|
| Status |
Public on Feb 01, 2019 |
| Title |
Drained_wounding_2 |
| Sample type |
RNA |
| |
|
| Source name |
tree stem_wounding in drained soil
|
| Organism |
Pinus sylvestris |
| Characteristics |
sample id: W2b
inoculated with: sterile pine wood dowels (wounding)
responses to pathogen infection:
field/growth condition: drained peatland
time point: 4 months after the inoculations
tissue: tree stem
|
| Treatment protocol |
The sample trees were inoculated either with sterile pine wood dowels (wounding), or with dowels pre-colonized by Heterobasidion annosum (infection). Autoclaved sterile dowels of Scots pine wood (diameter 10 mm) were placed on a two-week-old malt agar culture of H. annosum (homokaryotic isolate 03005 Kirkkonummi (Porkkala), courtesy of K. Korhonen, Finland) in Petri dishes incubated at room temperature in darkness for three weeks. Infected trees were inoculated with the pre-colonized dowels and wounded trees with the sterile dowels. Three inoculations were done in the stem and three in the roots of each tree in June 2010. Ten trees representing random genotypes were designated for both treatments. Stem inoculations were made at 50 cm, 100 cm and 150 cm height above the root neck. In the roots, the inoculations were placed at least 15 cm apart. After four months, the samples were harvested by falling the trees and by cutting the roots with chainsaw. Periderm tissues were removed with a knife and an axe, after which the length and width of the necrotic lesions formed in the phloem and xylem were measured. Phloem tissues were harvested from each inoculation using a knife and a chisel. After harvesting, the tissue samples were stored at -20ºC. Phloem tissue from unwounded tree served as a control. Phloem samples were frozen in liquid nitrogen, ground to powder using IKA® A11 basic analytical mill (IKA Werke) and stored at -80 ºC for further processing. RNA was extracted from the samples as described elsewhere (Chang et al. 1993). One microgram of total RNA from phloem in one stem inoculation was pooled into each RNA sample from infected, wounded and control sample trees.
|
| Growth protocol |
The sample plots were located in Lakkasuo, Orivesi (61°47’ N; 24° 18’ E; 150 m asl). Naturally regenerated mature Scots pine (Pinus sylvestris) trees representing random genotypes were growing in drained peatland (Vaccinium vitis-idaea type II, yield corresponding to Vaccinium type (VT) forest on mineral soil). Inoculations were conducted in the beginning of June 2010, and samples were harvested in the end of September 2010 (4 months after inoculation). Trees were cut down, and sample pieces close to the points of inoculation were cut out, and transported for storage at +4°C for 2 days, after which the samples were transported and stored at -20°C. Control samples were excised from untreated trees as 8 mm diameter core samples containing both the phloem and sapwood. Control samples were stored on dry ice after excision, and later stored at -20°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
1. Grind plant tissue in liquid nitrogen.
2. Transfer 2.0 g of the ground sample to a sterile 50 ml tube.
3. Heat Extraction buffer to 65ºC and add 2-Mercaptoethanol (final concentration 2%). Mix by inversion.
4. To each tissue sample, add 8 ml of extraction buffer. Vortex well. Incubate for 40 min at 65ºC, and vortex briefly every 10 minutes.
5. Add equal volume of chloroform:isoamyl alcohol (24:1). Mix by inversion and centrifuge at 8000 rpm for 30 min at 25ºC.
6. Collect the upper (aqueous) phase to a sterile 15 ml tube. Add equal volume of chloroform:isoamyl alcohol (24:1). Mix by inversion and centrifuge at 8000 rpm for 30 min at 25ºC. Repeat if the aqueous phase is very cloudy.
7. Transfer aqueous phase to a new sterile tube. Add 1/4 volume of 10M LiCl and mix by inversion. Precipitate RNA overnight at 4ºC. Centrifuge at 12 000 rpm for 40 min.
8. Pipet out the supernatant and dry the pellet briefly. Dissolve the pellet in 500 µl SSTE and transfer into a sterile 1.5 ml tube.
9. Add an equal volume of chloroform:isoamyl alcohol. Mix by inversion and centrifuge at 13000rpm for 30 min at 25ºC.
10. Transfer aqueous phase to a new 1.5 ml tube. Add 2 volumes of cold absolute ethanol. Mix by inversion. Precipitate at -20ºC for at least 2 hours. Centrifuge at 13000rpm for 30 minutes.
11. Pipet out the supernatant. Wash the pellet by adding approximately 100 μl cold 70% ethanol. Centrifuge at 7500 rpm for 10 min.
12. Carefully pipet out the supernatant and dry the pellet in the sterile hood. Re-suspend the pellet in DEPC-treated water.
EXTRACTION BUFFER
2% CTAB
2% Polyvinylpyrrolidone (PVP) K-30
2% 2-Mercaptoethanol
100 mM Tris-HCl
25 mM EDTA
2 M NaCl
DEPC-treated water
SSTE BUFFER
1 M NaCl
0.5% SDS
10 mM Tris-HCl
1 mM EDTA
DEPC-treated water
|
| Label |
Cy3
|
| Label protocol |
Labelling by NimbleGen Company.
|
| |
|
| Hybridization protocol |
Hybridization by NimbleGen Company.
|
| Scan protocol |
Scanning by NimbleGen Company.
|
| Data processing |
Expression values reflect across-array quantile normalization and gene calls generated using the Robust Multichip Average (RMA). For information on RMA, see Biostatistics 2003 Apr; 4(2): 249-264.
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| |
|
| Submission date |
Feb 20, 2015 |
| Last update date |
Feb 01, 2019 |
| Contact name |
Tommaso Raffaello |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Helsinki
|
| Department |
Forest Sciences
|
| Street address |
Latokartanonkaari 7 PO Box 27
|
| City |
Helsinki |
| ZIP/Postal code |
00014 |
| Country |
Finland |
| |
|
| Platform ID |
GPL19078 |
| Series (1) |
| GSE66168 |
Transcript profiling of Pinus sylvestris trees as a response to Heterobasidion annosum infection under field conditions |
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