Plant Epigenetics 2010 European Networking Summer School … · 2017. 9. 27. · All participants...

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Plant Epigenetics 2010

European Networking Summer School (ENSS)

September 20th to 24th 2010 Editors: Markus Kuhlmann and Michael Florian Mette Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany

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Content: page General information 3 Organisational: 7 Schedule 7 Group assignment 8 Lectures: Speakers 9 Laboratory work: 22 I) Chromatin analysis of mitotic and meiotic chromosomes Introduction (for I and II) 23 Protocol 27 II) Analysis of chromatin in flow-sorted interphase nuclei of plants Protocol 31 III) Transcriptional gene silencing in Arabidopsis and Drosophila A) Analysis of cytosine methylation patterns in plant DNA

Introduction 35 Protocol 40 B) PEV in Drosophila and heterochromatic gene silencing in Arabidopsis - a

comparison Introduction 45

Protocol 48 IV) Post-transcriptional gene silencing in plants: case studies A) Reverse genetics by transient RNA interference in plant epidermis cells

Introduction 50 Protocol 53 B) Detection of transgene silencing by histochemical GUS staining Introduction 75 Protocol 79

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General information

1. Summer school venue To those who have not yet been at IPK:

The Leibniz Institute of Plant Genetics and Crop Plant Research (IPK Gatersleben) is

a research institute of the Leibniz Association located at Corrensstraße 3, D-06466

Gatersleben, Germany. All lectures of the school will be held in the main lecture hall

(German: “Hörsaal”, see map below) of IPK Gatersleben. The lab sessions will be

held in several buildings of the institute. All relevant locations are in convenient

walking distance on the institute campus.

Gatersleben is a quaint village in an idyllic rural area in the state Saxony-Anhalt,

Germany, just north of the Harz mountains. Quedlinburg is an UNESCO world

heritage town, a preserved European city of medieval origin distinguished by its

exceptional architectural heritage of Romanesque and half-timbered buildings.

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2. Accommodation All participants of the Summer School will be accommodated in hotels in Quedlinburg

(please check the map on the next page for details) from Sunday, September 19th to

Friday, September 24th 2010. The hotel rooms in Quedlinburg will be not available for

the last night from Friday 24th to Saturday 25th 2010. Therefore, we will offer a bus

departing Friday 24th evening at 7:30 p.m. from IPK Gatersleben to the central

railway station of the close-by (50 km) city of Magdeburg from where trans-regional

train connections are available. Participants can choose to either immediately

continue their journey home by train on Friday evening or to stay for the night at the

local InterCityHotel in front of the central railway station and start off Saturday

morning.

Accommodation costs (“bed and breakfast”) will be directly settled from the summer

schools budget. All extra costs like phone calls, internet access, etcetera, are to be

covered by the participants themselves.

3. Transfers Every morning we organised bus transfers from two central places in Quedlinburg to

the institute in Gatersleben. For the pick-up sites, please consult the map on the next

page. The two pick-up sites are depicted with bus signs.

Daily Pick up times are:

– Carl-Ritter-Strasse: departure at 8:00 a.m. – Mathildenbrunnen (close to hotel Zur Goldenen Sonne): departure at 8:05 a.m. After the lab work and the social activities on Tuesday and Wednesday the bus will

drop all participants at those two sites, too.

4. WLAN access All hotels offer WLAN access at least in the lobbies. For further details, please check

with the reception in your hotel and ask for access information and associated costs.

Please, bear in mind that those costs must be borne by every participant and will not

be covered by the Summer School.

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1- Romantik Hotel Theophano, Markt 13/14, 06484 Quedlinburg 2- Hotel Zur Goldenen Sonne, Steinweg 11, 06484 Quedlinburg and bus

departure Mathildenbrunnen (Every morning 8:05 a.m.!) 3- Hotel Zum Bär, Markt 8, 06484 Quedlinburg 4- Bus departure Carl-Ritter-Straße (Every morning 8:00 a.m.!)

bus departures to IPK Gatersleben

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train station

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5. Social Events Tuesday, 21st September On Tuesday evening, a barbecue in the canteen of the institute will be held after the

lab sessions starting 6:30 p.m.. Wednesday, 22nd September For Wednesday, a trip to the nearby top of the mountain Brocken is planned. You

should consider a temperature drop of 20 °C on top of Brocken and bring shoes and

other clothes accordingly, especially for the way back in the evening. Rain showers

are also very common. The climb-up will be done in coachers of a historic railway.

The descent will be an opportunity for hiking. Take-off time from the institute will be

after lunch at 1:30 p.m. and the expected time of return to Quedlinburg will be about

9:00 p.m.. Thursday, 23rd September On the last evening in Quedlinburg, you will experience the special atmosphere of

the town while a guided tour through the small lanes and yards. Starting time is 8:30 p.m. and gathering point is the office of the tourist information next to the town hall.

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Schedule ENSS Plant Epigenetics 2010 - September 19th to 25th 2010

IPK Gatersleben, Corrensstraße 3, 06466 Gatersleben, Germany

day morning afternoon evening

Sunday Sept. 19th individual arrival in Quedlinburg

Monday Sept. 20th 8:00 bus from Quedlinburg

2 lectures 08:30 I. Schubert 10:00 V. Colot

lab work 12:30 - 18:00

A-IV, B-I, C-II, D-III

1 lecture 18:30 R. Aalen

21:00 bus back to

Quedlinburg

Tuesday Sept. 21st 8:00 bus from Quedlinburg

2 lectures 08:30 O. Mittelsten Scheid 10:00 A. Depicker

lab work 12:30 – 18:00

A-I, B-II,

C-III, D-IV

18:30 barbeque 22:00 bus

back to Quedlinburg

Wednesday Sept. 22nd 8:00 bus from Quedlinburg

3 lectures 08:30 G. Reuter 10:00 P. Meyer 11:00 R. Schmidt

Social 13:30 bus

departure to Wernigerode

14:55 HSB to Brocken

Event 18:31

departure Brocken 20:20 bus

Wernigerode back to Quedlinburg

Thursday Sept. 23rd 8:00 bus from Quedlinburg

2 lectures 08:30 W.H. Shen 10:00 A. Houben

lab work 12:30 – 18:00

A-II, B-III, C-IV, D-I

18:30 bus to Quedlinburg

Cultural Event 20:30 guided tour

historic town of Quedlinburg

Friday Sept. 24th 8:00 bus from Quedlinburg

2 lectures 08:30 P. Schweizer 10:00 M.F. Mette

lab work 12:30 – 18:00

A-III, B-IV, C-I, D-II

18:30 résumé 19:30 bus

to Magdeburg

Saturday Sept. 25th individual departure

Social and cultural events are indicated in red.

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Group assignment for lab sessions

I) Chromatin analysis of mitotic and meiotic chromosomes - Andreas Houben II) Analysis of chromatin in flow-sorted interphase nuclei of plants - Jörg Fuchs and

Ingo Schubert III) Transcriptional gene silencing in Arabidopsis and Drosophila - Michael Florian

Mette and Gunter Reuter IV) Post-transcriptional gene silencing in plants: case studies - Patrick Schweizer

and Ann Depicker

group A group B group C group D

Martin Antosch Maksym Danchenko Dagmara Kwolek Massimo Rainieri

Alok Arun Anna De Carlo Liliana Marii Omar Saleh

Michael Bartsch Federica Della

Rovere Carmen Martin Stefanie Seitz

Wubishet Abebe

Bekele Domenico De Paola Liliana Marum Reza Shirzadi

Georgi Bonchev Kirsten De Wilde Francesco Mercati Renatantonio

Tavano

Haroon Butt Bernardi Jamila Tunde Nyiko Silje Veie Veiseth

Anna Vittoria

Carluccio Sladjana Jevremovic Alice Pajoro Christina Vogiatzi

Marco Catoni Meglena Kitanova Nadine Petersen Igor Yakovlev

Aurelie Comte Viera Kovacova Alexandra Plotnikova Wojciech Zalewski

Mo 20th: IV Mo 20th: I Mo 20th: II Mo 20th: III

Tu 21st: I Tu 21st: II Tu 21st: III Tu 21st: IV

Th 23rd: II Th 23rd: III Th 23rd: IV Th 23rd: I

Fr 24th: III Fr 24th: IV Fr 24th: I Fr 24th: II

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Lectures Monday, September 20th 2010 08:30 Schubert, Ingo (IPK, Gatersleben, Germany)

Modifications of plant interphase chromatin at the microscopic level - an overview 10:00 Colot, Vincent (École Normale Supérieure, Paris, France)

A genome-wide view of chromatin dynamics and chromatin-based epigenetic processes in Arabidopsis

18:30 Aalen, Reidunn Brigitta (Dept. of Molecular Biosciences, University of Oslo, Norway) Readers and writers of the histone code

Tuesday, September 21st 2010 08:30 Mittelsten Scheid, Ortrun (Gregor Mendel Institute of Molecular Plant Biology,

Vienna, Austria) Stability and plasticity of transcriptional gene silencing

10:00 Depicker, Ann (Vlaams Interuniversitair Instituut voor Biotechnologie, Ghent, Belgium) Post-transcriptional gene silencing in plants - basics and relevance in biotechnology

Wednesday, September 22nd 2010 08:30 Reuter, Gunter (Martin Luther Universität Halle-Wittenberg, Halle, Germany)

PEV in Drosophila and silencing in Arabidopsis - a comparison 10:00 Meyer, Peter (University of Leeds, Leeds, United Kingdom)

Plant gene regulation via antisense transcripts 11:00 Schmidt, Renate (IPK, Gatersleben, Germany)

Transcript level-mediated post-transcriptional gene silencing Thursday, September 23rd 2010 08:30 Shen, Wen-Hui (Institut de Biologie Moléculaire des Plantes, Strasbourg,

France) Histone methylation in plants: insight into TrxG and PcG functions

10:00 Houben, Andreas (IPK, Gatersleben, Germany) Phosphorylation of histone H3 - a dynamic affair

Friday, September 24th 2010 08:30 Schweizer, Patrick (IPK, Gatersleben, Germany)

RNA interference as a tool in crop plant genetic analysis 10:00 Mette, Michael Florian (IPK, Gatersleben, Germany)

RNA-directed DNA methylation in plants

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Monday, Sept. 20th, 8:30

Schubert, Ingo (IPK, Gatersleben, Germany)

[email protected] Modifications of plant interphase chromatin at the microscopic level - an overview Fuchs et al. (2006) Trends Plant Sci. 11, 199-208 (review) Jasencakova et al. (2003) Plant J. 33, 471-480 (original article)

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Monday, Sept. 20th, 10:00 Colot, Vincent (École Normale Supérieure, Paris, France)

[email protected] A genome-wide view of chromatin dynamics and chromatin-based epigenetic processes in Arabidopsis Roudier et al. (2009) Trends Genet. 25, 511-517 (review) Teixeira and Colot (2010) Heredity. 105, 14-23 (review)

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Monday, Sept. 20th, 18:30 Aalen, Reidunn Brigitta (Department of Molecular Biosciences, University of Oslo, Norway)

[email protected] Readers and writers of the histone code Baumbusch et al. (2001) Nucleic Acids Res. 29, 4319-4333 (original article) Liu et al. (2010) Annu Rev Plant Biol. 61, 395-420 (review)

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Tuesday, Sept. 21st, 8:30 Mittelsten Scheid, Ortrun (Gregor Mendel Inst. of Molecular Plant Biology, Vienna, Austria)

[email protected] Stability and plasticity of transcriptional gene silencing Matzke and Mittelsten Scheid (2006) In: Epigenetics (eds. Allis CD, Jenuwein T, Reinberg D),

Cold Spring Harbour Laboratory Press, pp. 167-189 (review) Chinnusamy and Zhu (2009) Curr Opin Plant Biol. 12, 133-139 (review)

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Tuesday, Sept. 21st, 10:00 Depicker, Ann (Vlaams Interuniversitair Instituut voor Biotechnologie, Ghent, Belgium)

[email protected] Post-transcriptional gene silencing in plants - basics and relevance in biotechnology Fojtová et al. (2006) Nucleic Acids Res. 34, 2280-2293 (original article) De Buck et al. (2004) Cell Mol Life Sci. 61, 2632-2645 (original article)

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Wednesday, Sept. 22nd, 8:30 Reuter, Gunter (Martin Luther Universität Halle-Wittenberg, Halle, Germany)

[email protected] PEV in Drosophila and silencing in Arabidopsis - a comparison Naumann et al. (2005) EMBO J. 24, 1418-1429 (original article) Rudolph et al. (2007) Mol Cell. 26, 103-115 (original article)

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Wednesday, Sept. 22nd, 10:00 Meyer, Peter (University of Leeds, Leeds, United Kingdom)

[email protected] Plant gene regulation via antisense transcripts Borsani et al. 2005 Cell. 123, 1279-1291 (original article) Swiezewski et al. 2009 Nature. 462, 799-802 (original article)

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Wednesday, Sept. 22nd, 11:00 Schmidt, Renate (IPK, Gatersleben, Germany)

[email protected] Transcript level-mediated post-transcriptional gene silencing Meins (2000) Plant Molecular Biology 43, 261-273 (review) Schubert et al. (2004) Plant Cell 16, 2561-2572 (original article)

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Thursday, Sept. 23rd, 8:30 Shen, Wen-Hui (Institut de Biologie Moléculaire des Plantes, Strasbourg, France)

[email protected] Histone methylation in plants: insight into TrxG and PcG functions Xu and Shen (2008) Curr Biol. 18, 1966-1971 (original article) Zhao et al. (2005) Nat Cell Biol. 7, 1256-1260 (original article)

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Thursday, Sept. 23rd, 10:00 Houben, Andreas (IPK, Gatersleben, Germany)

[email protected] Phosphorylation of histone H3 - a dynamic affair Houben et al. 2007 Biochim Biophys Acta. 1769, 308-315 (review) Demidov et al. 2005 Plant Cell. 17, 836-848 (original article)

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Friday, Sept. 24th, 8:30 Schweizer, Patrick (IPK, Gatersleben, Germany)

[email protected] RNA interference as a tool in crop plant genetic analysis Himmelbach et al. (2010) Plant Cell. 22, 937-952 (original article) Dong et al. (2006) Plant Cell. 18, 3321-3331 (original article)

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Friday, Sept. 24th, 10:00 Mette, Michael Florian (IPK, Gatersleben, Germany)

[email protected] RNA-directed DNA methylation in plants Law and Jacobsen (2010) Nat Rev Genet. 11, 204-220 (review) Aufsatz et al. (2002) Proc Natl Acad Sci U S A. Suppl 4,16499-16506 (original article)

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Laboratory Work

I) Chromatin analysis of mitotic and meiotic chromosomes - Andreas Houben

II) Analysis of chromatin in flow-sorted interphase nuclei of plants - Jörg Fuchs and Ingo Schubert

III) Transcriptional gene silencing in Arabidopsis and Drosophila - Michael Florian Mette and Gunter Reuter

IV) Post-transcriptional gene silencing in plants: case studies - Patrick Schweizer and Ann Depicker

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I) Chromatin analysis of mitotic and meiotic chromosomes - Andreas Houben

II) Analysis of chromatin in flow-sorted interphase nuclei of plants - Jörg Fuchs and Ingo Schubert

Analyses of histone modifications at the microscopic level

Introduction

The organisation of DNA into a hierarchy of chromatin fibres facilitates the packaging

within the nucleus and regulates expression and maintenance of nuclear genetic

information. The basic units of chromatin are the nucleosomes with ~147 bp of DNA

wrapped around a histone octamer consisting of two molecules of each H2A, H2B,

H3 and H4. The core histones possess amino-terminal tails which extend from the

surface of the nucleosomes. These histone tails are subjected to many types of

covalent histone modifications, like acetylation, methylation, phosphorylation,

ubiquitination, sumoylation and ADP-ribosylation (Loidl, 2004). These post-

translational modifications constitute the 'histone code' (Fig. 1) or together with DNA

methylation the 'epigenetic code' (Strahl and Allis, 2000; Turner, 2000; Jenuwein and

Allis, 2001). They control the folding of nucleosome arrays into higher order

structures and mediate signalling for developmental processes. Although the

histones and their modifications are conserved among eukaryotes, chromosomal

distribution and/or the biological meaning of the individual modifications differ

between plants and non-plant eukaryotes (Fuchs et al. 2006).

While molecular analyses of chromatin modifications characterise either the total

chromatin or regions of individual DNA sequences, immunostaining with antibodies

specific for individual histone modifications allows to detect the subnuclear and

chromosomal distribution of these marks at the microscopic level (Houben et al.,

1996).

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Fig. 1: Histone code, scheme of the core histones with

the positions of selected histone modifications

Immunostaining or immunohistochemistry (IHC) refers to the process of localising proteins in

cells or isolated cell organelles exploiting the principle of antibodies binding specifically to

antigens in biological tissues. Visualising an antibody-antigen interaction can be

accomplished in different ways. In the most common instance, an antibody is conjugated to an

enzyme, such as peroxidase, that can catalyse a colour-producing reaction (see

immunoperoxidase staining). Alternatively, the antibody can also be tagged to a fluorophore,

such as fluorescein or rhodamine.

The antibodies used for specific detection can be polyclonal or monoclonal.

Monoclonal antibodies are generally considered to exhibit greater specificity.

Polyclonal antibodies are made by injecting animals with peptide antigen (Ag) and,

after a secondary immune response is stimulated, isolating antibodies from whole

serum. Thus, polyclonal antibodies are a heterogeneous mix of multiple different

antibodies that recognise several epitopes.

Antibodies can also be classified as primary or secondary reagents according to the

order in which they are used in IHC. Primary antibodies are raised against an antigen

of interest and are typically unconjugated (unlabelled), while secondary antibodies

are raised against primary antibodies. Hence, secondary antibodies recognise

immunoglobulins of a particular species and are conjugated to either biotin or a

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reporter enzyme such as alkaline phosphatase or horseradish peroxidase (HRP).

Secondary antibodies conjugated to fluorescent agents, such as the Alexa Fluor or

Dylight Fluor family, are also frequently used for detection of proteins in IHC

procedures. Protein (antigen) concentration is generally measured by densitometry

analysis, where the intensity of staining correlates with the amount of the protein of

interest.

There are two strategies used for the immunohistochemical detection of antigens, the

direct method and the indirect method. The direct method is a one-step staining

method, and involves a labelled antibody (e.g. FITC conjugated antiserum) reacting

directly with the antigen in tissue sections (Fig. 2). This technique utilises only one

antibody and the procedure is therefore simple and rapid. However, it can suffer

problems with sensitivity due to little signal amplification and is in less common use

than indirect methods.

The indirect method involves an unlabeled primary antibody (first layer) which reacts

with tissue antigen, and a labelled secondary antibody (second layer) which reacts

with the primary antibody (Fig. 3). (The secondary antibody must be raised against

the IgG of the animal species in which the primary antibody has been raised.) This

method is more sensitive due to signal amplification through several secondary

antibody reactions with different antigenic sites on the primary antibody. The second

layer antibody can be labelled with a fluorescent dye or an enzyme. The application

of secondary antibodies ladled with different fluorescent dyes allows the detection of

different target proteins if primary antibodies were raised in different host species.

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Fig. 2. Direct immunostaining

Fig. 3. Indirect immunostaining

The indirect method, aside from its greater sensitivity, also has the advantage that

only a relatively small number of standard conjugated (labelled) secondary antibodies

need to be generated. For example, a labelled secondary antibody raised against

rabbit IgG, which can be purchased "off the shelf", is useful with any primary antibody

raised in rabbit. Visualisation of signals occurs either via light, fluorescence or

electron microscopy [transmission (Houben et al., 2005) or scanning (Schroeder-

Reiter et al., 2003)].

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I) Chromatin analysis of mitotic and meiotic chromosomes

Analysis of histone modifications at the chromosomal level

Protocol

A: Preparation of plant material

Mitotic chromosomes from root apical meristems

1. For plants cultivated in pots, keep the soil at moderate moisture. For seeds, cover

the bottom of a Petri dish with several layers of filter paper and add distilled water

until the filter paper is wet adequately. Sow the seeds on the wet filter paper and

incubate for several days at 24 °C to get germination.

2. When roots growing in soil are used, gently remove the soil from the root tips and

cut root tips into lengths of 5 to 10 mm with forceps, place onto a glass slide and

wash with a drop of water. Seedlings grown in a Petri dish are preferable as no

care regarding contamination with soil is required.

3. The number of cells in mitosis can be enriched by inhibition of progression through

mitosis, e.g. by pretreating the unfixed root meristems with colchicine, 8-

hydroxyquinoline or ice water. Note that such a pretreatment can influence the

distribution of histone marks at the chromosomal level (e .g. see (Manzanero et

al., 2002)).

2 mM 8-hydroxyquinoline: Dissolve 290 mg of 8-hydroxyquinoline in 1 l of distilled

water. The solution may be stored at RT for at least 1 year. For synchronisation of

cell division treat the roots for 2 h at 20°C.

0.05% colchicine: Dissolve 0.05 g of colchicine in 100 ml of distilled water. For

synchronisation of cell division treat the roots for 2 h at 20°C.

ice water treatment: Treat roots for 12 - 16 h in ice water (0 – 4°C).

Meiotic chromosomes from anthers

In spite of the possibility that meiosis or some of its characteristics may differ

between the sexes, male meiocytes, which are found in hundreds within the anthers,

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are the usual choice for studying meiotic chromosomes. In cereal grains, such as

wheat, barley and rye, each flower contains three anthers at very similar

developmental stages. Thus, one of the anthers of a flower can be used to ascertain

the meiotic stage under the phase contrast microscope, while it is still possible to

spare the remaining two anthers for preparation of meiocytes when the tested one is

at or near the appropriate stage. In many other plants species, such as Arabidopsis

thaliana, meiocytes at definite stages can be collected from flower buds showing an

appropriate flower or anther size, respectively, since meiotic development is closely

related to overall flower development. Obtaining pollen mother cells at particular

meiotic stages is sometimes facilitated by the existence of meiotic gradients. In

cereals, the oldest flowers are located at or near to the middle of the spike and

developmental gradients from older to younger run toward both ends. Meiotic

gradients in the corn tassel develop along the branches, with the oldest flowers

located distal toward the tip of the branch.

B: Preparation of chromosome spreads

1. Fix pretreated or untreated root or anther material for 25 minutes in 10 ml ice-

cold 4% paraformaldehyde solution [dissolved in 1x PBS (phosphate-buffered saline,

for 1 litre take 8.00 g NaCl, 0.20 g KCl, 1.44 g Na2HPO4, 0.24 g oKH2PO4 and

dissolve in 800 ml of distilled H2O, adjust the pH to 7.4 with HCl or NaOH and add

distilled H2O to 1 liter), to dissolve paraformaldehyde it will be necessary to heat up

PBS ]. In case of penetration problems, employ a weak vacuum. In case of working

with big anthers (above 4 mm), cut-open the anthers for better fixation. Please be

careful with the preparation of the paraformaldehyde solution! Therefore, adjust pH of

1xPBS buffer to pH 11 with KOH and heat solution to 60°C and add parformaldehyde

powder and stir under fume hood until it is dissolved completely. After, cool solution

to room temperature, adjust pH to 6.9-7.0 with H2SO4.

For simultaneous detection of microtubules, the material should be fixed in 4%

paraformaldehyde solution dissolved in 1x MTSB [(microtubule-stabilising buffer):

50 mM Pipes, 5 mM EGTA, 5 mM MgSO4; pH 6.9 - 7.0 adjust with KOH ]

2. After fixation, wash the material 3x 15 minutes in 1x PBS on ice.

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3. Macerate root tips and anthers by dipping them into an enzyme cocktail (2.5%

Pectinase, 2.5% Cellulase R-10, 2.5% Pectolyase Y-2 dissolved in 1x PBS).

Between 100 and 200 µl of enzyme cocktail is enough for maceration of approx.

10 to 20 root tips. Incubate the material at 27°C for 20 – 50 minutes until the

material becomes soft. Note, the maceration time may vary considerable

depending on the materials and their conditions. It is possible to reuse the enzyme

cocktail.

4. Carefully wash the material 2x 15 minutes in 1x PBS on ice.

5. Squash the tissue over a well-cleaned slide using one cover slip in 1x PBS. Tap

gently over the cover slip with some appropriate tool (Lancet, forceps etc.) to

break the tissue. Pollen mother cells are set free from the anther tissues at this

stage and can be seen in a phase contrast microscope sitting alone or in small

groups. Use blotting paper or filter paper to remove the surplus of butter from

around the edges of the cover slip. Then press the cover slip firmly with a finger

without moving the cover slip.

6. To remove the cover slip, freeze the preparation (to completion!) by placing the

glass slide in a –80°C freezer or on dry ice or dipping into liquid nitrogen. Flick

away the cover slip using a razor blade in a way that the cover slip does not move

over the glass slide, as this would break the chromosome samples. Subsequently,

collect slides immediately in a Coplin jar with 1x PBS. For longer storage and

transfer, slides were kept in 100% glycerol at 4°C.

Note, put on gloves when handling liquid nitrogen and dry ice. Wear safety

goggles when flicking away the cover slips.

C: Detection of antigens

1. Rinse slides 3 x 5 min in 1xPBS (if previously stored in 100% glycerol)

2. Drop 30 µl blocking solution (4% BSA, 0.1% Tween 20 in 1x PBS) on slides, cover with Parafilm and incubate for 30 min at 37°C in a moisture chamber

3. Drop 30 µl of primary antibody on slides [diluted as indicated in Table 1 in AK buffer, 1x PBS (+1% BSA, 10% horse serum, 0.1% Tween 20)], cover with Parafilm and incubate for 1 h at 37°C in a moisture chamber.

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4. Wash slides 3 x 5 min in 1x PBS

5. Drop 30 µl of secondary antibody (anti-rabbit rhodamine, 1:200 in AK) on slides, cover with Parafilm and expose for 1 h at 37°C in a moisture chamber

6. Rinse slides 3x 5 min in 1x PBS

7. Counterstain with DAPI (0,5 – 1 µg/ml in Vectashield, 9 µl per slide), cover with cover slip (22 x 22 mm) and store slides (dark) in a refrigerator until use.

8. Ready for the fluorescence microscope and a bottle of beer

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II) Analysis of chromatin in flow-sorted interphase nuclei of plants

Analysis of histone modifications in flow-sorted interphase nuclei

Protocol

A: Generation of suspensions from leaf nuclei of Arabidopsis thaliana

1. Fix 10 mg leaf tissue in 10 ml ice-cold 4% paraformaldehyde in Tris-buffer for 20

min under vacuum

2. Rinse 2 x 10 min in ice-cold Tris-buffer

3. Chop tissue in 400 – 1000 µl in ice-cold LB01 buffer in a pre-cooled Petri-dish with

a fresh razor blade

4. Filter the suspension through a mesh of 35 µm pore size

5. For flow cytometry add DAPI (final concentration: 1 µg/ml)

Tris-buffer:

10 mM Tris-HCl, pH 7.5 10 mM Na2-EDTA

100 mM NaCl

LB01-buffer: 15 mM Tris-HCl, pH 7.5 2 mM Na2-EDTA

0.5 mM Spermin x 4HCl 80 mM KCl 20 mM NaCl 0.1 % Triton X-100

DAPI stock solution:

100 µg/ml DAPI in distilled water B: Preparation of slides from sorted nuclei of A. thaliana

Flow cytometry is a technique for counting, examining and sorting of microscopic particles, such as cells, nuclei or chromosomes. Isolated particles are running in a hydrodynamicly-focused stream of fluid and passing a beam of light (usually laser

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light) of a single wavelength. A number of photo detectors at the point where the stream is passing the light beam record scatter and fluorescence light information. This allows the analysis of physical and/or chemical characteristics of up to several thousands of particles per second. During this course a flow sorter will be used to separate nuclei according to their ploidy level from cell debris and other cellular compounds. 1. Flow-sort 10.000 nuclei according to their ploidy level (= DNA content) using a

FACSAria (BD Biosciences) into Eppendorf tubes, then put tubes on ice

2. Drop 15 µl sorting buffer on clean glass slides, add 15 µl of suspension containing nuclei and mix gently with the pipette tip

3. Let slides dry overnight

Sorting buffer: 100 mM Tris 50 mM KCl

2mM MgCl2 0.05 % Tween

5 % sucrose adjust to pH 8.0 and filter under sterile conditions

C. Immunostaining in situ of nuclei of A. thaliana with antibodies against methylated lysine residues of histone H3

1. Fix air-dried slide with sorted nuclei for 20 min in 4% paraformaldehyde/1xPBS

2. Rinse 3 x 5 min in 1xPBS

3. Drop 30 µl blocking solution (4% BSA, 0.1% Tween 20 in 1xPBS) on slides, cover with Parafilm and incubate for 30 min at 37°C in a moisture chamber

4. Drop 30 µl of primary antibody on slides (diluted in AK buffer as indicated in Table 1), cover with Parafilm and incubate for 1 h at 37°C in a moisture chamber.

5. Wash slides 3 x 5 min in 1xPBS

6. Drop 30 µl of secondary antibody (anit-rabbit rhodamine, 1:200 in AK) on slides, cover with Parafilm and expose for 1 h at 37°C in a moisture chamber

7. Rinse 3x 5 min in 1xPBS

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8. Counterstain with DAPI (0,5 – 1 µg/ml in Vectashield, 9 µl per slide), cover with cover slip (22 x 22 mm) and store slides (dark) in a refrigerator until use.

1xPBS (phosphate-buffered saline)

8 g NaCl 0.2 g KCl

1.44 g Na2HPO4 0.24 g KH2PO4

Dissolve in 800 ml distilled water, adjust to pH 7.4 with HCl, fill up to 1 l with distilled water

AK (antibody buffer): 1xPBS (+1% BSA, 10% horse serum, 0.1% Tween 20)

Table 1: Dilution of primary antibodies (Upstate, Millipore):

H3K4me1 07-436 1:200 H3K4me2 07-030 1:500 H3K4me3 07-473 1:200 H3K9me1 07-395 1:200 H3K9me2 07-441 1:100 H3K9me3 07-523 1:100 H3K27me1 07-448 1:100 H3K27me2 07-452 1:50 H3K27me3 07-449 1:100 H3T11ph 07-492 1:100 H3S10ph 06-570 1:300

References: Fuchs, J.; Demidov, D.; Houben, A.; Schubert, I. (2006) Chromosomal histone

modification patterns--from conservation to diversity. Trends Plant Sci 11, 199-208.

Houben, A., Belyaev, N.D., Turner, B.M., and Schubert, I. (1996). Differential immunostaining of plant chromosomes by antibodies recognizing acetylated histone H4 variants. Chromosome Res 4, 191-194.

Houben, A., Demidov, D., Rutten, T., and Scheidtmann, K.H. (2005). Novel phosphorylation of histone H3 at threonine 11 that temporally correlates with condensation of mitotic and meiotic chromosomes in plant cells. Cytogenet Genome Res 109, 148-155.

Jenuwein, T., and Allis, C.D. (2001). Translating the histone code. Science 293, 1074-1080.

Loidl, P. (2004). A plant dialect of the histone language. Trends Plant Sci 9, 84-90. Manzanero, S., Rutten, T., Kotseruba, V., and Houben, A. (2002). Alterations in the

distribution of histone H3 phosphorylation in mitotic plant chromosomes in

34

response to cold treatment and the protein phosphatase inhibitor cantharidin. Chromosome Res 10, 467-476.

Schroeder-Reiter, E., Houben, A., and Wanner, G. (2003). Immunogold labelling of chromosomes for scanning electron microscopy: A closer look at phosphorylated histone H3 in mitotic metaphase chromosomes of Hordeum vulgare. Chromosome Res 11, 585-596.

Strahl, B.D., and Allis, C.D. (2000). The language of covalent histone modifications. Nature 403, 41-45.

Turner, B.M. (2000). Histone acetylation and an epigenetic code. Bioessays 22, 836-845.

35

III) Transcriptional gene silencing in Arabidopsis and Drosophila A) Analysis of cytosine methylation patterns in plant DNA – M.F. Mette and

Markus Kuhlmann

Introduction

In contrast to the situation in mammals, where methylation of cytosines in nuclear

genomic DNA occurs mainly in CG context, DNA methylation in plants can affect

every cytosine in the genome. Methylation of cytosines in a symmetric context like

CG and CNG is mainly regulated via maintenance mechanisms that act during DNA

replication. Cytosines in an asymmetric context can be methylated in the presence of

homologous double stranded (ds) RNA by RNA directed DNA methylation (RdDM).

Methylation of DNA can lead to transcriptional gene silencing (TGS) and is enriched

in, but not restricted to, heterochromatic regions of the plant genome. Under normal

circumstances this mechanism is controlling the spreading of “selfish sequences” like

retroelements, transposons and (para)retroviruses.

For the genetic and molecular analysis of the mechanism of RNA directed DNA

methylation (RdDM) and transcriptional gene silencing (TGS), an experimental

system comprising two transgenes in Arabidopsis thaliana has proven to be very

useful (Figure 1).

Figure 1: Transgene-based two component experimental system.

36

Transgene based experimental system in Arabidopsis thaliana The silencer transgene (H) contains two copies of the NOPALIN SYNTHASE

promoter (NOSpro) arranged as an inverted repeat structure downstream of a single

copy of the Cauliflower Mosaic Virus 35S promoter (35Spro). The 35Spro drives

strong and ubiquitous transcription of the NOSpro inverted repeat structure (IR),

leading to the production of abundant transcripts that can fold to form double

stranded (ds) RNA with NOSpro homology. This dsRNA serves as a target for DICER

enzymes and is thereby cleaved to 21-24 nt short interfering (si) RNAs. The 24 nt

siRNAs can act in trans to induce RNA-directed DNA methylation and transcriptional

gene silencing at unlinked NOSpro sequences elsewhere in the genome.

The second transgene in our experimental setup is the target transgene (K) that

contains a kanamycin resistance gene (NPTII) under the control of the NOSpro. The

activity of this reporter gene can be monitored either by testing the resistance of the

plants against the antibiotic kanamycin or, at the molecular level, by quantitative

"real-time" RT-PCR for NPTII transcripts or by an ELISA for NPTII protein. In the

presence of 24 nt siRNAs with NOSpro homology, the NOSpro can be methylated

and NPTII expression can be transcriptionally repressed. The level of DNA

methylation and transcriptional repression differs for independent transgenic lines

containing the same reporter gene (Figure 2; Fischer et al., 2008).

Kchr3-3Kchr1-10 Kchr2-3K/K;-/- K/K;H/H K/K;-/- K/K;H/HK/K;-/- K/K;H/H

Figure 2: Silencing of NPTII expression as indicated by the inhibition of growth on medium containing 200 mg/l kanamycin.

37

In our approach, we will compare NOSpro DNA methylation levels at three defined

target transgenes: Kchr1-10 is located at the end of the lower arm of chromosome 1

and shows strong methylation of the NOS promoter combined with strong repression

of the NPTII reporter gene in the presence of the silencer. Kchr3-3 is located at the

upper arm of chromosome 3 and shows only weak NOSpro methylation and almost

no effect on the expression of the NPTII reporter gene in the presence of the silencer.

Finally, Kchr2-3 is located at the end of the lower arm on chromosome 2. This target

shows a moderate level of DNA methylation but also persisting strong expression of

the NPTII reporter gene. We will use these transgenes in an exemplary approach to

the quantitative comparison of cytosine methylation at a particular sequence.

Experimental approaches to DNA cytosine methylation For the analysis of DNA methylation various methods based on different principles to

discriminate unmethylated and methylated cytosines in DNA are available:

• Methylated DNA ImmunoPrecipitation (MeDIP)

o Quantification of precipitated DNA by PCR o Quantification of precipitated DNA by Slot blot o Genome wide analysis by micro array hybridisation or next generation

sequencing Based on the affinity of an antibody against methylated cytosines or of a methylated DNA binding protein.

• Bisulfite Sequencing

o PCR amplification and sequencing o Combined Bisulfite Restriction Analysis (COBRA), Xiong & Laird,

Nucleic Acids Research 1997) o Genome wide analysis by micro array hybridisation or next generation

sequencing Based on the protection of methylated cytosines against chemical modification with sodium bisulfite.

• Methylation sensitive restriction enzyme cleavage

o Detection of the fragments by Southern blot analysis o Methylation sensitive amplified polymorphisms (MSAP) o Quantitative “real time” PCR

Based on the inhibition (or dependence) of restriction enzyme cleavage by (on) the presence of methylated cytosines at the restriction recognition site.

38

DNA methylation assay based on cytosine methylation-sensitive restriction cleavage and quantitative PCR

We will perform an exemplary quantification of DNA cytosine methylation present at a

particular sequence by PCR-based quantification of that part of the respective DNA

that is resistant to cleavage by cytosine methylation-sensitive restriction enzymes.

Previous investigations by Southern blot analysis and bisulfite sequencing (Fischer et

al. 2008) in our two component experimental system found cytosine methylation

exclusively in the region homologous to the ds/si RNA derived from the IR region in

the silencer transgene. We selected three cytosine methylation sensitive enzymes,

Psp1406I, NheI, and Alw26I (Table 1) with single recognition sites in this region

(Figure 3). Additionally, NcoI, which is also cytosine methylation sensitive (Table1),

was selected as a control site outside the methylated region. PCR amplification is

performed with primers flanking the NOSpro region.

Psp1406I NheI

Alw26I NcoI

region with homology to the silencer

NOS for

NOS revPCR fragment (460 bp)

(282 bp)

pNOS NPTII

Figure 3: Cleavage sites of cytosine methylation-sensitive restriction enzymes and binding sites of PCR primers in the target NOSpro. The region of the target transgene with homology to ds/siRNA derived from the

transcribed inverted repeat in the silencer transgene is marked in blue. pNOS:

promoter region of the NOPALINE SYNTHASE; NPTII: coding region for

neomycinphosphotransferase type II, Kanamycin resistance mediating gene;

Psp1406I, NheI, Alw26I, NcoI: recognition sites for restriction enzymes.

39

The enzyme Psp1406I with recognition sequence AACGTT tests cytosine

methylation in symmetric CG context. NheI with recognition sequence GCTAGC

Alw26I with recognition sequence GTCTC tests in the NOSpro sequence context

dependent for methylation that is not in CG context. NcoI with recognition sequence

CCATGG is also cytosine methylation sensitive, but its cleavage site lies outside the

region that is methylated due to the presence of homologous ds/si RNA derived from

the silencer transgene. Thus, NcoI serves as a control for residual unmethylated DNA

that is resistant to restriction cleavage DNA in the assay.

Table 1: Effect of cytosine methylation on cleavage by restriction enzymes. Psp1406I

site cleaved cleavage impaired site not cleaved

A A C G T T T T G C A A

m5 A A C G T T T T G C A A

m5

NheI


G C T A G C C G A T C G

m5 G C T A G C C G A T C G

m5 m5


(20% cleavage)

m5 G C T A G C C G A T C G

m5


m5

Alw26I


G T C T C C A G A G

G T C T C C A G A G

m5 m5

G T C T C C A G A G

m5 G T C T C C A G A G

NcoI


C C A T G G G G T A C C

m5 C C A T G G G G T A C C

source: REBASE database http://rebase.neb.com/rebase/

40

Protocol Cleavage of genomic DNA Genomic DNA preparations from individual plants (from rosette leaf tissue of 9 week old plants grown under short day conditions using the QIAGEN DNeasy Plant Maxi kit): group black blue red (K/K) Kchr1-10 Kchr3-3 Kchr2-3 (K/K; H/H) Kchr1-10 H Kchr3-3 H Kchr3-3 H For each DNA preparation: 0,1 µg genomic DNA in 500 µl H2O Aliquot 90 µl in 5 tubes (0,0225 µg) tube I tube II tube III tube IV tubeV no enzyme Psp1406I NheI Alw26I NcoI Add 10 µl buffer Y (10x, Fermentas) Add 3 µl enzyme (30 U, 10 U/µl; Fermentas) Incubate for 3 h (or better overnight for more complete cleavage) at 37°C Inactivate restriction enzymes for 5 min at 85°C (do also for “no enzyme” control) Add 897 µl bidistilled water to achieve a final volume of 1000 µl Standards (st.) for quantitative PCR calibration gDNA serial dilution: 0.01 µg / 10 µl 0.001 µg / 10 µl 0.0001 µg / 10 µl 0.00001 µg / 10 µl water-control

41

Setup of quantitative “real-time” PCR Use 10 µl (~ 0,000225 µg) of cleaved or control DNA preparation per 25 µl PCR setup: 10 µl template 12,5 µl SyBr green Supermix (Bio-Rad cat.no. 170-8882) 1.25 µl 100 pmol/µl forward primer 1.25 µl 100 pmol/µl reverse primer 25 µl total volume per reaction Prepare a master-mix for all samples (SyBr green Supermix, for. & rev. primers), set up samples for PCR in 3 pieces of a 96 well plate (group black, blue and red).

1 2 3 4 5 6 7 8 9 10 11 12

A st.

0.0 1

st. 0.0 01

st. 0.0 001

st. 0.0

0001

H2O

no enz.

1

no enz.

1

no enz.

1

Psp 1406I

1

Psp 1406 I 1

Psp 1406I 1

B NheI

1

NheI 1

NheI 1

Alw 26I 1

Alw 26I 1

Alw 26I 1

NcoI1

NcoI1

NcoI1

no enz.

2

no enz.

2

no enz.

2

C Psp

1406I 2

Psp 1406 I 2

Psp 1406 I 2

NheI 2

NheI2

NheI2

Alw 26I 2

Alw 26I 2

Alw 26I 2

NcoI 2

NcoI 2

NcoI2

D no

enz. 3

no enz.

3

no enz.

3

Psp 1406I

3

Psp 1406I 3

Psp 1406I 3

no enz.

5

no enz.

5

no enz.

5

Psp 1406I

5

Psp 1406 I 5

Psp 1406I 5

E NheI

3

NheI 3

NheI 3

Alw 26I 3

Alw 26I 3

Alw 26I 3

NheI5

NheI5

NheI5

Alw 26I 5

Alw 26I 5

Alw 26I 5

F NcoI

3

NcoI 3

NcoI 3

no enz.

4

no enz.

4

no enz.

4

NcoI5

NcoI5

NcoI5

no enz.

6

no enz.

6

no enz.

6

G Psp

1406I 4

Psp 1406 I 4

Psp 1406 I 4

NheI 4

NheI4

NheI4

Psp 1406I

6

Psp 1406I 6

Psp 1406I 6

NheI 6

NheI 6

NheI6

H Alw 26I 4

Alw 26I 4

Alw 26I 4

NcoI 4

NcoI4

NcoI4

Alw 26I 6

Alw 26I 6

Alw 26I 6

NcoI 6

NcoI 6

NcoI6

transfer into Bio-Rad iQCycler real time PCR device

42

PCR in BioRad iQCycler program: “SyBrsoloLTR” 1: 5 min 95°C 2: 15 sec 95°C 3: 30 sec 62°C 4: 30 sec 72°C Detection 5: repeat 2-4 40 x Melting curve 6: 65°C Detection 7: +0.5°C 8: repeat 7 60 x Evaluation of data Evaluation of realtime PCR data should always be performed according to the MIQE

(Minimum Information for publication of Quantitative real-time PCR Experiments)

guidelines as a minimal standard (Bustin et al. 2009).

The calculation of the relative amount of amplified target is done according to Livak

and Schmittgen (2001) with the 2-ΔΔCT formula.

After the run of the PCR, the melting curve of the PCR products provides an

important control for the homogeneity of the amplification products (Figure 4). A

sharp, narrow peak is desired. Shoulders or signals beside the peak at higher

temperatures are hints for unspecific amplification “background“. As these unspecific

products also contribute to the fluorescence signal on which quantification is based,

PCR conditions should be carefully optimised for each primer combination.

The values for the correlation coefficient of the PCR should be around 0.9 and the

PCR efficiency around 100%. In routine operation, values between 80% and 110%

are acceptable and might vary according to pipetting errors or sample quality.

43

Figure 4: Melting-curve of the PCR product obtained with primers NOSpro for and NOSpro rev with 460 bp length and a melting point at 88.5-89°C.

Figure 5: “Real-time” fluorescence signals during PCR amplification. The CT (cycle threshold) is the value at which the fluorescence signal curve of a

sample crosses the pre-set threshold of amplification (orange line ~15000) in a

particular analysis. The CT should be set in the range of the exponential amplification

phase of the PCR for all samples.

The “CT value” of a PCR reaction is defined as the number of amplification cycles

until the fluorescence signal passes the CT line. Measurements are usually done in

triplicate and the mean value of CT values for each sample will be used for further

calculation. The CT values generated for the different samples can be compared with

the help of the “delta CT method” (Livak and Schmittgen 2001).

44

Relative amount of PCR target in sample1 compared to sample2: sample1 / sample2 = 2 – (CT sample1 – CT sample2) [ accordingly, percentage methylation = 100% x 2 – ( CT sample cleaved – CT sample uncleaved) ] References

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R,

Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE

guidelines: minimum information for publication of quantitative real-time PCR

experiments. Clin Chem. 55:611-622.

Fischer U, Kuhlmann M, Pecinka A, Schmidt R, Mette MF (2008) Local DNA features

affect RNA-directed transcriptional gene silencing and DNA methylation. Plant J.

53:1-10.

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-

time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408.

45

III) Transcriptional gene silencing in Arabidopsis and Drosophila B) PEV in Drosophila and heterochromatic gene silencing in Arabidopsis - a

comparison - Gunter Reuter

Institute of Biology/Genetics, Martin Luther University Halle-Wittenberg,

Weinbergweg 10, D-06120 Halle/S.

Introduction

Genetic dissection of chromatin regulation and epigenetic programming In principal any phenotypic change, which is due to a change in chromatin

organisation allows identification of factors controlling chromatin differentiation by

isolation of mutations modifying expressivity of the phenotypic effects. The isolated

mutants afterwards can be used for mapping the identified genes and for molecular

cloning. A pioneering role in this experimental strategy has position-effect variegation

(PEV) in Drosophila. Using PEV first important key functions of epigenetic

programming could be identified with dominant suppressor mutations. One of these is

the SU(VAR)3-9 histone methyltransferase and the heterochromatin protein HP1.

Heterochromatin and gene silencing in position-effect variegation In PEV euchromatic genes juxtaposed to heterochromatin become silenced

(variegated phenotype) by heterochromatisation (facultative heterochromatin). Muller

first discovered PEV in X-ray induced chromosomal rearrangements of Drosophila.

Heterochromatic gene silencing in PEV, therefore, reflects the repressive effect of

heterochromatin on active genes. Although PEV has been studied intensively in

Drosophila and also was demonstrated in mammals in plants only in Oenothera

blandina an unequivocal case of PEV has been described by Catcheside.

The silencing that occurs in PEV can be attributed to packaging of the reporter gene

in a heterochromatic form, indicating that heterochromatin formation, once initiated,

can spread to encompass nearby genes. Genetic, cytological and biochemical

analyses using this system in Drosophila melanogaster have led to the identification

of many potential components, and to characterisation of several proteins that play

key roles in establishing and maintaining heterochromatin (cf. Rudolph et al. 2007).

46

Transcriptional gene silencing (TGS) by heterochromatisation Phenomena comparable with position-effect variegation include homology-dependent

gene silencing in transgenic plants and paramutation. Generally, in homology-

dependent gene silencing, an increase in the copy number of particular sequences is

positively correlated with reduction of gene expression of both the endogenous and

the transgenic copies.

A newly developed TGS system, which does not depend on reactivation of genes

mediating antibiotic resistance is based on a transgene construct containing four

tandemly arranged 35S::luciferase repeats and allows direct visualisation of gene

silencing by monitoring luciferase activity (Naumann et al. 2005). This luciferase

transgene repeat system was used for isolation and functional analysis of

suppressors and enhancers of TGS in Arabidopsis.

Figure 1. Genetic dissection of epigenetic functions. A. Repeat dependent gene

silencing (TGS) of a luciferase transgene repeat. B. Modification of TGS after

overexpression and antisense knockdown of SUVH2. SUVH2 is an Arabidopsis

homolog of the Drosophila SU(VAR)3-9 H3K9 methyltransferase. C. The dosage

dependent effect of Su(var)3-9 on white gene silencing in a PEV rearrangement.

Su(var)3-9 was isolated first in Drosophila and by sequence comparison the

structural and functional Arabidopsis homolog SUVH2 could be identified.

Discussing experimental methods and the results received I will compare both

systems for genetic dissections of epigenetic processes. Finally the analysis allowed

47

identifying of conserved molecular processes controlling heterochromatic gene

silencing and chromatin differentiation in Drosophila and Arabidopsis.

Naumann K, Fischer A, Hofmann I, Krauss V, Phalke S, Irmler K, Hause G, Aurich AC, Dorn R, Jenuwein T, Reuter G (2005) Pivotal role of AtSUVH2 in heterochromatic histone methylation and gene silencing in Arabidopsis. EMBO J. 24: 1418-1429.

Rudolph T, Yonezawa M, Lein S, Heidrich K, Kubicek S, Schäfer C, Phalke S, Walther M, Schmidt A, Jenuwein T, Reuter G (2007) Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3. Mol. Cell 26: 103-115.

48

Transcriptional silenced Luciferase transgene repeats as a model system for

the epigenetic regulation of gene expression

Analysis of the silenced and transcriptional active state of a transgenic Luciferase

repeat in Arabidopsis thaliana plants containing mutations in chromatin remodelling

proteins

In eukaryotic organisms, processes of development and differentiation are

epigenetically controlled at the level of higher order chromatin structure. Changes in

packaging of chromatin domains lead to activation or repression of genes within such

domains. Numerous studies have shown that several phenomena of homology

dependent transcriptional gene silencing in transgenic plants are also the

consequence of chromatin remodelling. Isolation of mutants affecting such silencing

processes should lead to the identification of proteins involved in establishment and

regulation of epigenetic structures. According to this hypothesis, one aim of our work

was the isolation and characterisation of new suppressor mutants for transcriptional

gene silencing (TGS). Due to the lack of sensitive and facile test systems for TGS in

plants, a new silencing system based on transcriptionally silenced transgenic

Luciferase reporter genes was established. For this purpose, Arabidopsis-T-DNA

lines have been constructed containing one to four copies of the reporter gene driven

by the CaMV 35S promoter. Nearly all plants transformed with a single Luciferase

expression cassette show high Luciferase activity (LUC1 in Fig. 1). High variability in

Luciferase gene expression could be observed in plants containing two expression

cassettes whereas most plants with four Luciferase copies don’t show any transgene

expression (LUC7 and LUC9 in Fig 1). Line LUC2 (Fig. 1) also comprise four

Luciferase genes but display weak remaining transgene expression. This line is very

useful for the visualisation of changes in silencing in both directions: enhancer and

suppressor effects. Several studies suggest that TGS of the Luciferase transgene

repeats is controlled by a heterochromatic pathway comparable to processes

controlling the formation of heterochromatin in plants. The transgenes show a typical

pattern of DNA methylation regularly found at heterochromatic sequences like

retrotransposons. Beside symmetric CpG and CpNpG methylation also asymmetric

CpNpN DNA methylation is found. Furthermore high amounts of typical

heterochromatic histone methylation marks are detected at the Luciferase repeats by

49

chromatin immunoprecipitation. Using the Luciferase silencing system TGS

suppressor mutations have been systematically isolated after EMS mutagenesis.

Among other chromatin functions, mutants were isolated for two genes encoding

SWI2/SNF2-like proteins implicated in nucleosome remodelling. Three alleles were

identified for the DDM1 gene. Two mutations affect the chromatin remodelling factor

DRD1, which is involved in RNA-directed DNA methylation. Some of these mutants

are very strong suppressors of TGS. The aim of this analysis is to define the

influence of these mutants on the Luciferase silencing system.

The luciferase reaction emits yellow-green light (560nm) and requires, besides the

enzyme, a substrate, ATP, Mg2+ and O2. The substrate Na-luciferin (1,3mM in water)

where sprayed on the plants 10 to 30 minutes before the measuring period begins. In

this way the luciferase assay is non-invasive but luciferin availability can be a limiting

factor. Luciferase assays using cell-free extracts are 10- to 1000-fold more sensitive

than standard assays like GUS or GFP; as little as 10-20 moles of luciferase protein

can be detected.

Figure 1: Transgene silencing system based on Luciferase repeats. Transgene

expression in plants containing a T-DNA with four Luciferase expression cassettes is

strongly repressed.

50

IV) Post-transcriptional gene silencing in plants: case studies A) Reverse genetics by transient RNA interference in plant epidermis cells –

Patrick Schweizer and Dimitar Douchkov

Introduction High-throughput functional capabilities for testing candidate genes are rapidly

expanding. Functional validation of the candidate genes can be accomplished by

genetic mutation, overexpression, or gene silencing (Caldwell et al. 2004; Douchkov

et al. 2005; Hein et al. 2005; Scofield et al. 2005). A number of resources needed for

reverse genetic and functional analysis of candidate genes are now available for both

plants and pathogens.

The most promising functional analysis breakthroughs for molecular plant

pathologists are improvements in RNA interference (RNAi) techniques that alter gene

expression in local cells or tissues that are the targets of pathogen invasion. This

can be achieved by bombardment-based methods or by virus-induced gene silencing

(VIGS), beside generation of stable transgenic plants. Due to its sequence-homology

dependent mode of action, double-stranded RNA interference (dsRNAi) is unique in

its potential to overcome the problem of genetic redundancy, which is of critical

importance for the polyploid genomes of Triticeae species. Ten years ago, pioneering

work by Bushnell and colleagues demonstrated the principle usefulness of a single-

cell transient expression assay for powdery-mildew-attacked barley (Nelson and

Bushnell 1997). This assay was initially based on bombarded coleoptile tissue and

subsequently developed for use in detached barley leaves (Schweizer et al. 1999c;

Schweizer et al. 2000). This was possible because the microprojectile-mediated

transformation as well as powdery-mildew attack and potential development are cell-

autonomous events taking place in single epidermal cells. The reliability of results

obtained by using the transient assay has been verified by its ability to phenocopy

allele introgression or gene mutation in barley and wheat, and by transgenic plants

stably expressing genes of interest in leaf epidermis (Schweizer et al. 2000;

Schultheiss et al. 2005; Schweizer 2008; Eichmann et al. 2010).

The single-cell assay was further developed for transient induced gene-silencing

(TIGS) by the expression of RNAi hairpin constructs (Douchkov et al., 2005).

However, a limitation of the TIGS assay for barley- powdery mildew system has been

the laborious phenotyping required to identify transformed cells and assign an

51

infection severity rating based on haustorial features. Throughput is now significantly

enhanced through use of microscope robotics and automated image analysis (Fig. 1). Improvements in construct preparation using the GatewayTM technology have also

accelerated this technique, such that 300 genes per person per month can be

analyzed with the automated single-cell transient assay if two robotic microscopes

are used. Since the initial reports, a considerable amount of functional information

concerning defence-related barley and wheat genes has become available

(Summarised in Table 1). In addition to the single-cell haustorium assay, an

automated quantitative assessment of hyphal growth rate of powdery mildew has

been developed, which can be used to study transgene effects manifested later in

pathogenesis, permitting analysis of the slow mildewing phenomenon (Seiffert and

Schweizer 2005; Baum et al. 2010).

Table 1. Gene silencing using bombardment-based and virus-induced transient

assays has significant effects on Triticeae-fungal interactions. Gene namea Description RNAib Reference HvGER3; -4; -5

Germin-like proteins TIGS Zimmermann et al. 2006

HvMla alleles CC-NBS-LRR protein TIGS/ VIGS

Halterman and Wise 2006; Shen et al. 2003; Shen et al. 2007

HvSgt1; HvRar1

SCF complex TIGS/ VIGS

Azevedo et al. 2002; Hein et al. 2005

HvRacB Small GTP-binding protein

TIGS Schultheiss et al. 2002 and 2003

HvG (alpha) G protein subunit TIGS Kim et al. 2002 HvMlo 7-Transmembrane

protein TIGS Kim et al. 2002; Schweizer et al. 2000

HvBI-1 BAX inhibitor-like TIGS Eichmann et al. 2010 HvRBOH NADPH oxidase TIGS Trujillo et al. 2006 HvNAC6 NAC transcription

factor TIGS Jensen et al. 2007

HvSNAP34 Syntaxin-interacting protein

TIGS Collins et al. 2003; Douchkov et al. 2005

HvUbi Ubiquitin TIGS Dong et al. 2006 HvPrx40 Secreted peroxidase TIGS Johrde and Schweizer 2008 HvWIR1 Small cell-wall protein TIGS Douchkov, Johrde et al. 2010

aThe prefix “Hv” specifies barley (Hordeum vulgare). bTIGS designates Transient Induced Gene Silencing. VIGS designates “Virus

Induced Gene Silencing”.

Table 1 modified from Wise et al. 2009.

52

Fig. 1. High-throughput Transient Induced Gene Silencing (TIGS) pipeline in barley. (A) Summary of approaches and tools available for quantitative phenomics in the barley-powdery mildew interaction. Haustorial index, number of detected haustoria, divided by the number of observed GUS-positive, epidermal cells per bombardment. Hyphal growth, increase of pixel numbers attributed over time to growing fungal colonies, as determined by the HyphAREA software tool. File system, image files stored locally on a PC for data input into HyphAREA. AA-TIGS and HAU-software, Oracle database of microscopic images (input for the haustoria-recognition software) and quantitative microscopic data (output of the haustoria-recognition software). (B) Automated pattern recognition for quantitative assessment of fungal growth on the leaf surface. Upper panel, original image of a growing pustule, stained with Coomassie blue. Lower panel, final segmentation of hyphae for pixel quantification. Scale bar = 100 µm. (C) Example of transformed epidermal cell expressing the GUS reporter gene prior to and after automated segmentation of cell (black bordering line) and haustorium (red bordering line). (Figure 1 from Wise et al. 2009)

53

Protocol:

Transient expression and gene silencing by particle bombardment................. 55

1. Materials .......................................................................................................... 55 2. Solutions.......................................................................................................... 55

2.1 DNA............................................................................................................. 55 2.2 Phytoagar plates........................................................................................ 55 2.3 Benzimidazole solution............................................................................. 55 2.4 Gold particle .............................................................................................. 56 2.5 Calcium nitrate........................................................................................... 56 2.6 X – Gluc solution ....................................................................................... 56 2.7 Trichloroacetic acid solution.................................................................... 56 2.8 Coomassie staining solution.................................................................... 56

3. Procedure ........................................................................................................ 56 3.1 DNA Preparation........................................................................................ 56

3.2 Leaves............................................................................................................ 58 3.3 Shooting with the PDS-1000/He Particle Delivery System (BIORAD) ... 58 3.3.1 Power on ................................................................................................. 60 3.3.2 Helium Pressure ..................................................................................... 60 3.3.3 Coating Macro carriers with DNA.......................................................... 60 3.3.4 Macro carrier Launch Assembly ........................................................... 60 3.3.5 Loading the rupture disk ....................................................................... 60 3.3.6 Target tissue placement in chamber .................................................... 61 3.3.7 Chamber evacuation/hold...................................................................... 61 3.3.8 Bombard the sample .............................................................................. 61 3.3.9 Release vacuum from chamber............................................................. 61 3.3.10 Target cells removal from chamber .................................................... 62 3.3.11 Macro carrier and stopping screen removal from Micro carrier launch............................................................................................................... 62 3.3.12 Removal of spent rupture disk ............................................................ 62 3.3.13 Removal of Residual Helium Pressure—Shut Down......................... 62 3.3.14 Incubation of the leave samples ......................................................... 63

4. Inoculation....................................................................................................... 63 4.1 Pinning the leaves on big culture plates ................................................. 63

54

4.1 Inoculation ................................................................................................. 63 4.2 Incubation of the leaves after inoculation............................................... 63

5. X – Gluc staining............................................................................................. 64 6. TCA Distaining ................................................................................................ 64 7. Coomassie Staining........................................................................................ 64 8. Microscopy………………………………………………………………………….. 9. Appendix.......................................................................................................... 65

To 2.2 Phytoagar plates .................................................................................. 65 To 2.3 Benzimidazole ...................................................................................... 66 To 2.4 Gold particle ......................................................................................... 66 To 2.5 Calcium nitrate ..................................................................................... 67 To 2.6 X – Gluc Solution ................................................................................. 68 2.6.1 Solutions ................................................................................................. 68 Phosphate buffer, pH = 6,5 , V = 100 mL ....................................................... 68 Sodium EDTA Solution pH = 8,0 .................................................................... 68 2.6.2 Preparation for 500 mL X - Gluc ............................................................ 69 To 2.7 TCA........................................................................................................ 70 To 2.8 Coomassie Solution............................................................................. 70

10. Safety Information......................................................................................... 71 Calcium nitrate ................................................................................................ 71 Trichloroacetic acid......................................................................................... 71 X – Gluc (5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt)............................................................................. 71 Methanol........................................................................................................... 71 Potassium hydroxide ...................................................................................... 71 Sodium EDTA .................................................................................................. 71 Sodium hydroxide ........................................................................................... 72

55

Transient expression and gene silencing by particle bombardment

1. Materials

• 7-day-old barley plants Grown at 20 °C, 16 h light, 70 % humidity.

Pot size 13, 2 cm x 10, 4 cm.

~ Seeds amount: 20 mL for barley and 15 mL for wheat.

• Macro carriers and Rupture Disk washed in Ethanol and dried Per shot you need 7 Macro carriers and 1 Rupture Disk.

• Magnet stirrers cleaned with Ethanol

• Pins with numbers 7 pins per shot labelled with the number of the shot, same amount of pins

without labelling.

Pins can be reused.

2. Solutions

2.1 DNA

• Plasmid DNA prepared with the JETSTAR MIDI Kit from GENOMED (or

Qiagen)

For preparation see protocol from the kit.

• adjust to a concentration to 1 µg·µL-1 TE buffer.

2.2 Phytoagar plates (for details see page 65)

• For shooting: 0,5 % phytoagar and 0,01 mg·mL-1 benzimidazole in Petri

dishes of 9 cm diameter.

• For inoculation: 1 % phytoagar and 0,01 mg·mL-1 benzimidazole in square

culture plates of 20 x 20 cm.

2.3 Benzimidazole stock solution (for details see page 66)

• concentration of 40 mg·mL-1 in 96 % Ethanol

56

2.4 Gold particle (for details see page 66)

• concentration of 27,5 mg·mL-1 in glycerol

2.5 Calcium nitrate (for details see page 67)

• concentration of 1 mol·L-1 , pH = 10

2.6 X-Gluc solution (for details see page 68)

• solution for staining of gus positive cells

2.7 Trichloroacetic acid solution (for details see page 70)

• concentration of 7,5 % TCA and 50 % methanol

2.8 Coomassie staining solution (for details see page 70)

• for staining of the fungus

3. Procedure

3.1 DNA Preparation

• per shot use 7 µg of Plasmid DNA per construct Add 7 µL DNA (1 µg·µL-1) per construct of interest to a 1, 5 mL Eppendorf

tube.

• centrifuge briefly

• add 87,5 µL Gold Mix and ultrasonicate the gold well before you use it. Be sure that it’s good

dispensed.

• add the calcium nitrate drop wise while vortexing Volume of calcium nitrate is calculated as follows:

57

V (Ca (NO3)2) = V (total DNA) + V (Gold) Example:

You want to shoot the reporter gene pUbiGUS and one construct of

interest.

7 µL pUbiGUS (1 µg·µL-1)

7 µL construct of interest (1 µg·µL-1)

87, 5 µL Gold

101, 5 µL calcium nitrate

If your DNA concentration is below 1 µg·µL-1, calculate the Volume for 7 µg

and adjust the calcium nitrate.

Example:

You want to shoot the reporter gene pUbiGUS (1 µg·µL-1) and one

construct of interest, which has a concentration of 150 ng·µL-1.

7 µL pUbiGUS (1 µg·µL-1)

47 µL construct of interest (150 ng·µL-1) = total 7 µg

87, 5 µL Gold

141, 2 µL calcium nitrate

• incubate at least 20-30 minutes at room temperature by inverting from time to time Don’t vortex; just invert approx. every 2 minutes.

In the meantime you can prepare the leaves (see 3.2).

• centrifuge at 14 000 rpm for 30 seconds

• remove supernatant

58

Be sure to remove the calcium nitrate completely.

• wash with 1 mL 70 % ethanol Invert few times, don’t vortex vigorously.

• centrifuge at 14 000 rpm for 30 seconds


• wash with 1 mL 96 % ethanol

• remove supernatant Be sure to remove the ethanol completely.

• Resuspend pellet in 30 µL 96 % ethanol

• proceed with shooting (see 3.3)

3.2 Leaves

• Cut 7 leaves and fix them on a Petri dish (with phytoagar) with two

magnetic stirrer bars. The leaves will lay flat on the agar.

•

3.3 Shooting with the PDS-1000/He Particle Delivery System (BIORAD)

Figure 1 Unit components, front view

59

Figure 2 Front view of PDS-1000/He unit

Figure 3 The Biolistic bombardment process

60

3.3.1 Power on

• Press the ON switch of the gun

• Switch on the vacuum pump

3.3.2 Helium Pressure

• Confirm that the helium tank pressure regulator is set to 200 psi over the

selected rupture disk burst pressure (e.g., set the regulator to 1,100 psi when

working with a 900 psi rupture disk).

• Open the helium bottle and turn the Helium pressure regulator until the helium

regulator reaches 1100 psi.

3.3.3 Coating Macro carriers with DNA

• Place each macro carrier inside the macro carrier holder.

Fix them well with a tool or tweezers.

• Ultrasonicate the DNA briefly and distribute the 30 µL to the 7 macro carriers

Be sure that the DNA – Gold solution is mixed well.

Don’t sonicate the DNA too long, as this causes DNA sharing.

• Let the DNA-Gold solution on the discs until the Ethanol is evaporated.

3.3.4 Macro carrier Launch Assembly

• Put a stopping screen on the macro carrier holder.

• Close the Micro carrier Launch Assembly.

• Put the Micro carrier Launch Assembly into the gun (position is marked).

3.3.5 Loading the rupture disk

• Place the rupture disk in the recess of the rupture disk retaining cap.

• Screw the rupture disk retaining cap onto the gas acceleration tube using a left

-to-right motion.

61

3.3.6 Target tissue placement in chamber

• Place the Target Shelf at the desired level inside the bombardment chamber.

(position is marked)

• Place the sample (usually contained within a Petri dish) on the Target Shelf.

• Close and latch the sample chamber door.

3.3.7 Chamber evacuation/hold

• Set the vacuum switch on the PDS-1000/He (middle red control switch) to the

VAC position.

• Evacuate the sample chamber to the desired level

• When the desired vacuum level is reached, hold the chamber vacuum at that

level by quickly pressing the vacuum control switch through the middle VENT position to the bottom HOLD position.

3.3.8 Bombard the sample

• Press and hold the FIRE swich.

• Switch to allow helium pressure to build inside the gas acceleration tube that is

sealed by a selected rupture disk.

• Estimate rupture disk burst pressure by observing the helium pressure gauge

at the top of the acceleration tube.

• A small “pop” will be heard when the rupture disk bursts. The rupture disk

should burst within 10% of the indicated rupture pressure and within 11–13

seconds.

• Release the FIRE switch immediately after the disk ruptures to avoid wasting

helium gas.

3.3.9 Release vacuum from chamber

• Release the vacuum in the sample chamber by setting the VACUUM switch to

the middle VENT position.

62

3.3.10 Target cells removal from chamber

• After vacuum is released, the vacuum gauge should read 0 inches of mercury

(Hg) of vacuum.

• Open the sample chamber door.

• Remove the sample and treat as appropriate.

• Leave lids of plate slightly open for app. 30 minutes to allow water droplets on

leaves to dry.

3.3.11 Macro carrier and stopping screen removal from Micro carrier launch assembly

• Remove the Micro carrier launch assembly.

• Unscrew the lid and remove the macro carrier holder.

• Discard the used macro carrier and stopping screen

• Collect stopping screen for cleaning and reuse

3.3.12 Removal of spent rupture disk

• Unscrew the rupture disk retaining cap from the gas acceleration tube.

• Remove the remains of the rupture disk.

• The next bombardment may now be performed.

3.3.13 Removal of Residual Helium Pressure—Shut Down

• After completing all bombardment(s), remove the helium pressure from the

PDS-1000/He system and close the helium cylinder valve.

• Perform the following steps to remove helium pressure from the system.

• Close the helium cylinder valve and chamber door.

• With at least 5 inches of Hg of vacuum in bombardment chamber, remove

residual line pressure from the regulator, solenoid and PEEK tubing by

activating the FIRE swich.

• Release the FIRE swich on the apparatus, and remove all tension on the

pressure adjustment screw of the helium regulator, turning counter-clockwise,

until it turns freely.

63

• Vent any residual vacuum from the bombardment chamber by setting the

vacuum switch o the VENT position.

3.3.14 Incubation of the leave samples

• Incubate the plates with bombarded leaves in the air-conditioned sequencing

or robot lab on the window bench, under natural day-light conditions without

direct sunlight (northern exposure). These conditions are optimal for delaying

leaf senescence.

• Incubation time is depending on the experiment (from 4h up to 4 days).

4. Inoculation

Note: If you don’t have do inoculate, skip the following steps!

4.1 Pinning the leaves on big culture plates

• Pin one leaf per shot after the other on the big plates

• Fix the leaves on one site with a pin with number (according to the number of

the shot) and on the other site with a non labelled pin

• Put an object slide at one end of the big plate to measure the spore density

4.1 Inoculation

• Inoculate the leaves with Bgh or Bgt.

Put the big plates into the inoculation box and shake the spores over them or

blow them in by using our “hairdryer-like” device.

• Measure the spore density by counting one fourth of the slide under the

microscope (16x lens).

• Spore density should be between 150-200 Spores·mm-2.

4.2 Incubation of the leaves after inoculation

• Incubate the bombarded leaf segments in the sequencing or robot lab for 48 h.

64

5. X-Gluc staining

• Label 15 mL Falcon tubes with the number of shots.

• Fill the tubes to 7 mL with X-Gluc solution.

• Optional: remove the spores from the leaves with a wet cotton pad.

Don’t do this, if you have to stain and observe the fungus!

• Remove the pins from the leaves and cut ends on both sides.

• Collect the leaves. Finally you should have 7 leaves per shot inside the Falcon

tubes.

• Vacuum-infiltrate the falcon tubes approx. 3 times in the desiccator.

The colour of the leaves should turn to dark green; the leaves sink to the

bottom, if not: repeat infiltration.

• Fill the Falcon tubes to 14 mL with X-Gluc solution.

• Incubate 24 h at 37 °C.

6. TCA Destaining

• Remove the X-Gluc solution.

• Fill TCA solution to 14 mL.

• Invert the tubes a few times.

• Incubate approx. 10 minutes at room temperature.

The leaves should turn brownish, if not incubate longer.

• Remove the TCA and wash the leaves in the tubes 2 times with water.

• Store the leaves finally in water or put them on a microscope slide.

7. Coomassie Staining

Note: This is only necessary if you want to observe the fungus!

• Handle the leaves carefully not to remove the spores!

• Fill Coomassie solution to 14 mL.

• Invert the tubes a few times.

• Incubate approx. 5 to 10 minutes at room temperature.

• Remove the Coomassie and wash the leaves in the tubes once with water.

• Store the leaves finally in water or put them on a microscope slide.

65

8. Microscopy

The analysis of the interaction of powdery mildew with transformed epidermal cells

expressing GUS is performed by manual or automated light microscopy. For manual

microscopy, any good quality light microscope equipped with 10x and 20x objectives

for 100x and 200x magnification will work.

• Count the number of GUS-stained cells per shot without counting stomatal

cells or companion cells.

• Count the number of GUS-stained cells per shot containing at least one

normally-developed fungal haustorium.

• Calculate the percentage of haustoria-containing GUS-stained cells per shot.

This is referred-to as the susceptibility index (SI) per shot. SI of specific RNAi

constructs can be compared to internal empty-vector controls of experiments,

which leads to the relative SI.

In the case of automated microscopy, data will be generated at the PC by clicking

through series of regions of interest generated by the microscope-robot software.

Hotkeys or mouse clicks will add observation to a data table.

9. Appendix

To 2.2 Phytoagar plates

• plates for shooting – in Petri dishes (90 mm x 16 mm)

• 0,5 % phytoagar

1 plate 10 plates 12 plates 20 plates

Phytoagar 0,15 g 1,5 g 1,8 g 3,0 g

H2O 30 mL 300 mL 360 mL 600 mL

Benzimidazole 7,5 µL 75 µL 90 µL 150 µL

• plates for inoculation – big plates (23,2 cm x 23,2 cm)

• 1 % phytoagar

66

1 plates 2 plates 4 plates

Phytoagar 5 g 10 g 20 g

H2O 500 mL 1000 mL 2000 mL

Benzimidazole 250 µL 500 µL 1000 µL

• add phytoagar to a 1 L flask

• add the water

• cook it in a microwave until agar is dissolved

• cool it on a shaker

• add benzimidazole

• store plates at 4 °C until usage

To 2.3 Benzimidazole

ß (C7H6N2) = 40 mg·mL-1

• weight 40 mg of Benzimidazole into a Eppendorf tube

• at 1 mL of 96 % Ethanol

• vortex well

• store at – 20 °C

To 2.4 Gold particle

ß (Gold) = 27, 5 mg ·mL-1

• 27,5 mg 1.0 Micron Gold

• add 1 mL H2O

• mix well

• 30 sec ultrasonication

• 14000 rpm / 30 sec

67


• add 1 mL H2O

• mix well


• 14000 rpm / 30 sec


• add 1 mL Ethanol, w = 96 %

• mix well


• 14000 rpm / 30 sec


• dry pellet at 50 °C with open lid (in a Thermo mixer)

• add 1 mL Glycerol , w = 50 %

• mix well


• store at -20 °C

• mix and ultrasonicate before to use

To 2.5 Calcium nitrate

m (Ca(NO3)2 · 4 H2O) = c (Ca(NO3)2 · 4 H2O) · VLsg. · M (Ca(NO3)2 · H2O)

m (Ca(NO3)2 · 4 H2O) = 1 mol·L-1 · 0,25 L · 236,149 g·mol-1

m (Ca(NO3)2 · 4 H2O) = 59,037 g

• weight calcium nitrate into a 250 mL bottle

• add 150 mL H2O

• adjust pH with potassium hydroxide (c = 1 mol·L-1) to pH = 10,0

68

• Autoclave and store at 4 °C

To 2.6 X – Gluc Solution

2.6.1 Solutions

Phosphate buffer, pH = 6,5 , V = 100 mL

• add 50 mL of 0,5 mol·L-1 Sodium dihydrogen phosphate

• add 50 mL of 0,5 mol·L-1 di – Sodium hydrogen phosphate

• mix well

Sodium dihydrogen phosphate (NaH2PO4 ·H2O), c=0,5mol·L-1

m (NaH2PO4 ·H2O) = M (NaH2PO4 ·H2O) · c (NaH2PO4 ·H2O) · V (Lsg.)

m (NaH2PO4 ·H2O) = 1,0076 g·mol-1 · 0,5 mol·L-1 · 1 L

m (NaH2PO4 ·H2O) = 69 g

Di – Sodium hydrogen phosphate (Na2HPO4·2 H2O), c=0,5mol·L-1

m (Na2HPO4·2 H2O) = M (Na2HPO4·2 H2O) · c (Na2HPO4·2 H2O) · V (Lsg.)

m (Na2HPO4·2 H2O) = 177,99g·mol-1 · 0,5mol·L-1 · 1L

m (Na2HPO4·2 H2O) = 89 g

Sodium EDTA Solution pH = 8,0

m (C10H14N2Na2O8·2 H2O) = M · c · V (Lsg.)

m (C10H14N2Na2O8·2 H2O) = 372,24 g·mol-1 · 0,5 mol·L-1 · 0,5 L

69

m (C10H14N2Na2O8·2 H2O) = 93,06 g

• weight EDTA in a 500 mL bottle

• add 300 mL water

• solution becomes milky

• adjust the pH to 10 with a 10 mol·L-1 sodium hydroxide solution

• solution becomes clear

• fill up with water to 500 mL

2.6.2 Preparation for 500 mL X – Gluc

• weight 500 mg of X – Gluc (store at – 20 °C in darkness) into a 1 L tumbler

• add 100 mL Methanol

• dissolve with a magnetic stirrer

• add 100 mL phosphate buffer pH = 6,5

• solution becomes cloudy

• mix well with a magnetic stirrer


• add 10 mL of 0,5mol·L-1 Na2EDTA pH = 8,0

• add 500 µL Triton X-100

• add 0,231 g Potassium ferricyanide (K3[Fe(CN)6]

• add 0,296 g Potassium Hexacyanoferrate (II) (K4[Fe(CN)6] * 3 H2O

• solution becomes yellow

• check the pH and adjust if necessary

• pH should be between 6,8 – 7,2


• store X - Gluc solution at – 20 °C

• defreeze before use

If you see crystals, warm the X – Gluc in the microwave and dissolve again

70

To 2.7 TCA

Trichloroacetic acid, w (C2HCl3O2) = 7, 5 %

Methanol, (w (CH4O) = 50 %

For 250 mL

m (TCA) = w (TCA) · m (Lsg.)

m (TCA) = 0,075 · 250 g

m (TCA) = 18, 75 g

m (CH4O) = w (CH4O) · m (Lsg.)

m (CH4O) = 0,5 · 250 mL

m (CH4O) = 125 mL

• weight 18,75 g TCA in a 250 mL bottle

• add 125 mL Methanol

• dissolve


• store at room temperature

To 2.8 Coomassie Solution

0,3 % Coomassi R250

7, 5 % TCA (Trichloroacetic acid)

50 % Methanol

• weight Coomassie

• dissolve in Methanol

• weight TCA

71

• dissolve in H2O

• mix both solution and fill up with water

• filtrate

• store at room temperature

10. Safety Information

Calcium nitrate

O Oxidising Xi Irritant R 8-36

Trichloroacetic acid C Corrosive N Dangerous for the environment R 35-50/53 S 26-36/37/39-45-60-61

X – Gluc (5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt)

Methanol

F Highly flammable T Toxic R 11-23/24/25-39/23/24/25 S 7-16-36/37-45

Potassium hydroxide

C Corrosive R 22-35 S 26-36/37/39-45

Sodium EDTA

Xi Irritant

R 36-52/53 S 61

72

Sodium hydroxide

C Corrosive

R 35 S 26-37/39-45

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IV) Post-transcriptional gene silencing in plants: case studies B) Detection of transgene silencing by histochemical GUS staining – Ann

Depicker and Sylvie De Buck

Introduction Numerous studies have demonstrated that the introduction of transgenes into plants

can result in homology dependent gene silencing (HDGS). HDGS can occur at the

transcriptional (TGS) or post-transcriptional (PTGS) level. TGS refers to the silencing

process that suppresses transcription of the silenced genes based on homology in

the silencer locus-containing promoter region. PTGS needs homology in the

transcribed region, results in specific transcript degradation and/or specific repression

of translational initiation, and resets through sexual propagation (Depicker and Van

Montagu, 1997; Frizzi and Huang, 2010). PTGS is often correlated with the presence

of multicopy T-DNA loci, which are frequently found upon Agrobacterium-mediated

transformation (De Neve et al. 1997; De Buck et al. 1999; De Buck et al. 2009).

These multiple T-DNAs can be oriented both as an inverted repeat, and can act as in

trans silencers for homologous copies located elsewhere in the genome (Hobbs et al.

1993; De Buck et al. 2001). Also in natural examples of gene silencing, the

correlation with repeated genes is prominent.

A. The positive correlation between the presence of inverted repeats and transgene

silencing was clearly demonstrated by measuring the expression of the GUS gene

when present as an inverted repeat or as a single copy in otherwise isogenic lines

(De Buck et al. 2001; De Buck and Depicker, 2001). The parental line KH15

contains a single T-DNA locus with two inversely repeated and convergently

transcribed GUS genes, separated by a 732-bp palindromic sequence. Because

one of the GUS reporter genes is flanked by directly repeated lox sites, CRE-

mediated recombination made it possible to obtain derived deletion loci KH15d2

and KH15d6 with only one GUS gene (Figure 1A). In the parental line KH15, GUS

activity was low, both in hemizygous and homozygous seedlings, throughout

development (Figure 1B, De Buck et al. 2001), and the GUS transcribed region

and the centre of the inverted repeat were heavily methylated (De Buck and

Depicker, 2001). In the derived lines, KH15d2 and KH15d6, GUS activity

increased 50- to 100-fold at all developmental stages analyzed, and methylation

76

decreased significantly (Figure 1B, De Buck et al. 2001; De Buck and Depicker,

2001). As RNA-dependent-RNA polymerase 6 is required for triggering silencing

by inverted T-DNA repeat loci (Butaye et al. 2004), it is suggested that not

readthrough hairpin transcripts generate double stranded RNA but rather that

aberrant transcripts from the palindromic locus are efficient substrates for the

synthesis of double-stranded RNA.

A.

B.

Figure 1: Transgene silencing of invertedly repeated transgenes is released upon deletion of one of the transgenes involved. (adapted from De Buck et al. 2001). (A) Schematic outline of the CRE-mediated deletion. In one of the T-DNAs of the inverted repeat, the GUS gene is surrounded by two directly repeated loxP (•) sites. After CRE-mediated deletion, this GUS gene is removed, and an isogenic single copy line is generated (B) Expression levels of the GUS gene in homozygous (Ho) seedlings when present in inverted repeat (KH15) and as a single copy in an allelic position (KH15d) measured during development. Abbreviations: HPT, hygromycin phosphotransferase gene; NPTII, neomycin phosphotransferase gene; GUS, β-glucuronidase gene; BAR, phosphinotricin transferase gene; CRE, recombinase-coding sequence; lox, recognition site for the CRE recombinase.

77

B. Hairpin induced silencing

Since the discovery that double-stranded RNA is responsible for silencing induction,

various expression vectors for the production of self complementary hairpin RNA

(hpRNA) have been designed to obtain efficient PTGS in plants (Helliwell et al. 2002;

Karimi et al. 2007). The silencing efficiency by which a silencer locus can

downregulate the expression of a target gene seems to be strongly linked to the

levels of dsRNA and siRNA. Also, a particular 35S-GUS hairpin RNA construct

exhibits different in trans silencing efficiencies on a target 35S-GUS gene in different

plant tissues (Figure 2A, Marjanac et al. 2009). GUS histochemical staining patterns

were analyzed in all tissues of the parental line FK24 containing the constitutively

expressed 35S-GUS gene and of 9 super-transformants harbouring the hairpin

construct (Figure 2B, Marjanac et al. 2009). In the parental line FK24, the 35S

promoter displayed GUS activity in all plant organs, but staining was especially

intense in the root tips, rosette leaves and flowers. In three FK+hp super-

transformants (f.e. FK24/hp57), GUS staining was absent or very low in the

expanded roots. Almost all expanded leaves were GUS negative or showed a patchy

pattern of weak GUS staining, which may indicate some cell-to-cell variability in the

degree of silencing. In five FK+hp transformants, GUS staining was observed in

expanding roots and no or weak and patchy GUS staining was visible in the leaves.

The last super-transformant displayed GUS staining intensities in the roots similar to

those of the parental line, as well as a mixture of GUS-negative expanded leaves,

patchy stained leaves and uniformly stained leaves (Marjanac et al. 2009). In all

FK+hp super-transformants, GUS expression was suppressed in flowers, although

residual GUS activity was always observed in the style of the pistil. These results

clearly demonstrated that GUS activity levels decreased in all FK+hp super-

transformants, but that the efficiency of GUS suppression varied in different super-

transformants. As all the super-transformants harboured multiple hpUS T-DNA

copies, the locus structure might explain the variable hairpin RNA production and

thus variable GUS silencing in the different transformants.

78

Figure 2: Gene silencing induced by a hairpin construct (adapted from Marjanac et al. 2009). (A) Schematic representation of the pHhpUS vector and the T-DNA present in the FK24 parental line. (B) Histochemical analysis of the parental line and hairpin super-transformants. Abbreviations: P35S, cauliflower mosaic virus promoter; 3’35S, 35S terminator; Pnos, promoter of the nopaline synthase gene; 3’nos, 3’-end of the nopaline synthase gene; 3’ocs, 3’-end of the octopine synthase gene; US: 792 nucleotides of the 3’-end of the GUS coding sequence; GUS, β-glucuronidase gene; HPT, hygromycin phosphotransferase gene; NPTII, neomycin phosphotransferase gene; L, lox recognition site of CRE recombinase; LB, left border, RB, right border.

Figure 3: Principle of the Histochemical GUS staining. After addition of the colourless substrate X-Glu to the seedlings, a blue precipitate will be formed in the cells where the GUS gene is expressed.

79

Protocol:

The aim of this analysis is to determine whether a β-glucuronidase (GUS)

transgene is highly expressed or silenced in different tissues of an Arabidopsis

thaliana transformant by histochemical GUS staining (Figure 3; Jefferson et al. 1987).

Plant Material:

1) Col0 : non-transformed wild type plant

2) FK24 : contains 1 GUS transgene in homozygous condition

3) FK24/hp 57 : FK24 parental plant super-transformed with a 35S-GUS hairpin

construct ( a hemizygous seedstock: some of the plants will not contain the

hairpin construct)

4) KH15 : harbours two GUS transgene copies in inverted orientation

(homozygous)

5) KH15d6 : deletion variant of line KH15: one of the GUS genes is deleted

(homozygous)

Protocol: Reagents:

• 90 % Cold Acetone (keep at 4 degrees)

• 0.1 M phosphate buffer pH 7

For the preparation of phosphate buffer, you need two stock solutions:

Stock A: 0.2 M NaH2PO4 (= 2.76 g in 100 ml H2O)

Stock B: 0.2 M Na2HPO4.12H2O (= 7.16 g in 100 ml H2O)

Sterilisation through a filter, not in the autoclave!

Mix: 19.5ml stockA + 30.5ml stockB + 50 ml sterile H2O

• 0.5 M Na2EDTA/NaOH (= 18.6 g in 100 ml H2O, set to pH 8.0 with 10 M

NaOH)

• GUS assay buffer (per 1.0 ml):

0.97 ml phosphate buffer (100 mM NaH2PO4 / Na2HPO4 pH 7)

20 µl 0.5 M Na2EDTA/NaOH (gives 10 mM EDTA)

10 µl 50mg/ml X-gluc (5-Bromo-4-chloro-3-indolyl-beta-D-

glucoside) in DMSO (gives 0.5 mg/ml X-gluc; 1% DMSO)

• 90% ethanol

80

Method:

• Take 3 plants per transgenic line, put in a falcon tube and wash the

seedlings in 5 to 10 ml 90% ice-cold acetone: let it shake for 30 minutes

on ice (or on a shaker in the cold room)

• Wash the seedlings 1x10 minutes and 2x5 minutes in 5 to 10 ml

phosphate buffer (shake)

• Add 5 ml GUS-assay buffer to the seedlings, incubate at 37°C for 30

minutes to 1 hour (equal time for all samples!)

• Wash the seedlings 1x10 minutes and 2x5 minutes in 5 to 10 ml

phosphate buffer (shake)

• Wash the seedlings in 3x10 minutes (at least) in 5 to 10 ml 90 %

ethanol

• Spread the seedlings on a Petri dish and interpret the result

References

Butaye KMJ, Goderis IJWM, Wouters PFJ, Pues JMTG, Delauré SL, Broekaert WF,

Depicker A, Cammue BPA, De Bolle MFC (2004) Stable high-level transgene

expression in Arabidopsis thaliana using gene silencing mutants and matrix

attachment regions. Plant J. 39: 440-449.

De Buck S, Van Montagu M, Depicker A (2001) Transgene silencing of invertedly

repeated transgenes is released upon deletion of one of the transgenes involved.

Plant Mol. Biol. 46: 433-445.

De Buck S, Depicker A (2001) Disruption of palindromic arrangement leads to

selective loss of DNA methylation in gus transgenes. Mol. Gen. Genom. 265:

1060-1068.

De Buck S, Podevin N, Nolf J, Jacobs A, Depicker A (2009) The T-DNA integration

pattern in Arabidopsis transformants is highly determined by the transformed

target cell. The Plant Journal, 60: 134-145.

De Neve M, De Buck S, Jacobs A, Van Montagu M, Depicker A (1997) T-DNA

integration patterns in cotransformed plant cells suggest that T-DNA repeats

originate from cointegration of separate T-DNAs. Plant J. 11: 15-29.

Depicker A, Van Montagu M (1997) Post-transcriptional gene silencing in plants. Curr.

Opin. Cell Biol. 9: 373-382.

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Frizzi A, Huang S (2010) Tapping RNA silencing pathways for plant biotechnology.

Plant Biotech. J. 8: 1-23.

Helliwell CA, Wesley SV, Wielopolska AJ, Waterhouse PM (2002). High-throughput

vectors for efficient gene silencing in plants. Functional Plant Biology 29: 1217 –

1225.

Hobbs SLA, Warkentin TD, DeLong, CMO (1993) Transgene copy number can be

positively or negatively associated with transgene expression. Plant Mol. Biol.

21: 17-26.

Jefferson RA, Kavanagh TA, Bevan MW (1987). GUS fusions: β-glucuronidase as a

sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901-

3907.

Karimi M, Bleys A, Vanderhaegen R, Hilson P (2007). Building blocks for plant gene

assembly. Plant Phys. 145: 1183-1191.

Marjanac G, Karimi M, Naudts M, Beeckman T, Depicker A, De Buck S (2009) The

degree of silencing, induced by hairpin RNA or invertedly repeated sense

transgenes, varies in different cell types. New Phytologist, 851-864.

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