Clase2 Final

Clase 2
Métodos moleculares y aproximaciones “ómicas” usados para el

estudio de organismos filamentosos fitopatógenos.
1
Tools for the study of fungal pathogenesis
 Classical genetic
 Molecular genetics
 Cytological (microscopy)
 Biochemical (proteins, metabolites,
pharmacological approaches)
 “omics”: high through put data sets,
transcriptomics, proteomics, metabolomics…
http://www.fgsc.net/
2
3
Classical genetic
Screening for mutant phenotypes among survivors of radiation or chemical mutagenesis
A haploid phase for mutagenesis and screening.
A dikaryon or heterokaryon phase for complementation and epistasis analysis.
The ability to grow on defined media for selection and screening of nutritional and drug-
resistance markers.
The ability to grow at wide temperature ranges, which facilitates the isolation of
conditional lethal mutants.
Heterothallic strains for outcrossing.
Homothallic derivatives to allow haploid screens for mutants that are defective in
fundamentally diploid processes (for example, meiosis).
Small genomes (12–50 Mb) to facilitate mutant recovery and gene cloning.
The ability to carry out tetrad analysis, in which all four meiotic products are held together
in an ascus (in ascomycetes) or on the apex of a basidial cell (in basodiomycetes).
L. Casselton, M. Zolan, Nat. Rev. Genet. 3, 683–97 (2002). 4

Ron Morris (1975) in which he identified temperature-
sensitive, mitotic mutants of A. nidulans
L. Casselton, M. Zolan, Nat. Rev. Genet. 3, 683–97 (2002). 5

6
7
Fungal genetic transformation
 A plasmid or linear DNA carrying a selectable marker (usually drug
resistance) is introduced into the fungus
 The DNA molecule integrates into the genome
 Stable transformants are isolated following selection for the marker

gene or auxotrophic genes
Dominant positive selection markers
◦ Benomyl, Bialaphos, phleomycin, hygromycin
Complementation of auxotrophic mutants

◦ Nitrate, uridine
Mutant recipient strain required
8
Transformation of filamentous fungi
 Polyethylene (PEG)-mediated transformation of protoplasts
 Electroporation
 Biolistics
 Alkali metal ions (lithium acetate)
 Agrobacterium tumefaciens-mediated transformation
9
PEG-mediated transformation
 Generation of protoplasts using lytic enzymes (osmotic stabiliser
required)
 Transformation is carried out in the presence of PEG and CaCl2
 Circular or linear DNA may be used
 Protoplasts are mixed in molten agar and plated onto selective
medium
10
Regeneration of transformants
Liu, Zhaohui & Friesen, Timothy. (2012). Methods in molecular biology (Clifton, N.J.). 835. 365-75. 10.1007/978-1-
61779-501-5_21. 11
Transformation of filamentous fungi
 Polyethylene (PEG)-mediated transformation of protoplasts
 Electroporation
 Biolistics
 Alkali metal ions (lithium acetate)-YEAST
 Agrobacterium tumefaciens-mediated transformation
12
Agrobacterium tumefaciens
 Cause of crown-gall disease
 Unique naturally occurring trans-kingdom DNA transfer
 Has been exploited in plant research for many years
 Yeast (1995)
 A number of other fungi (de Groot et al., 1998; Nat. Biotechnol. 16:839-
842)
 Fungal transformation has been shown to depend on vir proteins
required for plant transformation (virB, virD1/D2)
13
AS
14
Selection of transformants
Transformed Control
15
Fungi that have been transformed using
Agrobacterium tumefaciens
1. Saccharomyces cerevisiae
2. Agaricus bisporus
3. Aspergillus niger
4. Aspergillus awamori
Can transform a variety of material –hyphae, spores,
5. Neurospora crassa protoplasts
6. Colleotrichum gloeosporiodes % single copy insertion events can be high
7. Fusarium venenatum
May be applicable to species recalcitrant to other
8. Fusarium oxysporum transformation methods (DNA protection?)
9. Magnaporthe grisea
10. Trichoderma reesei
11. Coccidioides immitis
12. Kluyveromyces lactis
16
Negative selection based on the thymidine kinase
- Thymidine kinase is a phosphotransferase that catalyses the reaction:
Thd + ATP → TMP + ADP
- Present in mammalian cells, TK have a key function in the synthesis of DNA
- 3'-Deoxy-3'-[(18)F]fluorothymidine is a thymidine analogue. When incorpororated

into cells, it is lethal.
LB TK RB
Hygr
17
Genetic transformation and its applications
 Gene inactivation
Inactivation of specific genes of interest
◦ Using homologous DNA to target the insertion event (REVERSE
GENETICS)
Random mutagenesis
◦ Generation of a collection of transformants that have undergone
insertion of non-homologous transforming DNA around the
genome (FORWARD GENETICS)
 Expression of transgenes
18
Targeted gene replacement
1) Clone the target gene EcoR1

5'
BamH1 BamH1
3' EcoR1
2) Construct a knock-out EcoR1

BamH1 BamH1
EcoR1
5' 3'
vector HygR
BamH1 BamH1
EcoR1 EcoR1
5' 3'
3) Transform linearised HygR
vector into fungus 5' 3'
TARGET GENE
BamH1 BamH1
4) Select for hygromycin 5' 3'
HygR
resistant transformants
19
Double-joint PCR: a PCR-based molecular tool for gene
manipulations
Fungal Genetics and Biology 41 (2004) 973–981

20
M. oryzae NADPH Oxidase Mutants Are Impaired in F-Actin Organization During
Appressorium Development
Split marker approach

- Generation of M. oryzae noxR null mutant
Supplemental Figure S1 Supplemental Figure S2
Ryder LS, Dagdas YF, Mentlak TA, Kershaw MJ, Thornton CR, et al. (2013) NADPH oxidases regulate septin-mediated cytoskeletal remodeling during plant
infection by the rice blast fungus. Proc Natl Acad Sci U S A 110: 3179-3184. 21
Reverse genetics:
An example of a homology-based approach
 Mitogen-activated (MAP) kinases are important signalling

components in yeast and other organisms
 MAP kinases have regions of highly conserved amino acids
 The M. grisea MAP kinase PMK1 was isolated using PCR
primers designed to match conserved regions of yeast MAP
kinases
 Targeted mutation of PMK1 was carried out
???
22
The Magnaporthe grisea kinase PMK1 is
required for pathogenicity
Wild type Pmk1-
Xu (2000) Fungal Genetics and Biology 31:137-152
23
Targeted genome editing using engineered nucleases
- Zn finger nucleases
- CRISPR/Cas system
- TALENs
The development of targetable DNA

cleavage reagents greatly enhanced the
efficiency of gene targeting
Genome Engineering with Targetable Nucleases Annu. Rev. Biochem. 2014. 83:409–39 24
2015
2015
25
Homologous recombination-
Yeast v filamentous fungi
Yeast (Saccharomyces cerevisiae)
◦ Highly efficient homologous recombination
◦ Only limited homology required (50 bp)
Filamentous fungi
◦ Inefficient homologous recombination (can be <1%)
◦ Frequency of homologous recombination increases with larger amounts of flanking
DNA (>1 kb generally used;5 kb better)
26
GENETICS)
Random mutagenesis
27
ATMT-mediated random integration into
the fungal genome
The origin of replication (ori) of E. coli allows the recovery of flanking sequences
28
29
TRANSFORMATION IN OOMYCETES
- Diploid organisms. Mutagenesis strategies (mutagen or targeted knockout)
for gene disruption must either target both copies of a gene or that
homozygosity of a mutation must be achieved through sexual outcrossing or
self-fertilization.
- Problem: However, some oomycete species such as P. ramorum do not
readily produce oospores in culture, whereas others such as P. infestans
produce oospores that are frequently recalcitrant to laboratory germination.
- In contrast to fungi, homologous recombination has not been reported in

oomycetes.
- Alternative: gene silencing or CRISPR
- In oomycetes, most studies that involve gene silencing have been

conducted in P. infestans.
30
Efficient disruption and replacement of an effector gene in the oomycete
Phytophthora sojae using CRISPR/Cas9
Molecular Plant Pathology

Volume 17, Issue 1, pages 127-139, 11 NOV 2015 DOI: 10.1111/mpp.12318
http://onlinelibrary.wiley.com/doi/10.1111/mpp.12318/full#mpp12318-fig-0001
31
Homology‐directed repair (HDR)‐mediated replacement of the
Avr4/6 open reading frame (ORF) with an NPT II ORF
Molecular Plant Pathology

Volume 17, Issue 1, pages 127-139, 11 NOV 2015 DOI: 10.1111/mpp.12318
http://onlinelibrary.wiley.com/doi/10.1111/mpp.12318/full#mpp12318-fig-0001
32
TRANSFORMATION IN OOMYCETES
- The number of oomycetes that have been transformed is low but increasing.
- Stable transformation has been achieved in several oomycete genera,
including Achlya, Phytophthora, Pythium, and Saprolegnia
Lamour, K & Kamoun, S. (2009). OOMYCETE GENETICS AND

GENOMICS. Diversity, Interactions, and Research Tools.
33
Gene silencing
1. RNA interference (RNAi) or RNA silencing is a gene
regulatory system, widely conserved in eukaryotes, that
Nicolás, et al. 2013 PLoS Pathogens represses gene expression through a homology-dependent
mechanism.
2. This repressive effect is mediated by small non-coding RNAs

(sRNAs) of about 20–30 nt, derived from double-stranded RNA
(dsRNA) precursors that are recognized and processed by the
RNaseIII Dicer.
3. These sRNAs are loaded into an RNA-induced

silencing complex (RISC), where the Argonaute protein plays a
main role.
4. Upon loading, the sRNAs selectively guide RISC to the target

RNAs, causing their degradation or preventing their translation.
5. Originally described as a defense mechanism against invasive

nucleic acids and viruses, RNAi and related pathways play many
fundamental roles in metazoans, including regulation of mRNA
levels and translation, chromatin silencing…
34
GENETICS)
Random mutagenesis
35
36
ADVANCES IN IMAGING THE CELL BIOLOGY
OF PLANT-MICROBE INTERACTIONS
-In vivo microscopy
using green fluorescent protein (GFP) as a marker
• Visualisation of fungal development in plants

• Reporter for gene expression
• Localisation of proteins
• Promoterless GFP gene can be used for random insertional
mutagenesis
• Reporter for host responses: use of fluorescent probes to
investigate changes in cytosolic calcium levels
37
Random integration of vector DNA into
the fungal genome
NcoI
(1898)
LB ToxA-Pr RB
SGFP
NOS-ter
Hygr TrpC-Pr
Cytosolic GFP
38
Reef Coral Fluorescent Proteins
GFP expressed in the cytosol
•Reef Coral Fluorescent Proteins

(AmCyan, ZsGreen, and
ZsYellow)
•Mixed population of germinating

conidia of Fusarium verticillioides
Bourett et al., Fungal Genetics and Biology 37 (2002) 211–220
39
Visualisation of fungal development in plants
fluorescence microscopy confocal laser scanning
30 mm
Fusarium
oxysporum -
tomato
Magnaporthe
oryzae- barley
40
41
The rice blast pathogen, Magnaporthe oryzae
42
-In vivo microscopy

mutagenesis
43
Reporter for gene expression
MPG1(p)::sGFP
MPG1(p)::sGFP
MPG1(pTr2)::sGFP
MPG1(pTr1)::sGFP
D.M. Soanes, et al. (2002) MPMI vol. 15 (12) p. 1253–1267 44

-In vivo microscopy

mutagenesis
45
PP2-LambdaN::3XGFP::NLS
PP2-LambdaN::3XGFP::NLS DIC
46
Pab1 is a poly-A binding proteins and has a cytoplasmic localization
Pab1::GFP DIC
47
Visualization of vacuoles in A. oryzae by expression of
CPY–EGFP
diferential interference microscopy fluorescence microscopy
48
PaNox1 locates both at the ER and the vacuolar system (VS)
I. Lacaze, H. Lalucque, U. Siegmund, P. Silar, S. Brun, Mol. Microbiol. 95, 1006–1024 (2015).
49
Mislocalisation of GFP fusion proteins in mutant backgrounds
N-WASP protein Las17, a component of the

actin-polymerizing Arp2/3 complex,
The ERM domain fusion protein Tea1-GFP
The localization of Rvs167-GFP a BAR-domain

protein
Ryder LS, Dagdas YF, Mentlak TA, Kershaw MJ, Thornton CR, et al. (2013) NADPH oxidases regulate septin-mediated cytoskeletal remodeling during plant
infection by the rice blast fungus. Proc Natl Acad Sci U S A 110: 3179-3184.
50
-In vivo microscopy

mutagenesis
51
Activation of GFP by promoters induced in specific fungal structures
Promoter trapping in Magnaporthe oryzae
Liu et al. / J Zhejiang Univ SCIENCE B 2006 7(1):28-33
52
-In vivo microscopy

mutagenesis
53
Reporter for host responses: changes in cytosolic
calcium levels during fungal penetration
susceptible cv resistant cv
0 hrs. cowpea epidermal cells – rust fungus
• microinjected with Calcium Green-1-dextran

linked with Texas Red (mRFP espectrum)
penetration • transient increase in cytosolic calcium level in

(d) shown by the greater yellow coloration.

lumen –Ann Rev Phytopath 2000 38:443–59
54
Quantification of light intensity
- Introduced a LifeAct-RFP gene fusion (14) into isogenic M. oryzae Δnox1, Δnox2,
Δnox1Δnox2 (15), and ΔnoxR mutants to observe organization of F-actin by live-cell
imaging
Figure 1
FRAP:
Fluorescence recovery
after photobleaching
Recovery of LifeAct-RFP ring after partial

photobleaching, with 75% recovery in fluorescence
after 3.5 min.
55
Scanning Electron microscopy Transmission Electron microscopy

(SEM) (TEM)
56
SEM images of oomycetes spores
57
58
Biological activity of drugs commonly used in cell biological research
Reference:
Nelson B. Cole. Compendium of Drugs Commonly Used in Cell Biology Research;
Current Protocols in Cell Biology (1998) Appendix 1.B.1-1.B.26
DNA replication inhibitors
59
60
Drugs affecting intracellular Ca2+
Drugs affecting oligosacharide biosynthesis/processing
61
Drugs affecting the pH of intracellular organelles
62
63
Kinase inhibitors (continued)
64
Phosphatase inhibitors
65
Protease inhibitors
66
Protein synthesis inhibitors
Antibiotic
Transcription inhibitors
67
68
69
Cell Cycle
• Cyclohexamide (CHX) and Hydroxyurea (HU) treatment

induced G1 and S-phase arrest
• Benomyl (Ben) treatment led to cell cycle arrest at G2/M
phase
70
71
“omics”
72
73
GENOMICS
74
POST-GENOMIC ERA
- Last 15 years rapid development of high trough put sequencing platforms → huge
advance in all fields of life sciences
 ABI 3730xL 1st generation
454 GS20 (Roche)

Illumina GAIlx (Solexa) 2nd generation
Illumina HiSeq (Solexa)
Pacific Biosystems 3rd generation

Ion Torrent
1 st generation 2 nd generation 3 rd generation
ABI 3730xl Roche 454 Illumina GAIlx Illumina PacBio Ion Torrent
GS/Titanium HiSeq 2000
read sequence lenght (bases) ~750 ~420 ~2 x 150 ~2 x 150 ~1,000 ~100-200
paralel reactions 96 1 million 600 millions 1,2 billions 160,000 500,000
bases (nt) number read per run <100,000 420 millions 90,000 millions (90GB) 450 GB 160 millions 50 millions
time of a complete run 2 hours 8 hours 12 days 8 days 15 minutes 1 hour
capacity of data storage MB GB TB TB GB MB
instrument cost €350,000 €500,000 €430,000 €750,000 €750,000 €50,000
cost per MB €500 €60 €1.2 €0.1 €1 €10
75
1000 fungal genomes project
http://1000.fungalgenomes.org/home/
http://genome.jgi.doe.gov/programs/fungi/index.jsf
76
Genome sizes of fungi relative to other
organisms
700
Genome size (Mb)
600
500
400
300
200
100
77
Main features of sequenced filamentous plant pathogen genomes
Raffaele S, Kamoun S (2012) Genome evolution in filamentous plant pathogens: why bigger
can be better. Nature Reviews Microbiology 10: 417-430. 78
PHYLOGENOMICS
- Appearance, duplication, and

loss of genes across the
ascomycetes (Wapinski, Nature 2007)
- It is not easy to study what makes

a fungus a plant pathogen
(Soanes et al.,, Plant Cell 2007) 79

Horizontal transfer of mobile chromosomes that confer
pathogenicity to Fusarium isolates
- four whole chromosomes present only in Fusarium oxysporum
- two of them → mobiles (horizontal transfer) and convert non-infective into pathogenic isolates
huésped
monocotiledóneas
monocotiledóneas
y
dicotiledóneas
monocotiledóneas
Li-Jun Ma, et al. Nature, 2010 80

Horizontal transfer
81
The rise of plant pathogenesis in fungal ascomycetes has involved
radically different evolutionary genomic changes
Magnaporthe oryzae (11,094 annotated genes)
- Mo has gained 3240 novel genes (20% genome)
- Mo has undergone: 193 unique duplications

1860 unique gene losses
Fusarium graminearum (13,332 annotated genes)
- Fg has gained 2513 novel genes (19% genome)
- Fg has undergone: 305 unique duplications

711 unique gene losses
Plant pathogenic ascomycetes

Broader pathogenic mechanisms
Species-specific pathogenic mechanisms
82
83
Emergence of wheat blast in Bangladesh was
caused by a South American lineage of
Magnaporthe oryzae
M. T. Islam et al., BMC Biol. 14, 84 (2016).
84
85
TRANSCRIPTOMICS
86
Transcriptome analysis using next-generation sequencing
To improve Genome annotation
87
Genome-transcriptome comparisons:
Identification of gene sets associated with particular attributes
 Yeast-like versus filamentous
 Saprophytes v pathogens
Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae
Galagan et al., (2005), Nature 438 p. 22-29
 Life style strategies Biotroph 1 v Necrotroph 2 v Hemibiotroph 3
Comparative genomic and transcriptomic analyses reveal the hemibiotrophic stage shift of
Colletotrichum fungi (2013) New Phytol. 197(4):1236–49 88
Genome-wide transcriptional profiling of appressorium development
by the rice blast fungus Magnaporthe oryzae.
Heatmaps showing levels of transcript abundance during time course of

appressorium development.
D. M. Soanes, A. Chakrabarti, K. H. Paszkiewicz, A. L. Dawe, N. J. Talbot, PLoS Pathog. 8, e1002514 (2012). 89
Estimation of the minimal sequencing depth required for
dual host–pathogen RNA-seq
Major RNA class

distribution
1%-5%
Westermann AJ, Gorski SA, Vogel J (2012) Dual RNA-seq of pathogen and host. Nat Rev Microbiol 10: 618-630. 90
Alternative polyadenylation also occurs in filamentous fungi
S7 (40S ribosomal subunit, MGG_00221)
5’ UTR 3’ UTR
wt
rbp35
91
Transcriptome analysis reveals the complexity of alternative
splicing regulation in the fungus Verticillium dahliae
L. Jin et al., BMC Genomics. 18, 130 (2017).

92
DNA-binding specificities of transcription factors
- Based in protein-binding microarray (PBM)
NATURE GENETICS 2004 VOLUME 36 (12), 1331
- Based in pull downs of tagged protein and chromatin

- Chromatin immunoprecipitation (ChIP-on-chip/Chip-SEQ)
93
What the transcriptome does not tell — proteomics and metabolomics are closer to the plants’ patho-phenotype (2015)
I. Feussner and A. Polle Current Opinion in Plant Biology 2015, 26:26–31
94
95
PROTEOMICS
96
Comparison of total protein extracts from wild type and mutant
(Two-dimesional gel;
27 differentially up- and
down-regulated)
M. oryzae wt / rbp35
97
Isobaric tags for relative and absolute quantitation (iTRAQ)
Current Status and Advances in Quantitative Proteomic Mass Spectrometry 98

- Pyrimorph is a novel fungicide from the carboxylic acid amide (CAA) family used to control
plant-pathogenic oomycetes such as Phytophthora capsici.
- The proteomic response of P. capsici to pyrimorph was investigated using the iTRAQ
technology to determine the target site of the fungicide and potential biomarker candidates of
drug efficacy.
- A total of 1336 unique proteins were identified from the mycelium of wild-type P. capsici
isolate (Hd3) and two pyrimorph resistant mutants (R3-1 and R3-2) grown in the presence or
absence of pyrimorph.
- Biochemical analysis using D-[U-14C]glucose verified the proteomics data, suggesting that
the major mode of action of pyrimorph in P. capsici is the inhibition of cell wall biosynthesis.
99
iTRAQ-MS/MS Proteomic Analysis Reveals Differentially Expressed
Proteins During Post-harvest Maturation of the White Button
Mushroom Agaricus bisporus.
M. Chen et al., Curr. Microbiol. 74, 641–649 (2017).

100
Tandem affinity purification (TAP; TAP tagging): a protein putification method
Nature Reviews Molecular Cell Biology 4, 74-80 (2003) 101

RBP35, a fungal specific RNA-binding protein, interacts
with Cleavage Factor I 25 kDa
- Immunoprecipitations experiments using RBP35 tagged with HA-FLAG
SDS-PAGE / Commassie blue
(Franceschetti et al., PLoS Pathogens 2011) 102

103
METABOLOMICS
104
Phylogenomics and evolution of secondary metabolism in plant-associated fungi
Fusarium
graminearum Claviceps purpurea (Pleurotus ostreatus) Alternaria mali
Cochliobolus heterostrophus
Secondary metabolites are small organic molecules that are not essential for an organism’s survival
J.W. Spatafora and K.E. Bushley. Current Opinion in Plant Biology 2015, 26:37–44 105
While all groups of fungi possess some form of secondary metabolism, it has diversified to
its greatest extent within Ascomycota and Basidiomycota
- genes involved in secondary

metabolism are
overrepresented in
saprotrophic,
necrotrophic, and
hemibiotrophic fungi,
whereas biotrophy is
associated with a
convergent loss of
secondary metabolites
- KOG annotation
J.W. Spatafora and K.E. Bushley. Current Opinion in Plant Biology 2015, 26:37–44 106
A Metabolic Profiling Strategy for the Dissection
of Plant Defense against Fungal Pathogens
Color-coded fluctuation of the soybean's seedling metabolic network caused

by Rhizoctonia solani infection at 24 h and 48 h post-inoculation
K. A. Aliferis, D. Faubert, S. Jabaji, PLoS One. 9 (2014), doi:10.1371/journal.pone.0111930. 107

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Clase 2

Métodos moleculares y aproximaciones “ómicas” usados para el

L. Casselton, M. Zolan, Nat. Rev. Genet. 3, 683–97 (2002). 4

L. Casselton, M. Zolan, Nat. Rev. Genet. 3, 683–97 (2002). 5

 The DNA molecule integrates into the genome

 Stable transformants are isolated following selection for the marker

Complementation of auxotrophic mutants

- Present in mammalian cells, TK have a key function in the synthesis of DNA

- 3'-Deoxy-3'-[(18)F]fluorothymidine is a thymidine analogue. When incorpororated

1) Clone the target gene EcoR1

2) Construct a knock-out EcoR1

vector into fungus 5' 3'

Fungal Genetics and Biology 41 (2004) 973–981

Split marker approach

Supplemental Figure S1 Supplemental Figure S2

 Mitogen-activated (MAP) kinases are important signalling

Wild type Pmk1-

Xu (2000) Fungal Genetics and Biology 31:137-152

The development of targetable DNA

- In contrast to fungi, homologous recombination has not been reported in

- Alternative: gene silencing or CRISPR

- In oomycetes, most studies that involve gene silencing have been

Molecular Plant Pathology

Molecular Plant Pathology

Lamour, K & Kamoun, S. (2009). OOMYCETE GENETICS AND

2. This repressive effect is mediated by small non-coding RNAs

3. These sRNAs are loaded into an RNA-induced

4. Upon loading, the sRNAs selectively guide RISC to the target

5. Originally described as a defense mechanism against invasive

-In vivo microscopy

using green fluorescent protein (GFP) as a marker

• Visualisation of fungal development in plants

•Reef Coral Fluorescent Proteins

•Mixed population of germinating

Bourett et al., Fungal Genetics and Biology 37 (2002) 211–220

fluorescence microscopy confocal laser scanning

-In vivo microscopy

using green fluorescent protein (GFP) as a marker

D.M. Soanes, et al. (2002) MPMI vol. 15 (12) p. 1253–1267 44

-In vivo microscopy

using green fluorescent protein (GFP) as a marker

diferential interference microscopy fluorescence microscopy

N-WASP protein Las17, a component of the

The ERM domain fusion protein Tea1-GFP

The localization of Rvs167-GFP a BAR-domain

-In vivo microscopy

using green fluorescent protein (GFP) as a marker

• Visualisation of fungal development in plants

Promoter trapping in Magnaporthe oryzae

Liu et al. / J Zhejiang Univ SCIENCE B 2006 7(1):28-33

using green fluorescent protein (GFP) as a marker

0 hrs. cowpea epidermal cells – rust fungus

• microinjected with Calcium Green-1-dextran

penetration • transient increase in cytosolic calcium level in

ADVANCES IN IMAGING THE CELL BIOLOGY

Recovery of LifeAct-RFP ring after partial

Scanning Electron microscopy Transmission Electron microscopy

DNA replication inhibitors

Drugs affecting oligosacharide biosynthesis/processing

• Cyclohexamide (CHX) and Hydroxyurea (HU) treatment

 ABI 3730xL 1st generation

454 GS20 (Roche)

Pacific Biosystems 3rd generation

- Appearance, duplication, and