Evaluation of the role of autophagy in fungal development and pathogenesis

In this study, an essential role for autophagy-assisted glycogen breakdown glycogen autophagy is described in the rice-blast fungus Magnaporthe oryzae.. The conidiation defect in autopha

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

TABLE OF CONTENTS

CHAPTER I INTRODUCTION 1

1.1 General introduction to autophagy 1

1.1.1 Process and classification of macroautophagy 1

1.1.1.1 Glycogen autophagy 2

1.1.1.2 The Cvt pathway 2

1.1.1.3 Pexophagy 4

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 8

1.3.3.3 Plants 9

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

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

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

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

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

LIST OF FIGURES Figure Page Figure 1 Schematic diagram of selective and non-selective autophagy 3 Figure 2 Schematic representation of Magnaporthe life cycle 17

Figure 3 Generation of atg8∆ mutant and genetic complementation strain 48

Figure 4 ATG8 is essential for autophagosome formation in response to

Nitrogen starvation 50

Figure 5 Growth characteristics, aerial hyphal development, and conidiation

in the atg8∆ mutant in Magnaporthe 52

Figure 6 Posttranslational modification and subcellular localization of

RFP-tagged Atg8p 54

Figure 7 Autophagy is naturally induced during conidiation in Magnaporthe 57

Figure 8 Suppression of conidiation defects by alternate carbon sources in atg8∆

mutant 59

Figure 9 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

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

LIST OF TABLES Table Page Table 1 List of M oryzae strains used in this study 23 Table 2 List of oligonucleotide primers used in this study 27 Table 3 List of plasmids used in this study 33

LIST OF ABBREVIATIONS

aa Amino acid

Atg8 Autophagy related gene 8

Cfu Colony forming unit

CM Complete medium

d Day

FA Fatty acid

G6P Glucose 6-phosphate

G1P Glucose 1-phosphate

h Hour

hpi Hours post inoculation

HPH Hygromycin phosphotransferase

LC3 Microtubule-associated light chain 3

LDL Low density lipoprotein

MDC Monodansyl cadavarine

MM Minimal medium

MM-N Minimal medium minus nitrogen

M6P Mannose-6-Phospate

NSF N-ethylmaleimide-sensitive factor

OL OLive oil

ORF Open reading frame

PA Prune agar

PAGE Polyacrylamide gel electropheresis

PCR Polymerase chain reaction

PE Phosphatidylethanolamine

PMSF Phenylmethylsulfonylfluoride

RFP Red fluorescent protein

RT-PCR Reverse transcription polymerase chain reaction

SDS Sodium dodecyl sulphate

Sga1 Sporulation-specific glucoamylase

SNAP Soluble NSF Attachment Protein

SNARE SNAp REceptor

TEM Transmission electron microscopy

TGN Trans-golgi network

TOR Target of rapamycin

WT Wild type

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