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Molecular and bioinformatic analysis of the

perA locus in Epichlo

This thesis is presented as a partial fulfilment of the requirements for the degree of

Master of Science (MSc)

in
Genetics

at Massey University, Palmerston North

New Zealand

Daniel Berry
2011

Abstract
Fungal endophytes of the Epichlo genus form largely mutualistic symbioses with coolseason grasses, systemically colonising the intercellular spaces of the host in a strictly
regulated fashion. The endophyte receives protection and sustenance from the host, and
in return provides benefits such as increased growth, drought resistance and protection
against herbivores. Protection against herbivory is mediated through the production of
bio-protective fungal secondary metabolites (SM). Examples of these SMs include
lolitrem B, the causative agent of ryegrass staggers in stock, and the insect feeding
deterrent peramine.
The genes responsible for the production of each of these SMs are usually found
clustered together in the genome, and are often closely associated with a range of
transposon relics. SM gene expression occurs only when the endophyte is growing in
planta, indicating the presence of plant-fungal signalling. This study investigated the
locus structure and organisation of the gene perA that encodes the non-ribosomal
peptide synthetase PerA, which is both essential and sufficient for production of
peramine. It was found that perA and its flanking intergenic sequences exhibit
considerable transposon-mediated variability across Epichlo, and that this transposon
activity is likely responsible for the taxonomically discontinuous production of
peramine both within and across Epichlo spp.
The major facilitator superfamily transporter gene EF102 is divergently transcribed
from and co-regulated with perA (EF103). Transcriptome data were used to identify
transcription start sites for both genes. Comparative analysis of the intergenic sequence
separating EF102/perA from 10 Epichlo isolates covering six different species refined
the perA translation start site, and identified conserved regions in the promoters of both
genes proposed to be important for regulation. A motif search identified a conserved
DNA motif present multiple times in the promoters of both genes.
Deletion analysis of EF102 revealed the gene probably does not encode a peramine
transporter, as was hypothesised; however the four independent EF102 mutants
exhibited a reduction in peramine production relative to wild type, resulting in an
alternative hypothesis that EF102 encodes a transporter for a PerA substrate precursor
molecule such as glutamate.

Acknowledgements
Firstly I thank my supervisor Barry Scott for his guidance and support (and funding!),
without which this would not have been possible.
Thanks to my collaborator Carolyn Young, who provided much of the material and
DNA sequences required for this study.
Thanks to the Scottbase members (both past and present) Ruth, Gemma, Carla, Emma,
Milena, Tetsuya, Yvonne, Sarah, Matt, Sanjay for their help and guidance around the
lab over the last two (and a bit) years, and to Murray for his help with the bioinformatics
aspects of this study. Thanks to Mike for getting me into this area in the first place.
Thanks to the lovely people at AgResearch who keep our plants watered, and especially
Wayne and Wade for their advice and work on the guttation analyses.
Thanks to IMBS and all the staff (esp. Ann, Pat and Cynthia) who have helped me
along the way.
Lastly thanks to Mum and Dad for their support both emotional and fiscal!

ii

Table of Contents
Abstract

Acknowledgements

ii

Table of Contents

iii

List of Tables

viii

List of Figures

ix

Common Abbreviations

1.

Introduction

1.1
Epichlo endophyte life cycle
1.1.1 Endophyte growth in planta
1.1.2 E. festucae and L. perenne as a model system
1.2
1.2.1

Epichlo taxonomy
Hybrid origins are common for Neotyphodium spp.

1.3
Benefits of the association
1.3.1 Bio-protective secondary metabolites are produced by epichlo
endophytes in planta
1.3.2 Fungal secondary metabolite gene clusters are plant-regulated
1.3.3 Peramine: distribution and genotype effects
1.3.3.1 Peramine synthesis pathway and PerA protein structure
1.3.3.2 Conservation and distribution of perA
1.3.3.3 PerA locus in E. festucae E2368
1.3.3.4 Detection of peramine in the host guttation fluid

2
2
4
4
5
7
7
8
9
9
10
13
13

1.4
Transcriptome analysis of a disrupted symbiosis E. festucae mutant
1.4.1 Transcriptome analysis of the E. festucae Fl1 sakA mutant
1.4.2 Transcriptome analysis identifies EF102 as a potential peramine
transporter

14
14
14

1.5
1.5.1

Regulation of epichlo secondary metabolite genes

Comparative analysis as a tool for regulatory motif identification

15
15

1.6

Aims

16

iii

2.

Materials & Methods

18

2.1

Biological material

19

2.2
Common stocks, growth media and conditions
2.2.1 Basic growth media
2.2.1.1 Potato Dextrose medium (PD)
2.2.1.2 Luria Bertani medium (LB)
2.2.1.3 Regeneration medium (RG)
2.2.1.4 Sterilization conditions
2.2.2 Growth conditions
2.2.2.1 Escherichia coli
2.2.2.2 Epichlo festucae
2.2.3 Common stock solutions
2.2.3.1 SDS loading dye
2.2.3.2 20x SSC Buffer

21
21
21
21
21
21
21
21
22
22
22
22

2.3
2.3.1
2.3.2
2.3.3
2.3.4

Standard E. coli cell methods

Cloning DNA fragments into an E. coli plasmid vector
E. coli transformations
Screening E. coli transformants
E. coli plasmid isolation

22
22
22
23
23

2.4
2.4.1
2.4.2

Standard E. festucae cell methods

Fungal DNA extraction
Preparation of E. festucae fungal protoplasts

23
23
24

2.5
2.5.1
2.5.2
2.5.3
2.5.4

Standard plant methods

Plant inoculation
Plant immunoblotting to confirm fungal infection
Preparation of infected ryegrass samples for confocal microscopy
Confocal microscopy

25
25
25
26
26

2.6
Standard DNA methods
2.6.1 DNA concentration measurement
2.6.2 Restriction enzyme digestion of DNA
2.6.2.1 Digestion of plasmid DNA
2.6.2.2 Digestion of genomic DNA for Southern blotting
2.6.3 DNA Sequencing
2.6.4 Polymerase Chain Reaction
2.6.4.1 Standard PCR
2.6.4.2 Difficult template PCR
2.6.4.3 Multiplex PCR
2.6.4.4 High fidelity PCR (Expand Hi-Fi)
2.6.4.5 High Fidelity PCR (Platinum Pfx)
2.6.5 Gel Electrophoresis
2.6.6 DNA purification following PCR or gel electrophoresis
2.6.7 A-tailing DNA fragments
2.6.8 DNA ligation
iv

27
27
27
27
27
28
29
30
30
30
31
31
32
32
32
33

2.6.9 Southern Blotting

2.6.10 Radioactive hybridization and visualization

33
34

2.7
2.7.1
2.7.2

Peramine analysis
Peramine HPLC-UV analysis
Peramine LC-MS analysis

34
34
35

2.8
EF102 deletion mutant creation
2.8.1 Creation of EF102 deletion construct
2.8.2 Transformation of Epichlo festucae
2.8.3 Screening for EF102 mutants

35
35
36
37

2.9
Bioinformatic methods
2.9.1 Multiple sequence alignments
2.9.2 DNA motif identification

38
38
38

3.

Epichlo perA Locus Comparison

39

3.1
3.1.1
3.1.2

PCR analysis of the perA locus in epichlo

Genes surrounding perA are highly conserved within epichlo
PerA and flanking intergenic sequences are highly variable
within epichlo

40
40
40

3.2
3.2.1

Southern blot analysis of the perA locus in epichlo

Southern blot analysis of the perA locus reveals considerable variation
within E. festucae
Southern blot analysis shows variation in the intergenic regions flanking
perA within the ephichlo species tested

44
44

3.3

Peramine production analysis of E. festucae isolates

52

3.4

Confocal analysis of E. festucae infected ryegrass samples

52

4.

Analysis of the perA Promoter

57

4.
4.1

Analysis of the EF102-perA intergenic sequence in epichlo

Refining the perA translation start site

58
58

4.2

Establishing the transcription start sites of EF102 and perA

61

4.3

EF102 and perA promoters share a common DNA motif

61

5.

Deletion Analysis of the Major Facilitator Superfamily

Transporter EF102

67

5.1

Deletion of EF102

68

5.2

EF102 chemotype analysis

68

6.

Discussion

74

3.2.2

45

6.1

The perA locus structure differs significantly within E. festucae

75

6.2

The perA locus structure is conserved between putative peramine

producing Epichlo spp

77

6.3

All E. festucae isolates are able to infect Lolium perenne

78

6.4

A new translation start site was identified for perA

78

6.5

Transcription start sites were identified for EF102 and perA

79

6.6

Identification of an extended region of sequence conservation

upstream of the perA transcription start site

79

6.7

Potential limitations of comparative analysis within epichlo

80

6.8

EF102 and perA share a common promoter motif

80

6.9

A peramine transport role for EF102 is not supported: alternative

substrate transport hypothesis proposed

81

6.10

Conclusions

85

7.

Appendices

86

7.1
7.1.1

Supplemental tables
Gene expression changes around the perA locus in the sakA mutant
relative to wild type Fl1 (Eaton et al., 2010)
Peramine concentrations guttation fluid samples for wild type
E. festucae Fl1 and EF102 mutant infected plants

87
87

Vector maps
pSF15.15
pCR4-TOPO

88
88
89

7.1.2

7.2
7.2.1
7.2.2
7.3
7.3.1

Multiple sequence alignments

MSA of the EF102-perA intergenic sequence from 10
epichlo isolates
7.3.2 MSA of EF102 and homologues from Periglandula Ipomea,
Gibberella zeae and Metarhizium anisopliae: N- and C-terminal
domains show significant sequence variation.
7.4
Supplemental Southern blots
7.4.1 Southern blot from Fig. 3.3 probed with EF102
7.4.2 Southern blot from Fig. 3.3 probed with EF104
7.4.3 Southern blot from Fig. 3.4 probed with EF102
7.4.4 Southern blot from Fig. 3.4 probed with EF104
vi

87

89
90-103
104

105
105
106
106
107

7.4.5
7.4.6
7.4.7
7.4.8
7.4.9
7.4.10
7.4.11

Southern blot from Fig. 3.5 probed with EF102

Southern blot from Fig. 3.5 probed with EF104
Southern blot from Fig. 3.6 probed with EF102
Southern blot from Fig. 3.6 probed with EF104
Southern blot from Fig. 3.7 probed with EF102
Southern blot from Fig. 3.7 probed with EF104
Southern blot from Fig 3.8 probed with EF102

107
108
108
108
109
109
110

Bibliography

111

vii

List of Tables
2.1
2.2
2.3:
3.1
5.1
6.1

Bacterial strains, fungal strains and plant material

Plasmids and DNA
Primers used in this study
Whole tiller peramine concentration in plants infected by different
E. festucae isolates
Peramine concentrations in whole tiller and guttation fluid samples
for wild type E. festucae Fl1 and EF102 mutant infected plants
Arginine and proline metabolism genes are not differentially
regulated in E. festucae

viii

19
20
29-30
53
73
84

List of Figures
1.1
1.2
1.3
1.4
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
4.1
4.2
4.3
4.4
4.5
4.6
5.1
5.2
5.3
6.1
6.2

Lifecycle of epichlo endophytes

Epichlo phylogenetic tree
Domain structure of PerA and the peramine synthesis pathway
Synteny analysis of the perA locus with other filamentous fungi
Gene map of the perA locus with PCR primers annotated
PCR based comparative analysis of the perA locus within epichlo
Southern hybridisation of BamHI digested gDNA from various
E. festucae isolates
Southern hybridisation of NdeI digested gDNA from various
E. festucae isolates
Southern hybridisation of SalI digested gDNA from various
E. festucae isolates
Southern hybridisation of BclI digested gDNA from various
E. festucae isolates
Southern hybridisation of BamHI digested gDNA from various
epichlo isolates
The E2368 and E189 perA locus show identical banding patterns
Confocal microscopy of E. festucae infected perennial ryegrass
samples
Refining the location of the perA start codon
Consensus Kozak sequence for epichlo
Transcriptome reads showing the transcription start sites of EF102
and perA
Sequence conservation in the promoters of perA and EF102
A conserved DNA motif identified in the promoters of EF102/perA
Potential regulatory motifs in the promoters of EF102 and perA
EF102 deletion construct
PCR screening of potential EF102 mutants
Southern blot analysis of EF102 mutants
Known and proposed physical map of the perA locus in different
E. festucae strains
Simplified arginine and proline metabolism pathways

ix

3
6
11
12
42
43
46
47
48
49
50
51
54-56
59
60
63
64
65
66
70
71
72
76
83

Common Abbreviations
A1/2
Amp
ASW
ATG
bp
C1
CDS
d
dATP
DMSO
DNA
dNTP
ECM
EDTA
EF
g
gDNA
h
HGT
HPLC
Hyg
Indel
kb
KO
LB
LB
LC
M
M1
min
MITE
mRNA
MFS
MS
MSA
NCM
ND
NRPS
NT
P5C
PBS
PCR
PD
PKS
R2/Rdom

NRPS adenylation domain from modules 1 or 2

Ampicillin
Argentine stem weevil
ATG translational start codon
base pairs
NRPS condensation domain from module 1
Coding sequence
Days
Deoxyadenine triphosphate
Dimethyl sulfoxide
Deoxyribonucleic acid
Deoxynucleotide triphosphate
Extracellular matrix
Ethylene diamine tetra-acetic acid
Epichlo festucae
Gravity
Genomic DNA
Hours
Horizontal gene transfer
High-performance liquid chromatography
Hygromycin
Insertion or deletion
Kilo base pairs
Knock out
Luria-Bertani medium
Left border
Liquid chromatography
Molar
NRPS methylation domain from module 2
Minutes
Miniature inverted transposable element
Messenger RNA
Major facilitator superfamily
Mass spectrophotometer
Multiple sequence alignment
Nitrocellulose membrane
Not detected
Non-ribosomal peptide synthetase
Not tested
1-pyrroline-5-carboxylate
Phosphate buffered saline
Polymerase chain reaction
Potato dextrose medium
Polyketide synthase
NRPS reductase domain from module 2
x

RB
RCF
RE
RG
RNA
ROS
RT-PCR
SDS
SSC
SM
SNP
T1/2
TBE
UV
WT

Right border
Relative centrifugal force
Restriction enzyme
Regeneration medium
Ribonucleic acid
Reactive oxygen species
Reverse transcription PCR
Sodium dodecyl sulfate
Saline sodium citrate
Secondary metabolite
Single nucleotide polymorphism
NRPS thiolation domain from module 1 or 2
Tris Borate EDTA buffer
Ultra-violet
Wild type

xi