PLANT PROTECTION FROM A PEST OR PATHOGEN BY EXPRESSION OF DOUBLE-STRANDED RNAs IN THE PLASTID
20170342429 · 2017-11-30
Assignee
Inventors
- Ralph Bock (Schwielowsee, DE)
- Jiang Zhang (Potsdam, DE)
- David G. Heckel (Jena, DE)
- Sher Afzal Khan (Jena, DE)
Cpc classification
C12N15/8218
CHEMISTRY; METALLURGY
C12N15/8279
CHEMISTRY; METALLURGY
Y02A40/146
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
The present invention lies in the field of plant protection, in particular in the field of controlling plant pests and pathogens that affect plants. The present invention relates to a plant comprising a plastid comprising a double-stranded RNA (dsRNA) capable of silencing at least one target gene of a pest of a plant or of an agent causing a disease of a plant. The present invention further relates to such a transplastomic plant, wherein said dsRNA comprises two (separate) complementary single-stranded RNA strands. The present invention further relates to a plastid as comprised in the plant of the invention and to a plant cell comprising said plastid. Moreover, the present invention relates to a method of producing a plant of the invention and to a method of controlling a pest of a plant or a plant disease-causing agent or of protecting a plant from said pest or agent. Furthermore, the present invention relates to the use of a dsRNA for controlling a pest of a plant or a plant disease-causing agent or for protecting a plant from said pest or agent.
Claims
1. A plant comprising a plastid comprising a double-stranded RNA (dsRNA) capable of silencing at least one target gene of a pest of a plant (plant pest) or of an agent causing a disease of a plant (plant pathogen), wherein said dsRNA comprises two complementary single-stranded RNA strands.
2. The plant of claim 1, wherein said dsRNA comprises two separate complementary single-stranded RNA strands.
3. The plant of claim 1, wherein said plastid is a chloroplast.
4. The plant of claim 1 which is a vascular plant.
5. The plant of claim 1, wherein the sense strand of said dsRNA is at least 60% identical to an RNA transcribed from a nucleotide sequence of at least 50 contiguous nucleotides of said target gene.
6. The plant of claim 1, wherein said plastid is genetically engineered so as to comprise a nucleotide sequence encoding said dsRNA, wherein said dsRNA is transcribed from said nucleotide sequence.
7. The plant of claim 1, wherein said dsRNA is expressed by transcription from a nucleotide sequence flanked by two convergent promoters.
8. The plant of claim 1, wherein said plant pest or plant pathogen is selected from the group consisting of: (i) an insect; (ii) a nematode; (iii) a mollusk; and (iv) a fungal plant pathogen.
9. The plant of claim 8, wherein said insect is a Colorado potato beetle (Leptinotarsa decemlineata), including any juvenile stage of said beetle.
10. The plant of claim 8, wherein said fungal plant pathogen is Phytophthora infestans.
11. The plant of claim 1, which is a potato plant or a tobacco plant.
12. The plant of claim 1, wherein said target gene is ACT, SHR, EPIC2B or PnPMA1.
13. A plastid as defined in claim 1.
14. A plant cell comprising a plastid of claim 13.
15. A method of controlling a plant pest or a plant pathogen as defined in claim 1 and/or of protecting a plant from said plant pest or plant pathogen comprising the steps of (i) growing a plant of claim 1; and (ii) allowing said plant pest or plant pathogen to affect said plant.
Description
[0156] The present invention is further described by reference to the following non-limiting figures and examples.
[0157] The Figures show:
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[0173] In this specification, a number of documents including patent applications are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
[0174] The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.
EXAMPLE 1: MATERIALS AND METHODS
Plant Material and Growth Conditions
[0175] To generate leaf material for biolistic plastid transformation experiments, tobacco plants (Nicotiana tabacum cv. Petit Havana) were grown under aseptic conditions on agar-solidified MS medium supplemented with 30 g/L sucrose (22). Potato (Solanum tuberosum cv. Desiree) plants for nuclear and chloroplast transformation experiments were grown on the same medium but at lower sucrose concentration (20 g/L). Transgenic and transplastomic lines were rooted and propagated on the same media in the presence of the appropriate antibiotic (spectinomycin or hygromycin). Rooted plantlets were grown in soil under standard greenhouse conditions. Inheritance patterns in transplastomic tobacco lines were analyzed by germination of surface-sterilized seeds on Petri dishes containing MS medium supplemented with spectinomycin (500 mg/L).
Construction of Transformation Vectors
[0176] The plastid transformation vectors constructed in this study are based on a modified version of the previously described plasmid pKP9 (23). The aadA cassette in pKP9 was replaced by a modified cassette consisting of the Chlamydomonas reinhardtii PpsbA promoter, the coding region of the selectable marker gene aadA and the 3′UTR of the rbcL gene from Chlamydomonas reinhardtii (24, 25). The cassette was excised from a plasmid clone with the restriction enzymes SpeI and SmaI, followed by a fill-in reaction with the Klenow fragment of DNA polymerase I to generate blunt ends, and then cloned into a progenitor clone of pKP9 that was cut with the restriction enzyme Ec113611. A clone was selected which contained the aadA cassette in the opposite orientation of the upstream trnfM gene, yielding plastid transformation vector pJZ100 (
[0177] Target gene selection for RNA interference was based on previous reports (12, 3). A DNA fragment covering 297 bp of the β-actin gene (ACT) and 220 bp of the Shrub gene (SHR) from Leptinotarsa decemlineata was chemically synthesized as a fusion (ACT+SHR) with a 5′ extension (5′-GCATGCCTGCAG-3′; introducing SphI and PstI restriction sites for cloning purposes) and a 3′ extension (5′-AGATCT-3′; introducing a BgIII restriction site for cloning), and ligated into vector pUC57 (GenScript, Piscataway, N.J., USA), generating plasmid pJZ191. The ACT fragment covers nucleotides −49 to +248 of the 5′UTR and coding region of the β-actin cDNA, the SHR fragment covers nucleotides+179 to +398 of the coding region of the Shrub cDNA.
[0178] To assemble the ptDP constructs for dsRNA expression from convergent promoters, two copies of the plastid Prrn promoter were amplified. One copy was amplified with primer pair Prrn(HindIII)-F/Prrn(SphI)-R, introducing HindIII and SphI restriction sites with the primer sequences (Table 1). The PCR product was cloned as HindIII/SphI fragment into the similarly cut cloning vector pUC19, generating plasmid pJZ11. Subsequently, the ACT+SHR fragment was excised from pJZ191 as SphI/BgIII fragment and cloned into pJZ11 digested with SphI and BamHI, resulting in plasmid pJZ19. The second Prrn promoter copy was amplified using primer pair Prrn(EcoRI)-F/Prrn(SacI)-R (Table 1). The PCR product was digested with EcoRI and SacI, and cloned into the similarly cut plasmid pJZ19, producing plasmid pJZ193. The dsRNA expression cassette was then excised from pJZ193 as EcoRI/HindIII fragment and subcloned into pBluescript KS(−) digested with the same enzymes, resulting in plasmid pJZ197. Finally, the dsRNA cassette was excised from pJZ197 as NotI/XhoI fragment and inserted into the similarly cut plastid transformation vector pJZ100, producing vector pJZ199. ACT and SHR gene fragments were obtained by PCR amplification with primer pairs actin(SbfI)-F/actin(SacI)-R and shrub(SbfI)-f/shrub(SacI)-R, respectively (Table 1), using plasmid pJZ191 as template. The resulting PCR products were digested with SbfI and SacI, and cloned into the similarly cut vector pJZ199 to replace with ACT or SHR, generating plastid transformation vectors pJZ237 and pJZ238, respectively. To assemble the ptSL construct (designed to express dsRNAs with flanking stem-loop structures), one of the two Prrn promoter copies (including a sequence folding into a 24 bp stem-loop structure at the RNA level) was amplified using primers Prrn(HindIII)-F and PrrnSL1 (PstI)-R (Table 1). The resulting PCR product was digested with HindIII and PstI and ligated into the similarly cut cloning vector pUC19, generating plasmid pJZ10. Subsequently, the ACT+SHR fragment was excised from pJZ191 as PstI/BamHI fragment and cloned into the similarly cut pJZ10, producing plasmid pJZ14. The second Prrn promoter copy (also including a sequence folding into a 24 bp stem-loop structure at the RNA level) was amplified with primer pair Prrn(EcoRI)-F/PrrnSL2 (BamHI)-R (Table 1). The PCR product was digested with EcoRI and BamHI and ligated into pJZ14 cut with the same enzyme combination, resulting in plasmid pJZ192. The dsRNA-SL expression cassette was then excised from pJZ192 as EcoRI/HindIII fragment and subcloned into pBluescript KS(−), generating plasmid pJZ196. Finally, the dsRNA-SL cassette was excised from pJZ196 as NotI/XhoI fragment and inserted into plastid transformation vector pJZ100, generating vector pJZ200.
[0179] To assemble the ptHP construct for dsRNA expression as a hairpin RNA structure, the first intron from the potato gibberellin 20 (GA20) oxidase gene was excised from a plasmid clone (pUC-RNAi; 26) as PstI/BamHI fragment and inserted into the similarly cut vector pJZ11, generating plasmid pJZ158. The rrnB terminator (TrrnB) from Escherichia coli was amplified with primer pair TrrnB(SacI)-F/TrrnB(EcoRI)-R (Table 1), using plasmid pNtcC1-TrrnB (27) as template. The obtained PCR product was cloned as SacI/EcoRI fragment into pJZ158, producing plasmid pJZ171. The ACT+SHR sequence was excised from pJZ191 as SphI/BgIII fragment and cloned into the similarly cut pJZ171, generating pJZ194. A second copy of the ACT+SHR sequence was amplified with primer pair act+shr(SacI)-F/act+shr(SmaI)-R (Table 1). The PCR product was cloned (in antisense orientation) as SacI/SmaI fragment into the similarly cut pJZ194, generating plasmid pJZ216. The hpRNA expression cassette was subsequently excised from pJZ216 as EcoRI/HindIII fragment and subcloned into pBluescript KS(−), generating plasmid pJZ219. Finally, the hpRNA cassette was excised from pJZ219 as NotI/XhoI fragment and inserted into plastid transformation vector pJZ100, generating vector pJZ222.
[0180] For expression of hairpin-type dsRNAs in the nucleus (nuHP constructs), the ACT and SHR fragments were amplified with primer pairs actin(XbaI)-F/actin(BgIII)-R and shrub(XbaI)-F/shrub(BamHI)-R, respectively (Table 1). The ACT PCR product was cloned as XbaI/BgIII fragment into vector pUC-RNAi (26) cut with XbaI and BamHI, generating plasmid pJZ249. The SHR PCR product was cloned as XbaI/BamHI fragment into the similarly cut vector pUC-RNAi, producing plasmid pJZ250. The second ACT fragment was amplified with primers actin(XhoI)-F and actin(BgIII)-R (Table 1), and ligated as XhoI/BgIII fragment (in antisense orientation) into the similarly disgested vector pJZ249, generating plasmid pJZ251. The second SHR fragment was amplified with primer pair shrub(XhoI)-F/shrub(BamHI)-R (Table 1). The obtained PCR product was then cloned (in antisense orientation) as XhoI/BamHI fragment into vector pJZ250 that had been digested with XhoI and BgIII, generating plasmid pJZ252. Finally, the ACT and SHR sequences were excised as XhoI/XbaI fragments from pJZ251 and pJZ252, respectively, and cloned into vector pEZR(H)-LN (a kind gift from Dr. Staffan Persson, MPI-MP) cut with Sail and XbaI, generating nuclear transformation vectors pJZ253 and pJZ254. The ACT+SHR sequence was excised as PstI/SacI fragment from pJZ216, followed by blunting with Klenow enzyme and cloning into the SmaI/XbaI digested and blunted vector pEZR(H)-LN. A clone was selected in which the GA20 intron has the same orientation as the CaMV35S promoter, yielding nuclear transformation vector pJZ202.
Construction of Plasmid Vectors for In Vitro Transcription
[0181] To construct vectors for in vitro synthesis of ssRNA, the ACT+SHR sequence was excised from pJZ191 as PstI/BamHI fragment and ligated into the similarly cut cloning vector pBluescript KS(−), resulting in plasmid pKS_ACT+SHR. The ACT sequence was amplified with primer pair actin(XhoI)-F/actin(BgIII)-R (Table 1). The PCR product was digested with XhoI and BgIII, and cloned into pBluescript KS(−) cut with XhoI and BamHI, generating plasmid pKS_ACT. The SHR sequence was amplified with primer pair shrub(XhoI)-F/shrub(BamHI)-R (Table 1). The PCR product was digested with XhoI and BamHI and cloned into the similarly cut pBluescript KS(−), generating plasmid pKS_SHR.
Plastid and Nuclear Transformation
[0182] For tobacco plastid transformation, young leaves from plants grown under aseptic conditions were bombarded with plasmid DNA-coated gold particles using a PDS1000/He particle delivery system equipped with a Hepta adaptor (BioRad, Hercules, Calif., USA). Primary spectinomycin-resistant lines were selected on RMOP medium containing 500 mg/L spectinomycin (28). For each construct, several independent transplastomic lines were subjected to two additional rounds of regeneration on spectinomycin-containing medium to select for homoplasmy.
[0183] For potato plastid transformation, a published protocol (13) was slightly modified. The basic media (BM) for potato regeneration contained MS salts supplemented with B5 vitamins (pH adjusted to 5.7), and were solidified with 0.6% Micro agar (Duchefa). Medium StM1 consists of BM, 3% sucrose, 0.1 M sorbitol and 0.1 M mannitol. Medium StM2 contains BM, 3% sucrose, 2 mg/L 2,4-diclorophenoxyacetic acid (2,4 D), 0.8 mg/L zeatin riboside and 400 mg/L spectinomycin. Medium StM3 contains BM, 1.6% glucose, 2 mgl/L indole-3-acetic acid (IAA), 3 mg/L zeatin riboside, 1 mg/L gibberellic acid (GA3) and 400 mg/L spectinomycin. Medium StM4 contains BM, 3% sucrose, 0.1 mg/L IAA, 3 mg/L zeatin riboside and 400 mg/L spectinomycin. For transformation, young leaves from aseptically grown potato plants were incubated for 24 h on StM1 medium in the dark. After biolistic transformation, leaves were incubated for up to 1 day in the dark, then cut into pieces of 3×3 mm, transferred to StM2 medium and incubated under dim light (˜10 μmol photons m.sup.−2 s.sup.−1) in a 16 h light/8 h dark regime for 1 month. Subsequently, the leaf pieces were transferred to StM3 medium and subcultured every 4 weeks until resistant calli or shoots appeared. Resistant material was transferred to StM4 medium, and incubated for 1 to 3 months to induce shoot regeneration and multiplication. To stimulate rooting, regenerated shoots were transferred to MS medium with 3% sucrose and 400 mg/L spectinomycin. Finally, rooted plantlets were transferred to soil and grown to maturity. Homoplasmy was confirmed by Southern blotting.
[0184] Nuclear transgenic potato plants were generated by Agrobacterium-mediated transformation (29). Transgenic plants were identified by hygromycin selection and initially tested for the presence of the transgene by PCR assays. The transgenic status was further confirmed by RNA gel blot analyses.
Isolation of Plant Nucleic Acids and Gel Blot Analysis
[0185] Total DNA from tobacco or potato plants was extracted from young leaves of soil-grown plants by a cetyltrimethylammonium bromide (CTAB)-based method (30). For DNA gel blot analysis, samples of 5 μg of total cellular DNA were digested with the restriction enzyme BgIII, separated by gel electrophoresis in 0.8% agarose gels and transferred onto Hybond nylon membranes (GE Healthcare, Buckinghamshire, UK) by capillary blotting. A 550 bp PCR product generated by amplification of a portion of the psaB coding region (31) was used as RFLP probe to verify plastid transformation and assess the homoplasmic status of transplastomic lines.
[0186] For RNA gel blot analysis, total cellular RNA was extracted using the peqGOLD TriFast reagent (Peqlab, Erlangen, Germany) from leaf samples of soil-grown tobacco or potato plants. Total RNA from potato tubers was isolated with the NucleoSpin RNA Plant kit (Macherey-Nagel, Duren, Germany) following the instructions of the supplier. RNA samples were separated by electrophoresis in 1% formaldehyde-containing agarose gels and blotted onto Hybond nylon membranes (GE Healthcare). For siRNA analysis, samples of 20 μg of total cellular RNA were separated in 14% polyacrylamide gels with 0.3 M sodium acetate and 7 M urea as gel buffer and 0.3 M sodium acetate (pH 5.0) as running buffer. The separated RNA samples were electroblotted onto Hybond nylon membranes in blotting buffer (10 mM Tris-acetate pH 7.8, 5 mM sodium acetate, 0.5 mM EDTA) at 40 V for 2 h at 4° C. (32) and subsequently cross-linked to the membrane by UV light.
[0187] PCR products generated by amplification with gene-specific primers were used as hybridization probes. [α.sup.32P]dCTP-labeled probes were generated using the Multiprime DNA labeling system (GE Healthcare). Hybridizations were performed at 65° C. for standard Southern and northern blots and at 42° C. for siRNA blot analysis.
In Vitro RNA Synthesis
[0188] For ssRNA synthesis by in vitro transcription from plasmids, 1 μg of plasmid DNA from clones pKS_ACT+SHR, pKS_ACT and pKS_SHR was linearized with XbaI. The linearized DNA fragments were purified using the NucleoSpinR Gel and PCR clean-up kit (Macherey-Nagel). In vitro transcription reactions were performed with T3 RNA polymerase (Thermo Scientific, Waltham, Mass., USA) following the manufacturer's instructions. The RNA yield was determined with a NanoDrop ND-1000 spectrophotometer.
[0189] In vitro synthesis of dsRNAs for insect feeding assays was carried out with the T7 RiboMAX™ express RNAi system (Promega, Mannheim, Germany) according to the manufacturer's protocol. pJZ191 plasmid DNA was used to amplify templates for in vitro transcription. The minimal T7 promoter sequence (5′-TAATACGACTCACTATAGG-3′) was added to the 5′ end of forward and reverse primers (Table 1).
Insect Bioassays
[0190] A strain of Colorado potato beetle (Leptinotarsa decemlineata) was kindly provided by the Julius Kuhn Institute, Federal Research Centre for Cultivated Plants, Kleinmachnow, Germany. The insects were reared in the lab on wild-type potato plants (Solanum tuberosum L., cv. Delana or Desiree). CPB larvae were hatched from eggs, and neonates were reared on potato leaves at 26° C. under a 16 h light/8 h dark cycle.
[0191] To obtain standardized larvae for growth and survival assays on transplastomic and transgenic potato plants, CPBs were fed on wild-type potato plants and adults were allowed to lay eggs. The eggs were collected and transferred onto fresh wild-type potato leaves for hatching. First instar larvae were allowed to feed on young leaves of two-month old transplastomic or transgenic potato plants and wild type plants as a control. For each feeding experiment, synchronized groups of larvae were selected, weighed individually and divided into three groups (each group containing 10-20 individuals and serving as a biological replicate). After feeding on detached potato leaves for 3, 5, 7 and 9 days, larvae were weighed, and midgut and carcass tissues were taken from dissected larvae for further analysis. Similarly, adult CPBs were used to feed on transplastomic plants (St-ptDP-ACT). To calculate the consumed leaf area, the leaves were photographed before and after feeding by CPB and the consumed area was determined using the Sigma Scan Pro5 software. Statistical analysis was performed with one-way ANOVA (SPSS software) and results are presented as means±standard deviation.
[0192] For larval performance assays onto potato leaves painted with dsRNA, in vitro synthesized dsRNA was painted on young potato leaves in defined amounts per leaf area. To this end, fresh potato leaflets were arranged in a circle of about 23 cm.sup.2 surface area and dsRNA (diluted in water) was painted onto the leaf surface to final concentrations of 4, 8 or 16 ng per cm.sup.2. Second instar larvae were weighed after 0, 3, 5, 7 and 9 days of feeding on dsRNA-painted leaves. The larvae were divided into three groups (for three biological replicates) and each group had 10-20 larvae per treatment. dsRNA derived from the gfp coding region was used as a control. The leaves were replaced with fresh dsRNA-painted leaves every 24 hours.
RNA Extraction from CPB Larvae
[0193] Larvae were dissected in ice-cold Schneider fs insect medium (Sigma-Aldrich, St. Louis, Mo., USA). Larval gut, Malpighian tubules and the rest of the body were isolated as described previously (33, 34) and placed in 100 μl of ice-cold Schneider's medium in separate Eppendorf tubes. Immediately after collection, the tissues were flash-frozen in liquid nitrogen and stored at −80° C. until use. RNA was extracted from tissue samples with Trizol (Invitrogen, Carlsbad, Calif., USA) following the manufacturer's instructions. RNA integrity and quantity were checked on an Agilent 2100 Bioanalyzer using the RNA Nano chips (Agilent Technologies, Santa Clara, Calif., USA). RNA was then precisely quantified with a NanoDrop ND-1000 spectrophotometer.
Quantitative Real-Time PCR (qRT-PCR)
[0194] qRT-PCR was used to assess transcript levels of ACT and SHR in gut tissues. The software Primer-3 (http://frodo.wi.mit.edu/) was used to design the primers for qPCR analysis (for primer sequences, see Table 1). Reverse transcription reactions were performed with 500 ng of total RNA and oligo d(T) primer using the First Strand cDNA Synthesis kit (Fermentas) according to the manufacturer's protocol. qRT-PCR was done in optical 96-well plates on a MX3000P Real-Time PCR Detection System (Stratagene) using the ABsolute qPCR SYBR Green Mix (Thermo Scientific) to monitor double-stranded DNA synthesis in combination with ROX Passive Reference Dye. Amplification conditions were 10 min at 95° C., followed by 40 cycles at 95° C. for 30 s, 60° C. for 30 s and 72° C. for 30 s. Melt curve analysis was performed in order to assess the specificity of amplification. Results were normalized to the mRNA levels of the CPB genes encoding ribosomal protein S18 (RPS18) and ribosomal protein S4 (RPS4) as housekeeping genes (Table 1), and relative mRNA accumulation levels were calculated according to the delta-delta Ct method. Each experiment was repeated with three independently isolated mRNA samples (biological replicates), and each reaction was repeated 3 times to minimize intra-experiment variation (technical replicates). All results were analyzed with the qBase software.
Histological Analysis of Actin Filaments in Larval Tissues
[0195] Third instar larvae fed on wild type and transplastomic potato plants (St-ptDP-ACT) were dissected on microscopic slides and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). Longitudinal cross sections of midgut (MG) tissue, hindgut (HG) tissue and Malpighian tubules (MT) were prepared according to published procedures (35). The larvae were processed for tissue preparation after 24, 48 and 96 h of feeding. The cross sections were incubated with 0.165 μM Alex FluorR 488 phalloidin (Invitrogen) in PBS containing 1% BSA for 20 min. After incubation, the samples were washed with PBS three times. The fluorescence of stained actin was viewed in a confocal laser-scanning microscope (TCS SP5; Leica, http://www.leica.com). The excitation wavelength was 488 nm and the barrier filter BP 530 (band pass, 515-545 nm) was used.
EXAMPLE 2: IN VIVO EVALUATION OF THE STRATEGY FOR DSRNA PRODUCTION IN THE PLASTIDS OF TOBACCO PLANTS—COMPARISON OF RNAI RESPONSES
[0196] To test the feasibility of stable dsRNA expression in plastids, we transformed the tobacco (Nicotiana tabacum) plastid genome with three different types of dsRNA constructs (
[0197] As insect target genes, the ACT and SHR genes from the Colorado potato beetle (Leptinotarsa decemlineata; CPB), a notorious insect pest of potato and other Solanaceous plants, were chosen based on their high efficacy in inducing mortality in feeding assays with in vitro-synthesized dsRNAs (12, 3). ACT encodes β-actin, an essential cytoskeletal protein, and SHR encodes Shrub (also known as Vps32 or Snf7), an essential subunit of a protein complex involved in membrane remodeling for vesicle transport. To also test longer dsRNAs and test for a possible synergistic action, we additionally produced an ACT+SHR fusion gene. To preliminarily compare the RNAi responses to these dsRNAs in CPBs, we synthesized dsRNAs (ACT, SHR, ACT+SHR fusion and GFP as a control) by in vitro transcription and painted them onto young potato leaves. Second instar CPB larvae were then allowed to feed on these leaves for up to 9 days. All three insect gene-derived dsRNAs strongly reduced the growth of CPB larvae (
[0198] The initial in vivo evaluation of the three strategies for dsRNA production (ptDP, ptSL and ptHP constructs;
[0199] To test if dsRNAs stably accumulate in chloroplasts, northern blot analyses were performed. The results revealed that all three types of expression constructs triggered production of substantial amounts of long dsRNAs (
EXAMPLE 3: STABLE PLASTID TRANSFORMATION IN POTATO—LONG DSRNAS ACCUMULATE TO HIGH LEVELS IN LEAVES OF TRANSPLASTOMIC POTATO LINES
[0200] We next optimized a protocol for biolistic plastid transformation in potato (13; see Example 1: Materials and Methods), the main host plant of CPB. The three target gene constructs (ACT, SHR and ACT+SHR, integrated into the ptDP cassette;
[0201] Northern blot analyses of transplastomic potato lines revealed that the accumulation levels of ACT dsRNAs were higher than those of SHR and ACT+SHR dsRNAs (
[0202] Since CPB larvae and beetles feed on leaves but not on below ground potato tubers, only the leaves need to be protected from herbivory. The expression of most plastid genes is drastically down-regulated in non-photosynthetic tissues (14, 15), which made it possible to prevent dsRNA production in the tuber where the accumulation of transgene-derived RNA is unnecessary and perhaps also undesired by the consumer. Comparative analyses of dsRNA accumulation in leaves and tubers revealed that indeed, dsRNA levels in tubers are nearly undetectably low (
EXAMPLE 4: DSRNA PRODUCTION IN THE CHLOROPLASTS OF POTATO PLANTS OFFERS PROTECTION AGAINST CPB
[0203] Having established that long dsRNAs accumulate to high levels in leaves of transplastomic potato lines, we next tested whether dsRNA production in the chloroplast offers protection against CPB. To this end, the mortality of first instar CPB larvae was determined upon feeding on detached leaves from wild-type, transplastomic and transgenic potato plants for 9 consecutive days (
[0204] To confirm that the killing of the CPB larvae by the transplastomic plants was due to induction of RNAi, expression of the target genes was determined in the gut of CPB larvae after three days of feeding (i.e., when the larvae fed on the transplastomic plants were still alive). Already at this early stage, expression of β-actin and Shrub was strongly suppressed in the insects (
[0205] CPB resistance of transplastomic potato plants was further assessed by determining the leaf area consumed by CPB larvae and adult beetles. Almost no visible consumption of leaf biomass occurred in St-ptDP-ACT leaves (
[0206] Our data reported here underscore the importance of producing large amounts of long dsRNAs to achieve efficient plant protection. While transplastomic ACT dsRNA-expressing plants cause 100% lethality to CPB larvae, SHR dsRNA-expressing plants are somewhat less efficient (70% mortality after 9 days;
EXAMPLE 5: PLASTID-EXPRESSED ACT DSRNA SILENCES THE ACTIN GENE IN CPB
[0207] To ultimately confirm that the plastid-expressed ACT dsRNA silences the actin gene in CPB larvae, we examined actin filaments in the larval midgut, hindgut and Malpighian tubules by staining with FITC-labeled phalloidin. Already after 1-2 days of feeding on transplastomic leaves, the larvae displayed disorganized actin filaments, which were particularly obvious in the columnar cells of the midgut (
[0208] Moreover, accumulation of ACT-derived siRNAs was detected in gut tissue of larvae fed with transplastomic leaves, whereas accumulation in larvae fed with nuclear transgenic leaves was below the limit of reliable detection (
[0209] The present invention refers to the following nucleotide sequences:
TABLE-US-00001 SEQ ID No. 1: Nucleotide sequence of the expression cassette of the construct Nt-ptDP- ACT + SHR, the sequences which encode the dsRNA representing the CPB ACT and SHR genes are underlined: gctcccccgccgtcgttcaatgagaatggataagaggctcgtgggattgacgtgagggggcagggatggctatattt ctgggagcgaactccgggcgaatacgaagcgcttggatacgcatgcctgcaggcacgaggtttttctgtctagtgag cagtgtccaacctcaaaagacaacatgtgtgacgacgatgtagcggctcttgtcgtagacaatggatccggtatgtg caaagccggtttcgcaggagatgacgcaccccgtgccgtcttcccctcgatcgtcggtcgcccaaggcatcaagg agtcatggtcggtatgggacaaaaggactcatacgtaggagatgaagcccaaagcaaaagaggtatcctcaccc tgaaataccccatcgaacacggtatcatcaccaactgggatgacatgcaatgtcatccatcatgtcgtgtacattgtc cacgtccaagtttttatcgctttcttaagagcttcagctgcatttttcatagattccaatactgtggtgttcgtactagctcc ctccagagcttctcgttgaagttcaatagtagttaaagtgccatctatttgcaactgatttttttctaatcgcttcttccgcttc agcgcttgcatggccgctcagatccccgggtaccgagctcgtatccaagcgcttcgtattcgcccggagttcgctccc agaaatatagccatccctgccccctcacgtcaatcccacgagcctcttatccattctcattgaacgacggcggggga gc SEQ ID No. 2: Nucleotide sequence of the expression cassette of the construct Nt-ptSL-ACT + SHR, the sequences which encode the dsRNA representing the CPB ACT and SHR genes are underlined: gctcccccgccgtcgttcaatgagaatggataagaggctcgtgggattgacgtgagggggcagggatggctatattt ctgggagcgaactccgggcgaatacgaagcgcttggatacgcatgcgggtgggtggaaaaccacccacccctg caggcacgaggtttttctgtctagtgagcagtgtccaacctcaaaagacaacatgtgtgacgacgatgtagcggctct tgtcgtagacaatggatccggtatgtgcaaagccggtttcgcaggagatgacgcaccccgtgcggtcttcccctatat cgtcggtcgcccaaggcatcaaggagtcatggtcctatcgacaaaaggactcatacgtaggagatgaagccc aaagcaaaagaggtatcctcaccctgaaataccccatcgaacacctatcatcaccaactcgatgacatgcaat gtcatccatcatgtcgtgtacattgtccacgtccaagtttttatgggctttcttaagagcttcagctgcatttttcatagattcc aatactgtggtgttcgtactagctccctccagagcttctcgttgaagttcaatagtagttaaagtgccatctatttgcaact gatttttttctaatcgcttcttccgcttcagcgcttgcatggcccctcagatcccgcacgctcctaatggagcgtgcggtat ccaagcgcttcgtattcgcccggagttcgctcccagaaatatagccatccctgccccctcacgtcaatcccacgagc ctcttatccattctcattgaacgacggcgggggagc SEQ ID No. 3: Nucleotide sequence of the expression cassette of the construct Nt-ptHP-ACT + SHR, the sequences which encode the dsRNA representing the CPB ACT and SHR genes are underlined: gctcccccgccgtcgttcaatgagaatggataagaggctcgtgggattgacgtgagggggcagggatggctatattt ctgggagcgaactccgggcgaatacgaagcgcttggatacgcatgcctgcaggcacgaggtttttctgtctagtgag cagtgtccaacctcaaaagacaacatgtgtgacgacgatgtagcggctcttgtcgtagacaatggatccggtatgtg caaagccggtttcgcaggagatgacgcaccccgtgccgtcttcccctcgatcgtcggtcgcccaaggcatcaagg agtcatggtcctatcgacaaaaggactcatacgtaggagatgaagcccaaagcaaaagaggtatcctcaccc tgaaataccccatcgaacacggtatcatcaccaactgggatgacatgcaatgtcatccatcatgtcgtgtacattgtc caccttccaagtttttatgggctttcttaagagcttcagctgcatttttcatagattccaatactgtggtgttcgtactagctcc ctccagagcttctcgttgaagttcaatagtagttaaagtgccatctatttgcaactgatttttttctaatcgcttcttccgcttc agcgcttgcatggccctctcagatctggtacggaccgtactactctattcgtttcaatatatttatttgtttcagctgactgca agattcaaaaatttctttattattttaaattttgtgtcactcaaaaccagataaacaatttgatatagaggcactatatatat acatattctcgattatatatgtaaatgagttaaccttttttttccacttaaaattatatagggggatccccggggagcccc atgcaagcgctgaagcggaagaagcgattagaaaaaaatcagttgcaaatagatggcactttaactactattgaa cttcaacgagaagctctggagggagctagtacgaacaccacagtattggaatctatgaaaaatgcagctgaagct cttaagaaagcccataaaaacttggacgtggacaatgtacacgacatgatggatgacattgcatgtcatcccagttg gtgatgatactttgttcgatggggtatttcagggtgaggatacctcttttgctttgggcttcatctcctaCgtatgagtccttt tgtcccataccgaccatgactccttgatgccttgggcgaccgacgatcgaggggaagacggcacggggtgcgtca tctcctgcgaaaccggctttgcacataccggatccattgtctacgacaagagccgctacatcgtcgtcacacatgttgt cttttgaggttggacactgctcactagacagaaaaacctcgtgcgagctcatcaaataaaacgaaaggctcagtcg aaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggattt gaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaa gcagaaggccatcctgacggatggcctttttgcgtttctac SEQ ID No. 4: Nucleotide sequence of the expression cassette of the construct St-ptDP-ACT, the sequence which encodes the dsRNA representing the CPB ACT gene is underlined: gctcccccgccgtcgttcaatgagaatggataagaggctcgtgggattgacgtgagggggcagggatggctatattt ctgggagcgaactccgggcgaatacgaagcgcttggatacgcatgcctgcaggcacgaggtttttctgtctagtgag cagtgtccaacctcaaaagacaacatgtgtgacgacgatgtagcggctcttgtcgtagacaatggatccggtatgtg caaagcccgtttcgcaggagatgacgcccccgtcttgcccctcttcccctcctatcgtcggtcgcccaaggcatcaagg agtcatggtcctatcgacaaaaggactcatacgtaggagatgaagcccaaagcaaaagaggtatcctcaccc tgaaataccccatcgaacacccgtatcatcaccaactgggatgacatgagctcgtatccaagcgcttcgtattcgccc ggagttcgctcccagaaatatagccatccctgccccctcacgtcaatcccacgagcctcttatccattctcattgaacg acggcgggggagc SEQ ID No. 5: Nucleotide sequence of the expression cassette of the construct St-ptDP-SHR, the sequence which encodes the dsRNA representing the CPB SHR gene is underlined: gctcccccgccgtcgttcaatgagaatggataagaggctcgtgggattgacgtgagggggcagggatggctatattt ctgggagcgaactccgggcgaatacgaagcgcttggatacgcatgcctgcaggcaatgtcatccatcatgtcgtgt acattgtccacgtccaagtttttatgggctttcttaagagcttcagctgcatttttcatagattccaatactgtggtgttcgtac tagctccctccagagcttctcgttgaagttcaatagtagttaaagtgccatctatttgcaactgatttttttctaatcgcttctt cccgcttcaggtcttqcatctttccctctcgagctcgtatccaagcgcttcgtattcgcccggagttcgctcccagaaatat agccatccctgccccctcacgtcaatcccacgagcctcttatccattctcattgaacgacggcgggggagc SEQ ID No. 6: Nucleotide sequence of the expression cassette of the construct St-ptDP-ACT + SHR, the sequences which encode the dsRNA representing the CPB ACT and SHR genes are underlined: gctcccccgccgtcgttcaatgagaatggataagaggctcgtgggattgacgtgagggggcagggatggctatattt ctgggagcgaactccgggcgaatacgaagcgcttggatacgcatgcctgcaggcacgaggtttttctgtctagtgag cagtgtccaacctcaaaagacaacatgtgtgacgacgatgtagccctcttgtcgtagacaatggatccctatgtg caaagccggtttcgcaggagatgacgcaccccttgctttcttcccctcgatcgtcggtcgcccaaggcatcaagg agtcatggtcggtatgggacaaaaggactcatacgtaggagatgaagcccaaagcaaaagaggtatcctcaccc tgaaataccccatcgaacacggtatcatcaccaactgggatgacatgcaatgtcatccatcatgtcgtgtacattgtc cacccttccaagtttttatgggctttcttaagagcttcagctgcatttttcatagattccaatactgtggtgttcgtactagctcc ctccagagcttctcgttgaagttcaatagtagttaaagtgccatctatttgcaactgatttttttctaatcgcttcttccgcttc agcgcttgcatggccgctcagatccccgggtaccgagctcgtatccaagcgcttcgtattcgcccggagttcgctccc agaaatatagccatccctgccccctcacgtcaatcccacgagcctcttatccattctcattgaacgacggcggggga gc SEQ ID No. 7: Phytophthora infestans (potato blight) sequences Nucleotide sequence of the expression cassette of the construct St-ptDP-EPI + PMA, the sequences which encode the dsRNA representing the Phytophthora infestans EPI and PMA genes are underlined: gctcccccgccgtcgttcaatgagaatggataagaggctcgtgggattgacgtgagggggcagggatggctatattt ctgggagcgaactccgggcgaatacgaagcgcttggatacgcatgcctgcagaaagaaggaagtcacgccaga ggacacggagctgctccagaaggcgcagagcaatgtgagcgcatacaacagcgacgtcacctcgcgcatctgct acctgaaggtcgacagtctcgagactcaagtcgtctccgcgagaactacaagttccacgtttcccttgcagcgtc aactccacaaggagctcgcggctgtgccaatcagaattgcgagtcatccaagtacgacatcgtcatctactcgc agtcgtggaccaacacgctgaaggtgacgtcgattacgcccgccaacgctggtgccgcaggtaactcgtacatgtc catggcgacgcccaacgacgtcaagaactacacgaacgacgttggccagatccagtgggcgcaggtgccgctg aacgccgcgcttgacaagctcaagtcgtcccgtgagggtctgacatccgatgaggctgagaagcgtctggccgag tacggcccgaacaagctgccggaggagaaggtgaacaagctgacgctgttcctgggcttcatgtggaacccgctg tcgtgggccatggaggtggccgctttctgtcgattgtgctgctggattacctctgatttcgcgctgatcctgttcctgctg ctgctaaacagatctcccgggtaccgagctcgtatccaagcgcttcgtattcgcccggagttcgctcccagaaatata gccatccctgccccctcacgtcaatcccacgagcctcttatccattctcattgaacgacggcgggggagc
[0210] The Present Invention Refers to the Following Tables:
TABLE-US-00002 TABLE 1 List of oligonucleotides used in the context of the invention. Recognition sequences of introduced restriction sites are underlined. The T7 promoter sequence is indicated in italics. SEQ ID Oligonucleotide Sequence 5′-3′ No. Description and Use Prrn(HindIII)-F AAAAAAGCTTGCTCCCCCGCCGTCGTTC 8 forward primer for amplification of the Prm promoter; introducing a HindlII site Prrn(SphI)-R AAAAGCATGCGTATCCAAGCGCTTCGTAT 9 reverse primer for amplification of the Prm promoter; introducing an SphI site Prrn(EcoRI)-F AAAAGAATTCGCTCCCCCGCCGTCGTTC 10 forward primer for amplification of the Prm promoter; introducing an EcoRI site Prrn(SacI)-R AAAAGAGCTCGTATCCAAGCGCTTCGTAT 11 reverse primer for amplification of the Prm promoter; introducing a SacI site PrrnSL1(PstI)-R AAAACTGCAGGGGTGGGTGGTTTTCCACCC 12 forward primer for amplification of the Prm promoter; introducing a PstI site and stem- ACCCGTATCCAAGCGCTTCGTAT loop sequence 1 PrrnSL2(BamHI)-R AAAAGGATCCCGCACGCTCCATTAGGAGCGT 13 reverse primer for amplification of the Prm promoter; introducing a BamHI site and GCGGTATCCAAGCGCTTCGTAT stem-loop sequence 2 actin(SbfI)-F AAACCTGCAGGCACGAGGTTTTTCTGTCTAG 14 forward primer for amplification of the ACT gene fragment; introducing an Sbfl site actin(SacI)-R AAAAGAGCTCATGTCATCCCAGTTGGTGAT 15 reverse primer for amplification of the ACT gene fragment; introducing a Sad 1 site shrub(SbfI)-F AAAACCTGCAGGCAATGTCATCCATCATGTC 16 forward primer for amplification of the SHR gene fragment; introducing an Sbfl site G shrub(SacI)-R AAAAGAGCTCGAGCGGCCATGCAAGC 17 reverse primer for amplification of the SHR gene fragment; introducing a Sad 1 site TrrnB(SacI)-F AAAAGAG19CTCATCAAATAAAACGAAAGGCT 18 forward primer for amplification of the E. coli rmB terminator; introducing a SacI site CAGTCG TrrnB(EcoRI)-R AAAAGAATTCGTAGAAACGCAAAAAGGCCAT 19 reserve primer for amplification of the E. coli rmB terminator; introducing an EcoRI CC site act + shr(SacI)-F AAAAGAGCTCGCACGAGGTTTTTCTGTC 20 forward primer for amplification of the ACT+30SHR fragment; introducing a SacI site act + shr(SmaI)-R AAAACCCGGGAGCGGCCATGCAAGC 21 reverse primer for amplification of the ACT+30SHR fragment; introducing a SmaI site actin(XbaI)-F AAAATCTAGACACGAGGTTTTTCTGTCTAG 22 forward primer for amplification of the ACT gene fragment; introducing an XbaI site actin(XhoI)-F AAAACTCGAGCACGAGGTTTTTCTGTCTAG 23 forward primer for amplification of the ACT gene fragment; introducing an XhoI site actin(BgIII)-R AAAAGATCTATGTCATCCCAGTTGGTGAT 24 reverse primer for amplification of the ACT gene fragment; introducing a BglI site shrub(XbaI)-F AAAATCTAGACAATGTCATCCATCATGTCG 25 forward primer for amplification of the SHR gene fragment; introducing an XbaI site shrub(XhoI)-F AAAACTCGAGCAATGTCATCCATCATGTCG 26 forward primer for amplification of the SHR gene fragment; introducing an XhoI site shrub(BamHI)-R AAAAGGATCCGAGCGGCCATGCAAGC 27 reverse primer for amplification of the SHR gene fragment; introducing a BamHI site PBT7actshrFwd TAATACGACTCACTATAGGCCTGCAGGCACG 28 forward primer for amplification of the ACT+30SHR fragment introducing the T7 AGGTTTTTCTGT promoter sequence; for in vitro dsRNA synthesis PBT7actshrRev TAATACGACTCACTATAGGGGCCCGGGATCC 29 reverse primer for amplification of the ACT+30SHR fragment introducing the T7 GATATGCC promoter sequence; for in vitro dsRNA synthesis PBT7actFwd TAATACGACTCACTATAGGATGTGTGACGAC 30 forward primer for amplification of the ACT fragment; introducing the T7 promoter GATGTAGCG sequence; for in vitro dsRNA synthesis PBT7actRev TAATACGACTCACTATAGGTTCCATGTCATCC 31 reverse primer for amplification of the ACT fragment; introducing the T7 promoter CAGTTGG sequence; for in vitro dsRNA synthesis PBT7 shrubFwd TAATACGACTCACTATAGGGAGTGGCCCTGC 32 forward primer for amplification of the SHR fragment; introducing the T7 promoter AAGCCCTCAA sequence; for in vitro dsRNA synthesis PBT7shrubRev TAATACGACTCACTATAGGGCAATGTCATCC 33 reverse primer for amplification of the SHR fragment; introducing the T7 promoter ATCATGTC sequence; for in vitro dsRNA synthesis T7GFPfwd TAATACGACTCACTATAGGAGGACGACGGCA 34 forward primer for amplification of the gfp gene; introducing the T7 promoter ACTACAAG sequence; for in vitro dsRNA synthesis T7GFPrev TAATACGACTCACTATAGGCTGGGTGCTCAG 35 reverse primer for amplification of the gfp gene; introducing the T7 promoter GTAGTGGT sequence; for in vitro dsRNA synthesis PBactinReTiFwd CCAGTCCTCCTCACTGAAGC 36 forward primer for gRT-PCR analysis of ACT expression PBactinReTiRev ACGACCAGAAGCGTACAAGG 37 reverse primer for gRT-PCR analysis of ACT expression PBshrubReTiFwd GATGATTTGGACGATGCTGA 38 forward primer for gRT-PCR analysis of SHR expression PBshrubReTiRev TAGCTGGTTTGACTGGCTTG 39 reverse primer for gRT-PCR analysis of SHR expression PBReTiRps18Fwd GCGGGAGAATGTACAGAGGA 40 forward primer for gRT-PCR analysis of RPS18 expression (as reference gene) PBReTiRps18Rev AAGTCTTCACGGAGCTTGGA 41 reverse primer for gRT-PCR analysis of RPS18 expression (as reference gene) PBRP4ReTiFwd CGTCAAAGAAACGAGCATTG 42 forward primer for gRT-PCR analysis of RPS4 expression (as reference gene) PBRP4ReTiRev TCGCTGACACTGTAGGGTTG 43 reverse primer for gRT-PCR analysis of RPS4 expression (as reference gene)
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