autophagy in fungal development and pathogenesis

EVALUATION OF THE ROLE OF AUTOPHAGY IN FUNGAL DEVELOPMENT AND PATHOGENESIS YIZHEN DENG A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE ACKNOWLEDGEMENTS The successful completion of my Ph.D program would not have been possible if no for the invaluable contributions for the following people, My supervisor, Dr Naweed I Naqvi, for his excellent scientific insights and his exemplary guidance and support through out the course of this work The member of my graduate committee: Dr Gregory Jedd, Dr Mohan Balasubramanian and Dr Markus Wenk, for their useful suggestions and constructive criticisms All the past and present membrers of the Fungal Patho-Biology Group: Shanthi Soundararajan, Li Xiaolei, Sun Chuanbao, Liu Hao, Angayarkanni Suresh, Marilou Ramou-Pamplona, Xue Yangkui, Ravikrishna Ramanuja, Patkar Rajesh, Yang Fan, He Yun long and Selvarai Poonguzhali, for making our lab a most pleasant and stimulating working environment The TLL community, especially the sequencing lab and the microscopy and imaging facility provided by Mr Ouyang Xuezhi, Miss Fiona Chia and Dr Graham Wright Temasek Life Sciences Laboratory and singapore millenium foundation, for providing the funding for this research Dear Family and Friends, for their unwavering support and affection i i TABLE OF CONTENTS CHAPTER I INTRODUCTION 1.1 General introduction to autophagy 1.1.1 Process and classification of macroautophagy .1 1.1.1.1 Glycogen autophagy 1.1.1.2 The Cvt pathway 1.1.1.3 Pexophagy 1.1.2 Molecular basis of autophagy .4 1.1.3 Physiological functions of autophagy .7 1.3.3.1 Yeasts .7 1.3.3.2 Filamentous fungi 1.3.3.3 Plants 1.3.3.4 Animals .10 1.2 General introduction of endosomal system .11 1.2.1 Classification and cellular roles of endosomes .11 1.2.2 Physiological function of endosomes .13 1.3 Magnaporthe oryzae, the rice blast pathogen 15 1.3.1 Life cycle of Magnaporthe oryzae 15 1.3.2 Genetic and biochemical regulation of M. oryzae conidiogenesis and pathogenesis .16 1.4 Carbohydrate metabolism and fungal sporulation / pathogenicity 19 1.5 Aims and objectives of this study 20 1.6 Significance of this study .20 CHAPTER II MATERIALS AND METHODS 22 2.1 Strains, reagents and genetic methods 22 2.1.1 Magnaporth oryzae strains 22 2.1.2 Media and growth conditions 22 2.2 Molecular methods 25 2.2.1 Plasmid vectors for gene deletion, gene tagging and genetic complementation .25 2.2.2 DNA techniques .32 2.2.2.1 DNA extraction .32 2.2.2.2 Recombinant DNA techniques 32 ii ii 2.2.2.3 Genomic DNA extraction from Magnaporthe 35 2.2.2.4 Southern blot .35 2.2.3 RNA techniques .37 2.2.3.1 RNA extraction .37 2.2.3.2 Reverse transcriptase PCR 37 2.2.3.3 Quantitative Real-time PCR (qRT-PCR) 37 2.2.4 Bacterial transformations .38 2.2.4.1 Transformation of E.coli by heat shock method .38 2.2.4.2 Electroporation of Agrobacterim (AGL1 strain) .38 2.2.5 Agrobacterium-mediated transformation of Magnaporthe .38 2.3 Protein and immunology related methods .39 2.3.1 Total protein lysates from Magnaporthe (denatured) .39 2.3.2 Immunoblot analysis 40 2.4 Evaluation of pathogenicity and pathogenicity-related traits 41 2.5 Microscopy 42 2.5.1 Fluorescence microscopy 42 2.5.2 Staining with fluorescent dyes 43 2.5.3 Transmission electron microscopy (TEM) .43 CHAPTER III AUTOPHAGY-ASSISTED GLYCOGEN CATABOLISM REGULATES ASEXUAL DIFFERENTIATION IN MAGNAPORTHE .46 3.1 Introduction 46 3.2 Results 47 3.2.1 Isolation and characterization of an atg8∆ mutant in Magnaporthe .47 3.2.2 ATG8 is essential for autophagy in Magnaporthe .49 3.2.3 Autophagy regulates asexual differentiation and conidiation in Magnaporthe 49 3.2.4 Post-translational processing and Atg8p targeting to autophagosomes in Magnaporthe 53 3.2.5 Induction and subcellular localization of RFP-Atg8 in Magnaporthe 56 3.2.6 Suppression of conidiation defects in atg8∆ by alternate carbohydrate sources .58 3.2.7 Rice leaf extracts and in planta growth suppresse the conidiation defects in atg8∆ 60 3.2.8 Gph1-catalyzed glycogen metabolism during Magnaporthe conidiogenesis 63 3.2.9 Sga1-catalyzed glycogen hydrolysis during Magnaporthe conidiogenesis .70 3.2.9.1 Sga1 is required for proper conidiation in Magnaporthe .70 3.2.9.2 Subcellular localization of Sga1 in Magnaporthe .73 iii iii 3.2.9.3 Sga1 catalyzes glycogen hydrolysis during Magnaporthe conidiation .76 3.2.10 Glycogen autophagy and Magnaporthe pathogenesis 79 3.2.10.1 Gph1-catalyzed glycogen catabolism and Magnaporthe pathogenesis 79 3.2.10.2 Sga1-catalyzed glycogen catabolism and Magnaporthe pathogenesis 81 3.3 Discussion .83 CHAPTER IV THE FUNCTION OF ATG20 IN MAGNAPORTHE ASEXUAL DIFFERENTIATION AND PATHOGENESIS .89 4.1 Introduction 89 4.2 Results 90 4.2.1 Generation and characterization of an atg20∆ mutant in Magnaporthe .90 4.2.2 Subcellular localization of Atg20 .90 4.2.3 Investigation of non-selective autophagy and the Cvt pathway in the atg20∆ mutant 98 4.2.4 Investigation of pexophagy in the atg20∆ mutant 100 4.2.4.1 Pexophagy is defective in the atg20∆ mutant 100 4.2.4.2 Pexophagy is not induced during Magnaporthe conidiogenesis and pathogenesis .102 4.2.4.3 Pexophagy is not required for Magnaporthe conidiogenesis and pathogenesis .105 4.2.5 Investigation of retrieval trafficking pathway in the atg20∆ mutant .112 4.2.6 Investigation of vacuolar morphology in atg20∆ .116 4.3 Discussion 118 CHAPTER V CONCLUSIONS 120 REFERENCES .124 APPENDIX .136 iv iv SUMMARY Autophagy is a conserved bulk degradation process in eukaryotic cells. It serves as a major survival function during starvation stress and is important for proper growth and differentiation. The targets for autophagic degradation can be non-selective or highly selective, depending on the specific biological circumstance and/or the specific inducer involved. Autophagy was first identified and characterized in yeast and in animal cells. In recent years, autophagy has been studied in filamentous fungi and evidence shows that it plays an important role at multiple stages of fungal development. Autophagy induction in differentiated structures (including fused filaments, aerial hyphae, germ tubes, appressorium) in fungi has been observed, but limited information is available on the mechanism and functional role of autophagy in such processes. In this study, an essential role for autophagy-assisted glycogen breakdown (glycogen autophagy) is described in the rice-blast fungus Magnaporthe oryzae. Glycogen is an important carbon store in the cell. During differentiation, increased demand for energy and/or cellular material may trigger large-scale glycogen breakdown. The conidiation defect in autophagy-deficient mutant, atg8∆ (ATG8 short for AuTophagy related Gene 8), could be significantly restored by exogenous addition of glucose or glucose phosphate-6 (G6P), indicating a role for carbon source utilization for autophagy in Magnaporthe. Characterization of a deletion mutant of GPH1 (Glycogen PHosphorylase 1), encoding glycogen phosphorylase, further suggests that vacuolar, but not cytosolic, degradation of glycogen is important for Magnaporthe conidiation. A vacuolar glucoamylase, Sga1 (Sporulation-specific GlucoAmylase 1), was identified as the enzyme carrying out v v vacuolar hydrolysis of glycogen and dependent on autophagy for getting access to the substrate. A cytosolic variant of Sga1 could bypass the requirement of autophagy for glycogen breakdown and thus restored conidiation in the atg8∆ mutant. Besides being important for conidiation, autophagy was also found to be essential for Magnaporthe pathogenesis. To uncouple the various functions of macroautophagy (such as pexophagy, cytoplasm to vacuole transport etc), the Atg20 (Autophagy related gene 20) protein was characterized and found to be essential for Magnaporthe conidiation and pathogenesis. Although mediated by Atg20, pexophagy was not important for Magnaporthe development. It was unclear whether the Cvt pathway exists in Magnaporthe. In this study, the specific target(s) for Atg20 mediated transport were uncovered and Atg20-dependent endosomal sorting and retrieval trafficking were key to conidiation and/or pathogenesis in Magnaporthe. Key words: Magnaporthe oryzae, autophagy, Cvt, pexophagy, endosomal sorting, retrieval trafficking, Atg8, Atg20. vi vi LIST OF FIGURES Figure Page Figure Schematic diagram of selective and non-selective autophagy .3 Figure Schematic representation of Magnaporthe life cycle .17 Figure Generation of atg8∆ mutant and genetic complementation strain .48 Figure ATG8 is essential for autophagosome formation in response to Nitrogen starvation .50 Figure Growth characteristics, aerial hyphal development, and conidiation in the atg8∆ mutant in Magnaporthe .52 Figure Posttranslational modification and subcellular localization of RFP-tagged Atg8p .54 Figure Autophagy is naturally induced during conidiation in Magnaporthe 57 Figure Suppression of conidiation defects by alternate carbon sources in atg8∆ mutant 59 Figure Suppression of conidiation defects by in planta growth .62 Figure 10 Identification and functional analysis of Gph1 in Magnaporthe 64 Figure 11 Growth characteristics and conidiation in the gph1∆ and gph1∆ atg8∆ mutant in Magnaporthe .65 Figure 12 Functional requirement of glycogen catabolism during Magnaporthe conidiation .67 Figure 13 Iodine staining for analysis of glycogen accumulation 69 Figure 14 Growth characteristics and conidiation in the sga1∆ and sga1∆ atg8∆ mutant in Magnaporthe .71 Figure 15 subcellular localization of Sga1 glucoamylase in Magnaporthe 74 Figure 16 Sga1-assisted hydrolysis of glycogen is required for proper asexual development in Magnaporthe .77 Firuge 17 Gph1 is not required for Magnaporthe pathogenesis .80 vii vii Figure 18 Sga1 is not required for Magnaporthe pathogenesis 82 Figure 19 A working model for glycogen metabolism and usage during asexual development in Magnaporthe 88 Figure 20 Characterization of Magnaporthe atg20∆ mutant 91 Figure 21 Subcellular localization of Atg20-GFP is independent of autophagy 93 Figure 22 Atg20-GFP is partially co-localized with RFP-Atg8 / autophagosome / autophagic vacuole 94 Figure 23 RFP-Atg8 localization does not depend on Atg20 .97 Figure 24 Atg20 is not required for non-selective autophagy or the Cvt pathway 99 Figure 25 Atg20 is important for Magnaporthe pexophagy 101 Figure 26 Visualization of pexophagy during Magnaporthe conidiation and pathogenic stages .103 Figure 27 Characterization of Magnaporthe atg26∆ mutant 107 Figure 28 N-terminal of Pex14 is important for pexophagy in Magnaporthe 110 Figure 29 Pexophagy is not required for Magnaporthe pathogenicity .111 Figure 30 Snc1 plays an important role for Magnaporthe conidiation .115 Figure 31 Investigation of vacuolar morphology .117 viii viii LIST OF TABLES Table Page Table List of M. oryzae strains used in this study 23 Table List of oligonucleotide primers used in this study 27 Table List of plasmids used in this study 33 ix ix LIST OF ABBREVIATIONS aa Atg8 Cfu CM d FA G6P G1P h hpi HPH LC3 LDL MDC MM MM-N M6P NSF OL ORF PA PAGE PCR PE PMSF RFP RT-PCR SDS Sga1 SNAP SNARE TEM TGN TOR WT Amino acid Autophagy related gene Colony forming unit Complete medium Day Fatty acid Glucose 6-phosphate Glucose 1-phosphate Hour Hours post inoculation Hygromycin phosphotransferase Microtubule-associated light chain Low density lipoprotein Monodansyl cadavarine Minimal medium Minimal medium minus nitrogen Mannose-6-Phospate N-ethylmaleimide-sensitive factor OLive oil Open reading frame Prune agar Polyacrylamide gel electropheresis Polymerase chain reaction Phosphatidylethanolamine Phenylmethylsulfonylfluoride Red fluorescent protein Reverse transcription polymerase chain reaction Sodium dodecyl sulphate Sporulation-specific glucoamylase Soluble NSF Attachment Protein SNAp REceptor Transmission electron microscopy Trans-golgi network Target of rapamycin Wild type x x PUBLICATIONS First author publication Deng, Y.Z., Ramos-Pamplona, M., and Naqvi, N.I. (2008) Methods for functional analysis of macroautophagy in filamentous fungi. Methods Enzymol 451: 295-310. Deng, Y.Z., Ramos-Pamplona, M., and Naqvi, N.I. (2009) Autophagy-assisted glycogen catabolism regulates asexual differentiation in Magnaporthe oryzae. Autophagy 5: 33-43. Deng, Y.Z., and Naqvi, N.I. (2010) A vacuolar glucoamylase, Sga1, participates in glycogen autophagy for proper asexual differentiation in Magnaporthe oryzae. Autophagy 6: 455 - 461. Co-author publication Sun, C.B., Suresh, A., Deng, Y.Z., and Naqvi, N.I. (2006) A multidrug resistance transporter in Magnaporthe is required for host penetration and for survival during oxidative stress. Plant Cell 18: 3686-3705. xi xi [...]... differentiation in Magnaporthe oryzae Autophagy 5: 33-43 Deng, Y.Z., and Naqvi, N.I (2010) A vacuolar glucoamylase, Sga1, participates in glycogen autophagy for proper asexual differentiation in Magnaporthe oryzae Autophagy 6: 455 - 461 Co-author publication Sun, C.B., Suresh, A., Deng, Y.Z., and Naqvi, N.I (2006) A multidrug resistance transporter in Magnaporthe is required for host penetration and for survival... Soluble NSF Attachment Protein SNAp REceptor Transmission electron microscopy Trans-golgi network Target of rapamycin Wild type x x PUBLICATIONS First author publication Deng, Y.Z., Ramos-Pamplona, M., and Naqvi, N.I (2008) Methods for functional analysis of macroautophagy in filamentous fungi Methods Enzymol 451: 295-310 Deng, Y.Z., Ramos-Pamplona, M., and Naqvi, N.I (2009) Autophagy- assisted glycogen... Low density lipoprotein Monodansyl cadavarine Minimal medium Minimal medium minus nitrogen Mannose-6-Phospate N-ethylmaleimide-sensitive factor OLive oil Open reading frame Prune agar Polyacrylamide gel electropheresis Polymerase chain reaction Phosphatidylethanolamine Phenylmethylsulfonylfluoride Red fluorescent protein Reverse transcription polymerase chain reaction Sodium dodecyl sulphate Sporulation-specific...LIST OF ABBREVIATIONS aa Atg8 Cfu CM d FA G6P G1P h hpi HPH LC3 LDL MDC MM MM-N M6P NSF OL ORF PA PAGE PCR PE PMSF RFP RT-PCR SDS Sga1 SNAP SNARE TEM TGN TOR WT Amino acid Autophagy related gene 8 Colony forming unit Complete medium Day Fatty acid Glucose 6-phosphate Glucose 1-phosphate Hour Hours post inoculation Hygromycin phosphotransferase Microtubule-associated light chain 3 Low density lipoprotein... Autophagy 6: 455 - 461 Co-author publication Sun, C.B., Suresh, A., Deng, Y.Z., and Naqvi, N.I (2006) A multidrug resistance transporter in Magnaporthe is required for host penetration and for survival during oxidative stress Plant Cell 18: 3686-3705 xi xi . EVALUATION OF THE ROLE OF AUTOPHAGY IN FUNGAL DEVELOPMENT AND PATHOGENESIS YIZHEN DENG A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK. Yun long and Selvarai Poonguzhali, for making our lab a most pleasant and stimulating working environment The TLL community, especially the sequencing lab and the microscopy and imaging facility. differentiation and conidiation in Magnaporthe 49 3.2.4 Post-translational processing and Atg8p targeting to autophagosomes in Magnaporthe 53 3.2.5 Induction and subcellular localization of RFP-Atg8 in