[0002]Method for producing eukaryotic organisms with enhanced pathogen resistance and/or resistance to stress and eukaryotic transgenic organisms with enhanced pathogen resis- tance and/or resistance to stress
[0003]The present invention relates to a method for producing eukaryotic organisms having enhanced pathogen resistance and/or resistance to stress as well as the respective eukaryotic transgenic organisms showing enhanced pathogen resistance and/or resistance to stress.
[0004]Organisms use a wide range of mechanisms to resist pathogens and stress, which are usually complicated and difficult to control. However, as organisms with enhanced resistance are desirable in particular in the fields of agricultural crops, useful plants or domestic animals, a method for enhancing pathogen resistance and/or resistance to stress would be of great economic inter- est.
[0005]Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) catalyses the first committed step of the oxidative pentose phosphate pathway (OPPP), an important catabolic route for the provision of NADPH and sugar phosphates. G6PDH is present in every eukaryotic organism and is nor- mally contained in the cytosol as well as in various organelles. In plants, G6PDH activity is present at least in both cytosol and plastids, and possibly also in peroxisomes. The chloroplastic enzyme is known to be reductively inactivated in the light by the ferredoxin/thioredoxin system to avoid futile interactions with the Calvin cycle during photosynthesis. The main role of G6PDH in plastids during the night or in heterotrophic tissues is the supply of reducing equiva- lents in the form of NADPH required for multiple anabolic reactions (e.g. amino acid or fatty acid synthesis). Moreover, sugar-phosphate intermediates that serve as precursors of nucleotides and secondary plant products are generated.
[0006]As mentioned above, enzyme reactions of the oxidative pentose phosphate pathway (OPPP) pro- vide reduction power for anabolic biosyntheses in the form of NADPH. This also plays a role during early defence reactions, i.e. upon elicitation of NADPH oxidase at the plasma membrane ("oxidative burst"), an early and evolutionary conserved attempt of eukaryotic cells to interfere with pathogen invasion (see Figs. 1 and 2).
It is an object of the present invention to obtain eukaryotic organisms having enhanced fitness, i.e. enhanced pathogen resistance and/or resistance to stress.
[0007]This object is solved by a method according to claim 1 as well as a transgenic organism accord- ing to claim 10.
[0008]Accordingly, the present invention is directed to a method for producing eukaryotic organisms having enhanced pathogen resistance and/or resistance to stress, comprising the steps of:
[0009]Expressing in the cytosol of said organism a glucose-6-phosphate dehydrogenase with increased NADPH tolerance compared to the endogenous cytosolic glucose-6-phosphate dehydrogenase, and reducing, eliminating or suppressing the activity of the endogenous cytosolic glucose-6- phosphate dehydrogenase.
[0010]In this context, increased NADPH tolerance means that the K,[NADPH] of the expressed G6PDH is enhanced compared to the endogenous cytosolic G6PDH. Furthermore, stress means abiotic as well as biotic stress and shall include every kind of stress which is always characterized by a primary oxidative burst at the plasma membrane. In present invention, resistance to stress shall also include stress tolerance.
[0011]The present inventors surprisingly found that when they introduced and expressed a glucose-6- phosphate dehydrogenase having increased NADPH tolerance in the cytosol of for example transgenic plants (a so-called ectopic expression), these organisms showed enhanced pathogen resistance as well as resistance to other stresses. In particular, the replacement of cytosolic G6PDH by a kinetically superior isoenzyme was able to enhance resistance in the progeny of a susceptible plant variety.
[0012]While overexpression of G6PDH in chloroplasts or the expression of hundreds of different enzymes including G6PDH in various organisms is generally described in the prior art (see for example Debnam et al., Plant Journal, vol. 38, no. 1, 2004, pages 49-59, and WO 03/000898 A or WO 02/16655 A), the expression of a G6PDH having increased NADPH tolerance in the cytosol of an eukaryotic organism with a concurrent reduction, elimination or suppression of the activity of the endogenous cytosolic G6PDH has not been published before the priority date of the present invention.
As a further surprise, the present inventors found that the inventive organisms with a replacement of cytosolic G6PDH by a kinetically superior isoenzyme showed an increased harvest yield as shown by increased 1000-grain weights (see Fig. 5 and Table 2).
[0013]In addition to producing complete organisms, the inventive method may also be used to produce only parts of an organism, e.g. organs, tissues or cells. Therefore, for the purpose of the present application the term "organism" is meant to also include parts of an organism as mentioned above. While complete organisms are nonhuman organisms, the parts of an organism also include human parts. As parts of an organism, cells are particularly preferred.
[0014]Preferably, the G6PDH with increased NADPH tolerance is an exogenous G6PDH, i.e. an isoenzyme from a different organism. However, if available, it is also possible to introduce and express a non-cytosolic G6PDH with increased NADPH tolerance from the same organism. Furthermore, the G6PDH with increased NADPH tolerance may be a mutated natural, an artificial and/or a genetically engineered modified enzyme.
[0015]In this regard, the present inventors found that the improvement in pathogen resistance and resistance to stress increased when the phylogenetic relationship was more distant (Solanaceae versus Arabidopsis G6PDH: 75% identity at the amino acid level).
[0016]In a further preferred embodiment of the present invention, the kinetic data of the G6PDH with increased NADPH tolerance fulfil the following relationship: K,[NADPH] > Km[NADP+], preferably K1[NADPH] ≥ 2 x K01[NADP+], in particular KI[NADPH] ≥ 5 X Km[NADP+] and most preferred KltNADPH] ≥ 10 x Km[NADP+]- In this regard, it is particularly preferred to use a genetically engineered enzyme fulfilling the relationship K,[NADPH] ≥ 10 X Km[NADP+]-
[0017]In the context of the present specification, K,[NADPHJ and Km[NADP+] are empirically determined kinetic enzyme parameters. Methods for determining these parameters are standard methods known to a person skilled in the art. As an example, these parameters may be determined as described in Wendt et al. (2000) for potato G6PDH and in Wakao & Benning (2005) for Arabidopsis isoenzymes.
Furthermore, it is also preferred that the G6PDH with increased NADPH tolerance is an extra- cytoplasmic G6PDH (e.g. plastidic or peroxisomal) that naturally shows these kinetic properties. Particularly preferred in this regard is one of the two plastidic P2 isoenzymes (and possibly also the Pl isoform) derived from Arabidopsis, which is described in more detail below.
[0018]In another preferred embodiment, the G6PDH with increased NADPH tolerance is overex- pressed to reach a concentration in the cytosol of at least twofold (in relation to the concentration of the endogenous enzyme).
[0019]With regard to the step of reducing, eliminating or suppressing the activity of the endogenous cytosolic G6PDH, it is particularly preferred to replace the endogenous cytosolic G6PDH by an isoenzyme with increased NADPH tolerance in such a way that substantially no activity, and even more preferred no activity of the endogenous cytosolic G6PDH remains.
[0020]Reducing, eliminating or suppressing the activity of endogenous cytosolic G6PDH is preferably carried out by targeted knock-out mutation or gene silencing, co-suppression, antisense or RNA interference, which are all standard methods belonging to the general knowledge of a person skilled in the art.
[0021]The eukaryotic organism is preferably selected from plants, animals or fungi, wherein the term "animals" includes vertebrates as well as invertebrates, and the term "plants" includes monocotyledons (monocots) as well as dicotyledons (dicots). As already mentioned above, the term "organism" also includes parts of an organism, and in particular also human cell lines.
[0022]Since all kinds of stress are characterized by an oxidative burst, and this mechanism is present in every eukaryotic organism, it is considered that the present invention is in fact applicable to all kinds of eukaryotic organisms like plants, animals or fungi.
[0023]In case that the eukaryotic organism is a plant, in particular a higher plant species, this plant is preferably selected from the group consisting of Solanaceae, soy bean, maize, rice, wheat, barley, rye, sugar cane, canola (rapeseed), cotton, sugar beet, switchgrass, Arabidopsis or else, particularly preferred a plant selected from tobacco, tomato, potato, pepper or else. Grain crops are also particularly preferred.
The present invention also relates to eukaryotic transgenic organisms having enhanced pathogen resistance and/or resistance to stress, characterized in that they express in their cytosol a G6PDH with increased NADPH tolerance compared to the endogenous cytosolic enzyme, while the activity of its endogenous cytosolic glucose-6-phosphate dehydrogenase is reduced, eliminated or suppressed.
[0024]These eukaryotic transgenic organisms are obtainable by the inventive method as described above, and they preferable are characterized by one or more of the features described above with respect to the inventive method.
[0025]Moreover, the present invention relates to the use of the inventive method described above for increasing the harvest yield of the respective eukaryotic organism.
[0026]As a preferred embodiment, this invention describes the benefits of enzyme replacement in the cytosol using an antisense approach for eliminating endogenous glucose-6-phosphate dehydrogenase activity combined with expression of an N-terminally truncated plastidial isoenzyme of P2 class, with enhanced K,[NADPH]-
[0027]The invention will now be described and explained with respect to the included figures and the following examples. These examples, however, are not intended to limit the scope of the present invention.
[0029]Fig. 1 : Scheme of the G6PDH "enzyme replacement" strategy. Abbr.: GIc, glucose; G6P, glucose-6-phosphate; Mito, Mitochondrium; NADP+/H, nicotinamide-dinucleotide phosphate, oxidized or reduced form; 3PGA, 3-phosphoglycerate; 6PG, 6-phosphogluconate; PPP, pentose- phosphate pathway; Ru5P, ribulose-5 -phosphate; Triose-P, triose phosphates DHAP and GA3P (dihydroxyacetone phosphate and glyceraldehyde-3-phosphate). For further explanation see text above.
[0030]Fig. 2: G6PDH and NADPH oxidase inhibitors interfere with necrosis formation. Top, G6PDH activity increases dramatically in the resistant tobacco wild type variety SNN after infection with Phythophthora nicotianae (P. nicotianae; see also Scharte et al., 2005) compared to susceptible
wild type Xanthi plants. This suggested a key role for G6PDH in successful plant defence. Bottom, Interference of Glucosamin-6-P (G6PDH inhibitor, infiltrated area marked red) and Diphenylene iodonium (DPI, NADPH oxidase inhibitor) with defence-induced H2O2 production (visualized by DAB-staining - after removal of chlorophyll) and the formation of hypersensitive lesions indicate that efficient plant defence depends upon NADPH availability in the cytosol, stemming mostly from the OPPP. Abbr.: w/o, without.
[0031]Fig. 3 : Construct design for the conducted studies.
[0032]Fig. 4: Disease evaluation of parental cP2 lines versus their corresponding super-transformed RNAi progeny after pathogen challenge with P. nicotianae.
[0033]Side-by side analysis of weak and strong parental cP2 lines versus their corresponding super- transformed RNAi progeny compared to SNN and Xanthi wild type varieties. The extent of hypersensitive lesions formed at fifty infiltration sites with zoospores of P. nicotianae on 5 plants (three or more independent rounds of infection) was evaluated after 2 days: High resistance, 80-100% lpa (lesions per area); intermediate resistance, 35-80% lpa; susceptible response, 0-10% lpa at infiltration site. The data obtained and illustrated in Fig. 4 are also summarized in Table 1 below:
[0035]
[0036]Fig. 5: Tobacco 1000-grain weights (mg) of different seed batches (n>5) were determined upon collection in pre-weighed Eppendorf tubes using an analytical balance.
Mean values, standard deviations (SD) and standard errors (SE) were calculated with Excel 2003 (vl 1.0, MICROSOFT, Redmond, USA). Differences described as significant were calculated by the Mest algorithm incorporated into Microsoft Excel. Data are shown as mean mg ± SE of at least five individual seed batches. Asterisks indicate significant differences compared to Xanthi wild type as determined by the student's Mest (*, p<0.05, **; p<0.01; ***, pO.001). The data obtained and illustrated in Fig. 5 are also summarized in Table 2 below:
[0038]
SNN = resistant, Xanthi = susceptible Nicotiana tabacum variety.
[0040]Tobacco Nicotiana tabacum lines of the susceptible variety Xanthi (Way et al., 2002) were engineered to replace endogenous cytsolic G6PDH activity by an N-terminally truncated plastidial isoenzyme with superior biochemical characteristics (see Fig. 3). Transgenic lines expressing Arabidopsis thaliana G6PD isoform Atlg24280 (P2 class) without transit peptide were additionally transformed with an RNAi construct eliminating expression of endogenous tobacco G6PD isoforms in the cytosol. "Enzyme replaced" Xanthi super-transformants reacted strongly {hypersensitive) after zoospore infection with Phytophthora nicotianae comparable to the natural tobacco wild type variety Samsun NN. Moreover, extent of necrosis formation was independent of the response displayed by the parental lines (weak to intermediate responses, see Fig. 4). This is an example that effective metabolic channelling ensues only when competing enzyme activities are eliminated in the same cellular compartment.
[0041]Example 1: Measurement of G6PDH and specific inhibitor studies.
[0042]Photometric determination of G6P DH activity
Freshly cut leaf discs were frozen in liquid N2 and ground to a fine powder. Extraction and determination of G6PDH activity was according to the method described by Fickenscher & Scheibe (1986).
[0044]Evidence for the involvement of NADPH oxidase as the source of ROS-formation is provided by the inhibitory effect of the flavoprotein inhibitor diphenylene iodonium (DPI). DPI is a well- known inhibitor of the mammalian neutrophil oxidase and inhibits also plant NAD(P)H oxidases (Pugin et al., 1997 and references cited therein). NADPH oxidase was shown to be the main source for extracellular ROS production (oxidative burst) in plants upon elicitation (Pugin et al., 1997). This leads to the formation of hypersensitive lesions in leaf tissue of resistant varieties after pathogen infection (incompatible interaction).
[0045]When tobacco leaves were treated with 25 -100 mM DPI, infection with P. nicotianae did not induce the formation of hypersensitive lesions.
[0046]The involvement of the oxidative pentose phosphate pathway (OPPP) in the oxidative burst of plants was shown by inhibitor studies with Glucosamine-6 phosphate (Glucosamine-6-P). This is a well-known competitive inhibitor for G6PDH (Glaser and Brown, 1955), the first enzyme of the OPPP, which transforms G6P into 6-phosphogluconolactone. When tobacco leaves are treated with 25 - 50 mM GN6P, infection with P. nicotianae did not result in detectable ROS production.
[0047]Example 2: Zoospore infiltration, H2O2 detection, and evaluation of leaf necroses formation.
[0048]Oomycete Growth, Zoospore Production, and Inoculation
[0049]Phytophthora nicotianae van Breda de Haan isolate 1828 (DSMZ, Braunschweig, GER) was cultivated at 24°C on clarified tomato agar as described by von Broembsen and Deacon (1996). Zoospores were produced under aseptic conditions according to von Broembsen and Deacon (1996). Source leaves from 8- to 10-week-old tobacco plants were infiltrated with a suspension containing 500 to 1,000 zoospores μL"1 according to Colas et al. (2001). This zoospore-leaf
infiltration assay was chosen to achieve a rapid, synchronized infection start in all parenchymatic cells of the infiltrated leaf area. For mock-inoculation, sterile tap water was infiltrated, and is further referred to as control. To take into account individual plant or developmental variations, samples from control and infection sites were excised from adjacent intercostal areas of the same source leaf. Plant inoculation was always performed at the beginning of the photoperiod.
[0050]Histochemical detection ofU2θ2
[0051]Hydrogen peroxide (H2O2) accumulation was detected by in situ staining with 3,3-diaminoben- zidine (DAB) following a modified protocol of Thordal-Christensen et al. (1997). Leaves were placed in DAB solution (1 mg mL'1, pH 3.8) for 6 h. A dark-brown polymerization product formed at sites where DAB reacts with H2O2 produced by the tissue. Incubations were stopped and leaf tissue was simultaneously cleared from chlorophyll by boiling in ethanol for 10 min.
[0052]Determination of the extent of hypersensitive lesions
[0053]The extent of hypersensitive lesions formed at fifty infiltration sites with zoospores of Phytophthora nicotianae on 5 plants (three or more independent rounds of infections) were evaluated after 2 days: High resistance, 80-100% lpa (lesions per area); intermediate resistance, 35-80% lpa; susceptible response, 0-10% lpa at infiltration site.
[0054]Example 3: Cloning strategy of cP2 plant expression constructs.
[0055]Total RNA was isolated from Arabidopsis leaves (Logemann et al., 1987) and reverse transcribed from mRNA using a polyA primer mix (5'-A30-C/G/T-3!) and Superscript II
[0056](Invitrogen) according to a protocol of the supplier. Truncated Arabidopsis P2 cDNA fragments (Atlg24280, 5' delta 195 bp termed cP2) were amplified from 1st strand cDNA using primers ZM_S2 and ZM S3 and Pful DNA polymerase (Stratagene), and directly cloned into BamHI and Sail opened pBluescript SK vector (Stratagene) yielding pZM3. Similarly, BamHI and Sail digested cP2 fragments were introduced into the multiple cloning site of pA35 (plant expression cassette assembled in pUC18, see Hόfte et al., 1991) between CaMV 35S promoter and OCS polyadenylation signal yielding pZM4. The entire plant expression cassette was transferred by complete HindIII and partial PvuII digest into HindIII and SnaBI opened vector pGSC1704
[HygR] (Plant Genetic Systems), yielding final binary construct pZM5 suited for Agrobacterium-mediated stable plant transformation (see Example 5 below).
[0057]Primers for amplification of Arabidopsis cP2 cDNA fragments:
[0058]ZM_S2 sense (BamHI recognition site underlined, start codon bold) (SEQ ID NO:1) 5'-Nn-GGA TCC AAG ATG GTT GTC GTG CAA GAT GGA TCA GTA GCC ACC-3'
[0059]ZM_A3 antisense (Sail recognition site underlined, stop codon bold) (SEQ ID NO:2) 5'-Nn-GTC GAC TCA CTG ATC AAG ACT TAG GTC TCC CCA TTG-3'
[0060]Example 4: G6PDH activity test of the recombinant cP2 enzyme in E. coli.
[0061]For cloning into E. coli expression vector pETlόb (Novagen) cP2 cDNA fragments were amplified from pZM4 (Fig. 3) using primers pET-cP2 sense and ZM_A3 (antisense, see above) and Phusion DNA polymerase (Finzymes). PCR products were digested with Ncol and Sail and cloned in E. coli XLl blue (Stratagene) after ligation to Ncol-Xhol opened vector pETlόb (Novagen), yielding pET-cP2. G6PDH activity of the cP2 enzyme was determined in a G6PDH- deficient E. coli strain. Host strain BL21(DE3) pLysS (Novagen) was modified by Pl trans- duction using E. coli zw/minus strain SU294 (Lee & Levy, 1992) resulting in BL21G6PDm™s zw/:TnlO[TetR] (Christian Schwoppe and Antje von Schaewen, unpublished). After retrans- formation, cP2 expression was induced in logarithmically growing BL21G<5/>Dmιnus: pET-cP2 cultures by adding IPTG (1 mM f.c) and allowed to grow for 2-3 h at 37°C. E. coli cells were harvested by centrifugation and adjusted to 10 OD6O0. Extraction was in 100 mM NaH2PO4, 10 mM Tris-NaOH pH 8 supplemented with 0.1 mM Pefabloc SC and 0.02 mM NADP (to stabilize G6PDH) by 3 times sonication for 10 sec at 50 W (Branson sonifier). G6PDH activity of cP2 was characterized by Km[o6p] = 0.58 mM, and KJ[NADPH]= 4.6 μM > Km[NADP+]= 2.4 μM. The latter values differ slightly from those previously reported by Wakao & Benning (2005) for the recombinant enzyme with C-terminal Strep-tag (KJ[NADPH] = 22 μM > Km[NADP*]= 17 μM).
[0062]Specific primer for cloning a pET16b-cP2 expression construct (without His-tag).
[0063]pET-cP2 sense (Ncol recognition site underlined, start codon bold) (SEQ ID NO:3) 5'-NNNCC ATG GTT GTC GTG CAA GAT GGA TCA G TA G-3'
Example 5: Generation of tobacco plants that overexpress cP2 (Atlg24280) in the cytosol.
[0064]Binary construct pZM5 (Example 4, see above) was directly transformed into Agrobacterium strain GV2260 (Deblaere et al., 1988) according to a protocol described by Hδfgen & Willmitzer (1985). Generation of transgenic tobacco plants was by Agrobacterium cocultivation of Nicotiana tabacum var. Xanthi leaf discs with GV2260:pZM5 followed by a combined callus- shoot regeneration protocol as described by Voelker et al. (1987). Xanthi transformants were selected for high expression of cP2 in TO using Northern-blot analyses, and by immunoblot analyses in Tl and all following generations (not shown) using a G6PDH antiserum specific for plant P2 isoenzymes (Wenderoth et al., 2000).
[0065]Example 6: Generation of Xanthi-cP2 plants with reduced levels of endogenous cytosolic G6PDH activity by supertransformation with a cytGdPD-dsRNAi construct.
[0066]Cloning of a cytG6PD-dsRNAi construct
[0067]The cytGdPD-dsRNAi construct was designed based on tobacco cyXG6PD isoforms (not shown). Approximately 400 bp were amplified by RT-PCR from total leaf RNA isolated from the Nicotiana tabacum variety Xanthi. The resulting fragment was inserted twice into vector pUC- RNAi (Chen et al., 2003) flanking the central first intron of potato GA20-Oxidase. The first insertion was via Sall/BamHI and the second insertion via Xhol/Bglll compatible ends.
[0068]Primers for cloning a tobacco G5PZ)-dsRNAi construct in pUC-RNAi (Chen et al., 2003)
[0069]cytG6PD-s (sense, Sail site underlined) (SEQ ID NO:4) 5'-CACCGTCGACAATATGAAGGCTATAAGGATGACC-S'
[0070]cytG6PD-as (antisense, BamHI site underlined) (SEQ ID NO:5) 5'-GGATCCTATATGACAGGTCTAATTCACTTTGAAC-3'
[0071]Then, the entire dsRNAi region was released by restriction digest with Pstl and the expression cassette was inserted (between the strong CaMV 35S promoter and OCS polyadenylation signal) of Sdal opened binary vector pBinAR[Kan] (Hδfgen & Willmitzer, 1990).
Supertransformation ofXanthi-cP2 lines using the binary cytG6P D-RNAi construct
[0072]Leaf discs of two independent Tl Xanthi-cP2 lines (weak cP2 83-1 and strong cP2 67-3) were transformed by the leaf-disc method (as in Example 5 above) using Agrobacterium strain
[0073]GV2260 carrying the pBinAR-cytG6P/>RNAi construct. Regeneration of supertransformants was on double selective media containing 100 mg/1 Kanamycin and 40 mg/1 Hygromycin B.
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