Novel nucleic acid sequences and their use in methods for achieving a pathogenic resistance in plants
A nucleic acid sequence, pathogen technology, applied in the field of expression cassettes and vectors, partially or transgenic propagating material, to generate or enhance plant pathogen resistance, in the field of culture
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Embodiment 1
[0359] Example 1: Plants, pathogens and inoculation
[0360] Ingrid barley var was obtained from Patrick Schweizer at the Institute of Plant Genetics and Crop Plants in Gatersleben. The Pallas variant and the backcross line BCIngrid-mlo5 were provided by Lisa Munk, Department of Plant Pathology, Royal Veterinary and Agricultural University, Copenhagen, Denmark. Its preparation has been described (Kölster P et al. (1986) Crop Sci 26:903-907).
[0361] Unless otherwise stated, seeds pregerminated for 12 to 36 hours on wet filter paper in the dark were placed 5 per well on the rim of square pots (8 x 8 cm) in type P Fruhstorfer soil, covered with soil and watered regularly with tap water. All plants were kept at 16 to 18°C, 50 to 60% relative atmospheric humidity and 3000 and 5000 lux (50 and 60 μmols- 1 m- 2 Photon flux density) of 16 hours light / 8 hours dark photoperiod cultured for 5 to 8 days, and used for embryo stage experiments. In experiments performed using primary l...
Embodiment 2
[0365] Example 2: RNA Extraction
[0366] Total RNA was extracted from 8 to 10 primary leaf nodes (length 5 cm) with "RNA extraction buffer" (AGS, Heidelberg, Germany).
[0367] For this purpose, 5 cm long central primary leaf nodes were harvested and homogenized in liquid nitrogen in a mortar. Homogenates were stored at -70°C for RNA extraction.
[0368] Total RNA was extracted from deep-frozen leaf material by RNA extraction kit (AGS, Heidelberg). For this, 200 mg of deep-frozen leaf material in a microcentrifuge tube (2 mL) was overlaid with a layer of 1.7 mL of RNA extraction buffer (AGS) and mixed immediately and thoroughly. After adding 200 μL of chloroform, mix thoroughly again and shake at room temperature for 45 min at 200 rpm on a horizontal shaker. Then, centrifuge at 20000g and 4°C for 15min to separate the phases, transfer the upper aqueous phase to a new microcentrifuge tube, and discard the lower layer. The aqueous phase was washed again with 900 μL of chlor...
Embodiment 3
[0371] Example 3: Cloning of the HvCSL1 cDNA sequence from barley
[0372] To isolate HvCSL1 cDNA, the cDNA fragments required for its cloning, sequencing and probe preparation were obtained by RT-PCR using the "One-Step RT-PCR Kit" (Life Technologies, Karlsruhe, Germany or Qiagen, Hilden, Germany). For this, total RNA from barley seedlings was used as template. RNA isolation from Ingrid var. 7 days after germination. Additionally, RNA from the Ingrid variety and the mlo5 backcross line was isolated at days 1, 2 and 5 after inoculation with BghA6 at day 7 post germination. For RT-PCR, use the following primers:
[0373] Hei131 5'-GTTCGCCGTTTCCTCCCGCAACT-3' (SEQ ID NO: 40)
[0374] and
[0375] Gene Racer 5'-Nested Primer, Invitrogen
[0376] 5'-GGACACTGACATGGACTGAAGGAGTA-3' (SEQ ID NO: 41)
[0377] For each reaction (25 μL mixture), use 1000 ng of total RNA, 0.4 mM dNTP, 0.6 mM each of OPN-1 and OPN-2 primers, 10 μl of RNase inhibitor and in 1×RT buffer (one-step RT-PCR ...
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