Patent Application
20080047033
The relative penetration efficiency (RPE) was determined in five individual experiments with inoculation with Bgh from barley cv Pallas. The RPE is calculated as the difference between the penetration efficiency of pNAox-dsRNA-transformed cells and the penetration efficiency of control-dsRNA-transformed cells (here: average penetration efficiency 38.74%). The percent RPE (% RPE) is calculated from the RPE minus 1, multiplied by 100.
The columns (1) to (5) represent the % RPE (i.e. the deviation of the penetration efficiency from the average of the penetration efficiency of the control) when evaluating at least 100 interaction sites for in each case one independent experiment. The column (m) represents the average % RPE of the experiments. The error bar indicates the standard error.
“Control dsRNA” represents the parallel experiments with a control dsRNA. “pNAox” dsRNA represents the experiments with the dsRNA of the barley NADPH oxidase.
In cells which have been bombarded with pNAox-dsRNA, the % RPE was markedly (significance p=0.0054) reduced in comparison with cells bombarded with a control dsRNA (TR: human thyroid receptor dsRNA).
General methods:
The chemical synthesis of oligonucleotides can be effected for example in the known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The cloning steps carried out within the scope of the present invention, such as, for example, restriction cleavages, agarose gel electrophoresis, purification of fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking fragments, transformation of E. coli cells, bacterial cultures, phage multiplication and sequence analysis of recombinant , are carried out as described by Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules are sequenced with a laser fluorescence sequencer from MWG Licor following the method of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA 74:5463-5467).
The variety Pallas was provided by Lisa Munk, Department of Plant Pathology, Royal Veterinary and Agricultural University, Copenhagen, Denmark. Its production is described (Kolster P et al. (1986) Crop Sci 26: 903-907).
Unless otherwise described, the seed, which had been pregerminated for 12 to 36 hours in the dark on damp filter paper, was placed at a rate of 5 kernels along the edge of a square pot (8×8 cm) in Fruhstorfer soil, type P, covered with soil and watered regularly with tap water. All plants were grown in controlled-environment cabinets or chambers at 16 to 18° C., 50 to 60% relative atmospheric humidity and a 16-hour-light/8-hour-dark photoperiod at 3000 or 5000 lux (photon flux density 50 or 60 μmols-1m-2) for 5 to 8 days and used in the experiments during the seedling stage. In experiments in which primary leaves were treated, the latter were fully developed.
Prior to carrying out the transient transfection experiments, the plants were grown in controlled environment cabinets or chambers at 24° C. daytime temperature, 20° C. nighttime temperature, 50 to 60% relative atmospheric humidity and a 16-hour-light/8-hour-dark photoperiod at 30 000 lux.
Powdery mildew of barley Blumeria graminis (DC) Speer f.sp. hordei Em. Marchal race A6 (Wiberg A (1974) Hereditas 77: 89-148) (BghA6) was used for the inoculation of barley plants. The fungus was provided from the Department of Biometry, JLU GieBen. The inoculum was propagated in controlled-environment chambers under identical conditions as described above for the plants by transferring the conidia of infected material to regularly grown 7-day-old barley plants cv. Golden Promise at a density of 100 conidia/mm2.
The inoculation with BghA6 was carried out using 7-day-old seedlings by shaking off the conidia from infected plants in an inoculation tower at approximately 100 conidia/mm2 (unless otherwise specified).
The c fragments required for the isolation of the HvpNAox c, its cloning, sequencing and generation of probes were obtained by RT-PCR using the “One Step RT-PCR Kit” (Life Technologies, Karlsruhe, Germany, or Qiagen, Hilden, Germany). To this end, a total RNA from barley seedlings was used as template. The RNA was isolated from Pallas 3, 5 and 7 days after germination. In addition, RNA was isolated from Pallas and from the backcrossed lines with mlo5, Mlg or Mla12 1, 2 and 5 days after inoculation with BghA6 on day 7 after germination. The RT-PCR was carried out using primers which are derived from conserved regions of the gp91phox homologs from rice and Arabidopsis thaliana (GenBank Acc. No.: X93301 and AB008111):
In each case 1000 ng of total , 0.4 mM dNTPs, in each case 0.6 mM OPN-1 and OPN-2 primer, 10 μl of RNase inhibitor and 1 μl of enzyme mix in lx RT buffer (one step RT-PCR Kit, Qiagen, Hilden) were employed for the reaction.
The following temperature program is used (PTC-100TM model 96V; MJ Research, Inc., Watertown, Mass.):
The PCR products were separated by means of 2% w/v agarose gel electrophoresis. This gave a 378 bp RT-PCR product (SEQ ID NO: 1) which encodes a part of the open reading frame of the barley NADPH oxidase. The corresponding cDNA was isolated from an agarose gel and cloned in the pGEM-T vector (Promega, Mannheim, Germany) by means of T-overhang ligation. The cDNAs were sequenced starting from the plasmid DNA using the “Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing Kit” (Amersham, Freiburg, Germany). The construct was named pGEM-T-pNAox.
The plasmid, which had been employed for the in-vitro RNA transcription, comprises the T7 and SP6 promoters at the respective ends of the inserted nucleic acid sequence, which makes possible the synthesis of sense RNA and antisense RNA. The plasmid can be linearized with suitable restriction enzymes (ApaI for SP6 polymerase and PstI for T7 polymerase) in order to ensure correct transcription of the inserted nucleic acid sequence and to prevent read-through into vectorial sequences. To this end, in each case 10 μg of pGEM-T-pNAox plasmid were cut with ApaI for SP6 polymerase and with and PstI for T7 polymerase. The cut plasmids are extracted in 200 μl of water with the same volume phenol/chloroform/isoamyl alcohol, transferred into a fresh Eppendorf vessel (RNAse-free) and centrifuged for 5 minutes at 20 000 g. 180 ρl of the plasmid solution were treated with 420 μl of ethanol, placed on ice and subsequently precipitated by centrifugation for 30 minutes at 20 000 g and −4° C. The precipitate was taken up in 10 μl of TE buffer. The preparations in question were employed directly in an in-vitro transcription with T7-RNA polymerase and with SP6-RNA polymerase, respectively. RNA polymerases were obtaied from Roche Molecular Biology, Mannheim, Germany.
Each transcription mixture contained the following in a volume of 40 μ:
2 μl linearized plasmid (1 ∝g)
2 μl NTPs (25 mm) (1.25 mM of each NTP)
4 μl 10× reaction buffer (Roche Molecular Biology),
1 μl RNAsin RNAsin (27 units; Roche Molecular Biology),
2 μl RNA polymerase (40 units)
29 μl DEPC water
After 2 hours of incubation at 37° C., in each case some of the reaction mixtures from the transcription of the sense and antisense strands were mixed, denatured for 5 minutes at 95° C. and thereafter hybridized with one another (annealed) by cooling over 30 minutes to a final temperature of 37° C. As an alternative, the mixture of sense and antisense strand can also be cooled for 30 minutes at −20° C. after the denaturation. The protein precipitate which formed during denaturation and hybridization was removed by briefly centrifuging at 20 800 g, and the supernatant was used directly for coating tungsten particles (see hereinbelow). For the analysis, in each case 1 μl of each RNA strand and of the dsRNA were separated on a non-denaturing agarose gel. Successful hybridization is evident by a band shift towards higher molecular weight in comparison with the individual strands.
4 μl of the dsRNA were precipitated with ethanol (by addition of 6 μl of water, 1 μl of 3M sodium acetate solution and 25 μl of ethanol, and centrifugation for at least 5 minutes at 20 000 g and 4° C.) and resuspended in 500 μl of water. The absorption spectrum between 230 and 300 nm was measured or the absorption at 280 and 260 nm was determined to determine the purity and the concentration of the dsRNA. As a rule, 80 to 100 μg of dsRNA with an OD260/OD280 ratio of 1.80 to 1.95 were obtained. If desired, a digestion with DNase I may be carried out, but this has no substantial effect on subsequent results.
The dsRNA of the human thyroid receptor (starting vector pT7beta-Sal (Norman C et al. (1988) Cell 55(6):989-1003), provided by Dr. Baniahmad, Department of Genetics, Giegen, Germany; the sequence of the insert is described under the GenBank Acc. No.: NM—000461) acted as control dsRNA. The plasmid was digested with PvuII to generate the sense RNA and with HindIII to generate the antisense RNA, and the RNA was then transcribed using T7 or SP6 RNA polymerase. The individual process steps for the generation of the control dsRNA are carried out analogously to those described above for the pNAox-dsRNA.
Barley cv Pallas leaf segments were transformed with a pNAox dsRNA together with a GFP expression vector. Thereafer the leaves were inoculated with Bgh and the result was analyzed after 48 h by means of light and fluorescence microscopy. The penetration into GFP-expressing cells was assessed by detecting haustoria in live cells and by assessing the fungal development in precisely those cells. In all five experiments, the bombardment of barley cv Pallas with pNAox dsRNA resulted in a reduced number of cells which were successfully penetrated by Bgh in comparison with cells which had been bombarded with foreign control dsRNA (human thyroid hormone receptor dsRNA, TR). The resistance-inducing effect of the pNAox dsRNA resulted in an average reduction of the Bgh penetration efficiency by 35% (
A method which had already been described for the biolistic introduction of dsRNA into epidermal cells of barley leaves was employed for the transient transformation (Schweizer P et al. (1999) Mol Plant Microbe Interact 12:647-54; Schweizer P et al. 2000) Plant J 2000 24: 895-903). Tungsten particles 1.1 μm in diameter (particle density 25 mg/ml) were coated with dsRNA (preparation see above) together with plasmid of the vector pGFP (GFP under the control of the CaMV 35S promoters) as transformation marker. To this end, the following amounts of dsRNA and reporter plasmid were used for the coating per shot: 1 μg pGFP and 2 μg dsRNA. Double-stranded RNA was synthesized by annealing sense and antisense RNA in vitro (see above).
To prepare microcarriers, 55 mg of tungsten particles (M 17, diameter 1.1 μm; Bio-Rad, Munich) were washed twice with 1 ml of autoclave-distilled water and once with 1 ml of absolute ethanol, dried and taken up in 1 ml of 50% strength glycerol (approximately 50 mg/ml stock solution). The solution was diluted with 50% glycerol to 25 mg/ml, mixed thoroughly prior to use and suspended in an ultrasonic bath. To coat microcarriers, 1 μg of plasmid, 2 μg of dsRNA (1 μl), 12.5 μl of tungsten particle suspension (25 mg/ml), 12.5 μl of 1 M Ca(NO3)2 solution (pH 10) per shot were combined dropwise with constant mixing, left to stand for 10 minutes at RT, centrifuged briefly, and 20 μl of the supernatant were removed. The remainder with the tungsten particles is resuspended (ultrasonic bath) and employed in the experiment.
Barley primary leaf segments approximately 4 cm in length were used. The tissues were placed on 0.5% Phytagar (GibcoBRL™ Life Technologies™, Karlsruhe) supplemented with 20 μg/ml benzimidazole in Petri dishes (diameter 6.5 cm) and, immediately before the particle bombardment, the edges were covered with a stencil with a rectangular opening of dimensions 2.2 cm×2.3 cm. One after the other, the dishes were placed on the bottom of the vacuum chamber (Schweizer P et al. (1999) Mol Plant Microbe Interact 12:647-54) over which a nylon mesh (mesh size 0.2 mm, Millipore, Eschborn) had been inserted on a perforated sheet to act as diffusor (5 cm above the bottom, 11 cm underneath the macrocarriers, see hereinbelow) in order to diffuse particle clumps and to slow down the particle stream. The macrocarrier attached at the top of the chamber (plastic sterile filter holder, 13 mm, Gelman Sciences, Swinney, UK) was loaded with 5.8 μl of -coated tungsten particles per shot (microcarriers, see hereinbelow). Using a diaphragm vacuum pump (Vacuubrand, Wertheim), the pressure in the chamber was reduced by 0.9 bar, and the tungsten particles were fired at the surface of the plant tissue at a helium-gas pressure of 9 bar. Immediately thereafter, the chamber was aerated. To label transformed cells, the leaves were bombarded with the plasmid (pGFP; vector on pUC18-basis, CaMV 35S promoter/terminator cassette with inserted GFP gene; Schweizer P et al. (1999) Mol Plant Microbe Interact 12:647-54; provided by Dr. P. Schweizer, Department of Plant Genetics IPK, Gatersleben, Germany). Each time a different plasmid was used for the bombardments, the macrocarrier was cleaned thoroughly with water beforehand. After incubation for four hours after the bombardment with slightly open Petri dishes at RT and with daylight, the leaves were incubated with 100 conidia/mm2 of powdery mildew of barley (race A6) and incubated under identical conditions for a further 40 to 48 hours.
Leaf segments were bombarded with the coated particles using a article inflow gun. For each shot, 312 μg of tungsten particles ere applied. 4 hours after the bombardment, the leaves were inoculated with Blumeria graminis f.sp. hordei mildew (race A6) and, after a further 40 hours, evaluated for symptoms of infection. The result (for example the penetration efficiency, defined as percentage of attacked cells with a mature haustorium and a secondary elongating hypha were analyzed by means of fluorescence and light microscopy. An inoculation with 100 conidia/mm2 results in an infection frequency of approximately 50% of the transformed cells. A minimum of 100 interaction sites was evaluated for each individual experiment. Transformed (GFP-expressing) cells were identified under excitation with blue light. Three different categories of transformed cells were distinguished:
1. Penetrated cells containing a readily recognizable haustorium. A cell with more than one haustorium was considered as one cell.
2. Cells which, while attacked by a fungal appressorium, contain no haustorium. A cell which has been attacked more than once by Bgh, but which contains no haustorium, was considered as one cell.
3. Cells which are not infected by Bgh.
Stomatal cells and guard cells were excluded from the assessment. Surface structures of Bgh were analyzed by means of light microscopy or fluorescence staining of the fungus with 0.1% Calcofluor (w/v in water) for 30 seconds. The fungal development can be evaluated readily by fluorescence microscopy following staining with Calcofluor. In pNAox-dsRNA-transformed cells, the fungus develops a primary and apressorial germ tube, but no haustorium. The development of a haustorium is a condition for the development of a secondary hypha.
The relative penetration efficiency (RPE) is calculated as the difference between the penetration efficiency of transformed cells (transformation with pNAox or control dsRNA) and the penetration efficiency of untransformed cells (here: average penetration efficiency 38.74%). The percent RPE (% RPE) is calculated from the RPE minus 1, multiplied by 100.
The % RPE value (deviation of the average penetration efficiency of the control) is used to determine the susceptibility of cells transfected with pNAox-dsRNA (
In the case of the control dsRNA, five different experiments reveal no difference between the transfection with the control dsRNA and water with regard to the penetration efficiency of Bgh.
To rule out an effect of the dsRNA and the transformation rate or survival rate of the attacked cells, the number of GFP-expressing cells in control experiments and pNAox-dsRNA experiments was compared. The pNAox-dsRNA had no effect on the total number or the number of the attacked GFP-expressing cells.
The results were supported by further experiments with the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI; table 1). In general, the experiments were carried out as described by Hückelhoven and Kogel, 1998.
aThe DPI treatment was carried out 12 hours after inoculation with the pathogen and the evaluation 36 hours after inoculation.
bControll with 10 mM potassium phosphate buffer, pH 7.8, with DMSO content as in the DPI treatment.
cDPI dissolved in 10 mM potassium phosphate buffer, pH 7.8, starting from a 10 mg/ml DPI stock solution in DMSO.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10233327.0 | Jul 2002 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/07589 | 7/14/2003 | WO | 00 | 7/26/2007 |
