Patent Application
20180245085
The present invention relates to a process for producing a temperature-sensitive conditionally mutant multicellular organism using a low-temperature N-terminal degradation cassette or a vector comprising it to transfect or transform at least one cell or part of a multicellular organism, thereby obtaining the temperature-sensitive conditional mutant of the multicellular organism or part thereof. Further, the present invention relates to a low-temperature N-terminal degradation cassette comprising nucleotide sequences encoding at least a temperature-sensitive DHFR protein, a destabilizing amino acid, a ubiquitin moiety, as well as a protein of interest.
Temperature-sensitive mutants are a powerful tool with which to study gene function in vivo. Such temperature-sensitive mutants are ones in which there is a marked drop in the level or activity of the gene product when the gene is expressed above a certain temperature, the so-called restrictive temperature. Below this temperature, at so-called permissive temperatures, the activity or phenotype of the mutant is similar to that of the wildtype. Thus, temperature-sensitive mutants provide an extremely powerful tool for studying protein function and assembly in vivo, because these mutants provide a reversible mechanism to lower the level of a specific gene product at any stage in the growth of the organism simply by changing the temperature of growth.
Thus, phenotypes can be generated on demand by controlling activation and accumulation of proteins of interest which are invaluable tools to analyze and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are difficult to identify in multicellular species.
Although there are other inducible systems for gene expression, temperature-sensitive mutants have several unique advantages, including fast temporal response, high reversibility, and the applicability to any tissue type or developmental stage of an organism. To date, temperature-sensitive mutants of a protein of interest are generated by random mutagenesis, typically with a chemical mutagen, often followed by laborious screening of large numbers of progeny. Thus, this procedure is usually limited to fast-growing monocellular organisms where mutated populations can be analyzed simultaneously at the restrictive and the permissive temperatures.
In an alternative approach, heat-sensitive mutants are generated using a temperature-inducible N-degron, where a protein of interest is fused to a portable N-terminal degron. In particular in yeast, it was shown that temperature-sensitive mutants can be generated by fusing a protein of interest to a temperature-sensitive degron. Temperature-sensitive degrons represent specific degradation signals and take advantage of the N-end rule pathway of targeted protein degradation (NERD). The fundamental principles of the N-end rule and the proteolytic pathway that implements it are well-established in the literature (see, e.g., Bachmair et al., Science 234:179 (1986); Varshaysky, Cell 69:725 (1992)). The classical yeast N-degron is based on a temperature-sensitive mutant of mouse dihydrofolate reductase (DHFR) containing a single amino acid substitution at position 67 (U.S. Pat. No. 5,705,387). This temperature-sensitive DHFR mutant is fused to an N-terminal destabilizing amino acid according to the N-end rule degradation pathway which dramatically decreases the in vivo half-life of a protein. This degron construct containing the mutant DHFR protein is heat-labile at temperatures above 37° C., leading to exposure of an internal lysine residue, the site of poly-ubiquitination. A protein of interest fused to this degron is targeted via poly-ubiquitination for proteasome dependent proteolysis upon shifting to a restrictive temperature.
Thus, by using this N-degron technique, proteins of interest can be expressed conditionally to establish phenotypes on demand by controlling activation and inactivation of these proteins via temperature shift from permissive to restrictive temperatures. This method can thus be used as a rapid on/off switch for protein expression based on temperature, e.g. for supplementing a central gene product in its mutated background. However, to date this system has only been used at the single-cell level in yeast and cell culture where it requires restrictive temperatures of 37 or 42° C. These high temperatures are beyond the physiological range of many multicellular organisms, e.g. plants. Accordingly, up to now it is not possible to provide multicellular organisms, in particular plants, which as a whole allow temperature-dependent conditional protein expression or wherein specific reaction compartments, in particular specific cells or specialized tissues thereof, are susceptible to a temperature-dependent switching of protein expression. Up to now, there are no multicellular organisms, in particular plants, available which are capable of conditional, i.e. temperature dependent, development of reaction compartments, that means specific cells or tissues whose presence and/or development can be controlled by switching protein expression on or off in a temperature-dependent way, whereby the protein expression controls and allows the development of said reaction compartment. Said presence and/or development advantageously can be controlled by switching protein expression on or off in a temperature-dependent way, advantageously under physiological conditions.
The technical problem underlying the present invention is thus to overcome the aforementioned disadvantages and in particular to provide methods, multicellular organisms and means to produce temperature-sensitive conditional mutants of multicellular organisms with restrictive temperatures tolerable by multicellular organisms like plants or animals. One particular technical problem underlying the present invention is further to provide multicellular organisms, in particular plants, which comprise conditional reaction compartments which can be generated via temperature-dependent protein expression and used for example as conditional bioreactors. This technical problem is solved by the subject-matter of the independent claims, in particular by a process for producing a temperature-sensitive conditional mutant of a multicellular organism comprising the steps of:
The present invention therefore uses as starting material, a low-temperature N-terminal degradation cassette or a vector comprising a low-temperature N-terminal degradation cassette and at least one cell or part of a multicellular organism. Said at least one cell or part of the multicellular organism, such as suitable animal cells or a plant part, in particular root, or a callus, is preferably a wildtype, in particular non-mutated, cell or part of a multicellular organism. By transfecting or transforming the at least one cell or part of the multicellular organism with the low-temperature N-terminal degradation cassette or the vector containing it, a temperature-sensitive conditional mutant of said multicellular organism or part thereof is obtained. Preferably, the whole multicellular organism can be generated from the at least one transfected or transformed cell or the transfected or transformed part of the multicellular organism. According to a preferred method of the present invention, the at least one cell or part of the multicellular organism is stably transfected or transformed with the low-temperature N-terminal degradation cassette or a vector comprising it to obtain a stable, genetically modified organism being a temperature-sensitive conditional mutant of the multicellular organism or a part thereof. The low-temperature N-terminal degradation cassette is preferably stably integrated into the genome of every cell of a multicellular organism by transfecting or transforming undifferentiated cells, preferably gametes, of a multicellular organism, growing and cultivating it to obtain a stable integration of the cassette into every cell of the obtained organism.
Thus, in a preferred embodiment, the present invention relates to stably transfecting or transforming at least one undifferentiated cell, in particular gamete, of a multicellular organism with the low-temperature N-degradation cassette or a vector comprising it, growing and cultivating it so as to obtain a stable genetically modified multicellular organism being a temperature-sensitive conditional mutant and comprising in every cell the low-temperature N-terminal degradation cassette.
Thus, the present invention advantageously allows generating developmental effects and phenotypes. Furthermore, the present invention allows for the temperature-dependent accumulation of proteins of interest. The present invention, therefore, advantageously allows for the conditional expression of proteins of interest.
Advantageously, by using the present low-temperature N-terminal degradation cassette it is possible to create temperature-sensitive conditional mutants of multicellular organisms because the restrictive temperatures are lower and thus tolerable by multicellular organisms. The present invention therefore presents a versatile and transferable genetically stable system based on a low-temperature-controlled N-terminal degradation signal (N-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. It is thereby possible to trigger developmental effects and to generate phenotypes on demand.
In a preferred embodiment, the low-temperature N-terminal degradation cassette used in the present process comprises:
i) a nucleotide sequence encoding a temperature-sensitive DHFR protein, which is a DHFR protein, in which at least one amino acid selected from the group consisting of the residues corresponding to threonine 39, valine 51, isoleucine 52, methionine 53, valine 75, valine 113, tryptophan 114, isoleucine 115, leucine 134, phenylalanine 135, valine 136, isoleucine 139 and glutamic acid 173 of the wildtype mouse DHFR protein according to SEQ ID No. 1 is substituted,
ii) a codon for a destabilizing amino acid selected from the group consisting of arginine, lysine, histidine, phenylalanine, tyrosine, tryptophan, leucine, isoleucine, methionine, aspartic acid, glutamic acid, asparagine, glutamine and cysteine which codon is located 5′ of the nucleotide sequence encoding the temperature-sensitive DHFR protein identified in i), and
iii) a nucleotide sequence encoding a ubiquitin moiety which is located 5′ of the codon for the destabilizing amino acid identified in ii).
In a preferred embodiment of the present invention, the N-terminal degradation cassette used in the present process comprises:
i) a nucleotide sequence encoding a temperature-sensitive DHFR protein, which is a DHFR protein, in which at least one amino acid selected from the group consisting of the residues corresponding to threonine 39, valine 51, isoleucine 52, methionine 53, valine 75, valine 113, tryptophan 114, isoleucine 115, leucine 134, phenylalanine 135, valine 136, isoleucine 139 and glutamic acid 173 of the wildtype mouse DHFR protein according to SEQ ID No. 1 is substituted,
ii) a codon for a destabilizing amino acid selected from the group consisting of arginine, lysine, histidine, phenylalanine, tyrosine, tryptophan, leucine, isoleucine, aspartic acid, glutamic acid, asparagine, glutamine and cysteine which codon is located 5′ of the nucleotide sequence encoding the temperature-sensitive DHFR protein identified in i), and
iii) a nucleotide sequence encoding a ubiquitin moiety which is located 5′ of the codon for the destabilizing amino acid identified in ii).
Thus, advantageously, the present low-temperature degradation cassette comprises a temperature-sensitive DHFR protein as identified in i) above, in which at least one of the above identified amino acid residues is substituted. Surprisingly, the inventors found that a DHFR protein comprising at least one of those amino acid substitutions is temperature-sensitive and becomes unstable at much lower temperatures compared to the prior art. Thus, an N-terminal degradation cassette comprising the present temperature-sensitive DHFR protein is degraded at lower temperatures, that means the restrictive temperature is lower, than that of known temperature-sensitive N-degrons. It is thus possible to enable conditional, temperature-dependent protein expression in multicellular organisms under physiological conditions. As with the classical yeast N-degron, degradation of the N-terminal degradation cassette is effected at restrictive temperatures via the N-end rule pathway and the proteasome. Thus, transcript levels of the low-temperature degradation cassette are unaffected by temperature shifts but the resulting protein is rapidly degraded by the proteasome at restrictive temperatures, whereas the protein is stable at permissive temperatures.
In particular, the permissive temperature at which the present degradation cassette is expressed and the protein is stable in the transfected or transformed cells is below 24° C., whereas the restrictive temperature at which the degradation cassette is unstable and the protein is degraded is as low as 24° C. and above.
As specified in ii) above, the present degradation cassette further comprises a codon for a destabilizing amino acid which is located 5′ of the nucleotide sequence encoding the temperature-sensitive DHFR protein. In particular, the destabilizing amino acid is an amino acid according to the N-end rule pathway, preferably type I primary destabilizing amino acids such as arginine, lysine or histidine; type II primary destabilizing amino acids such as phenylalanine, tyrosine, tryptophan, leucine, isoleucine or methionine; secondary destabilizing amino acids such as aspartic acid or glutamic acid; or tertiary destabilizing amino acids such as asparagine, glutamine or cysteine. Preferably, the destabilizing amino acid is selected from the group consisting of arginine, phenylalanine and leucine. Particularly preferred, the destabilizing amino acid is phenylalanine.
Furthermore, as specified in iii) above, the present degradation cassette comprises a nucleotide sequence encoding a ubiquitin moiety which is located 5′ of the codon for the destabilizing amino acid. The ubiquitin moiety is removed cotranslationally from the expressed fusion protein by deubiquitinating enzymes and the destabilizing amino acid is exposed. Thus, by using an N-terminal ubiquitin moiety the destabilizing amino acid of choice can be produced at the N-terminus of the temperature-sensitive DHFR. Concomitantly with deubiquitinating, previously internal lysine residues of the temperature-sensitive DHFR protein become surface exposed and act as poly-ubiquitin acceptors at restrictive temperatures, which in turn targets the whole protein for proteasome-dependent degradation. Particularly preferred, the nucleotide sequence encoding the ubiquitin moiety is codon optimized for the specific multicellular organism used. Thus, e.g. the nucleotide sequence encoding the ubiquitin moiety is a plant optimized ubiquitin moiety nucleotide sequence with an optimized sequence according to the codon usage of the respective species.
As discussed above, the present low-temperature degradation cassette comprises as component i) a nucleotide sequence encoding a temperature-sensitive DHFR protein, which comprises at least one of the above identified amino acid substitutions. Said nucleotide sequence encoding the DHFR protein can be derived from any suitable species, for instance animals, such as mammals, in particular rat, human or mouse, plants or protozoa, provided that the protein has dihydrofolate reductase activity and comprises at least the amino acid residues corresponding to the specifically identified amino acid residues in feature i) to be substituted. Said specifically identified amino acid residues are those from which at least one, preferably two, three or more, are substituted by another amino acid. The position of said specifically identified amino acid residues which are to be substituted in the DHFR protein is given with respect to a reference DHFR protein, namely the wildtype mouse DHFR protein according to SEQ ID No. 1. Thus, the mouse DHFR amino acid sequence SEQ ID No. 1 referred to in feature i) serves only as reference for the position of the amino acids to be substituted and does not limit the invention to the particular DHFR sequence. Due to the high level of conservation one skilled in the art is able to find the nucleotide and amino acid sequence corresponding to the mouse DHFR sequence in other species.
In a preferred embodiment, the degradation cassette comprises a nucleotide sequence encoding the wildtype mouse DHFR protein comprising at least one of the above identified amino acid substitutions. Such a preferred temperature-sensitive DHFR protein is represented by SEQ ID No. 2, wherein all putatively substituted amino acid residues, which are threonine 39, valine 51, isoleucine 52, methionine 53, valine 75, valine 113, tryptophan 114, isoleucine 115, leucine 134, phenylalanine 135, valine 136, isoleucine 139 and glutamic acid 173, are replaced by Xaa.
In a preferred embodiment, the temperature-sensitive DHFR protein comprises at least one amino acid substitution of one of the amino acids residues specified above, wherein in case any of the valine residues 51, 75, 113 and 136 is substituted, then it is substituted with an amino acid selected from the group consisting of glycine, asparagine and threonine; in case any of the residues isoleucine 52, methionine 53, tryptophan 114, isoleucine 115, leucine 134, phenylalanine 135 and isoleucine 139 is substituted, then it is substituted with an amino acid selected from the group consisting of glycine, glutamic acid and asparagine; in case the threonine residue 39 is substituted, then it is substituted with alanine and in case the glutamic acid residue 173 is substituted, then it is substituted with aspartic acid. Particularly preferred, at least one of the amino acids threonine 39 and glutamic acid 173 are substituted. Most preferred the amino acid glutamic acid 173 is substituted.
In a preferred embodiment of the present invention, the threonine residue 39 of the DHFR protein is substituted with alanine and/or the glutamic acid residue 173 is substituted with aspartic acid, preferably both amino acids are substituted as represented by SEQ ID No. 3. Most preferred only the amino acid glutamic acid 173 is substituted with aspartic acid.
By the substitution of at least one amino acid residue specified above, advantageously a temperature-sensitive DHFR protein is obtained suitable for the use in a low-temperature controlled N-terminal degradation signal (N-degron) allowing a reversible and switch-like tuning of proteins fused to the low-temperature N-terminal degradation cassette in a multicellular organism and under physiological conditions.
In a preferred embodiment of the present invention, the degradation cassette used in the present process further comprises:
iv) a nucleotide sequence encoding a protein of interest A, B or a combination thereof which is located 3′ of the nucleotide sequence encoding the temperature-sensitive DHFR protein identified in i).
Thus, the present degradation cassette preferably also comprises a nucleotide sequence encoding a protein of interest A, B or a combination thereof. This protein of interest A or B is thus expressed and accumulated in a cell which has been transfected or transformed with the present degradation cassette at a permissive lower temperature, whereas it is degraded at a restrictive higher temperature because it is fused to the temperature-sensitive DHFR protein of the present invention.
Thus, in a preferred embodiment, the present invention allows it to accumulate proteins of interest in cells of a multicellular organism, in particular in stably transformed transgenic cells of a multicellular organism.
Thus, in a preferred embodiment, the protein of interest A, B or the combination thereof is degradable upon shifting the temperature-sensitive conditional mutant of the multicellular organism from a permissive lower temperature to a restrictive higher temperature for a period of time sufficient to facilitate degradation of the protein of interest A, B or the combination thereof in the cells of the mutant multicellular organism.
In a preferred embodiment, the degradation cassette thus comprises from 5′ to 3′ nucleotide sequences encoding: the ubiquitin moiety (iii), the destabilizing amino acid (ii), the temperature-sensitive DHFR protein (i) and preferably the protein of interest A and/or B (iv). At permissive temperatures, this degradation cassette is expressed in the transfected or transformed cells of the multicellular organism and the protein of interest A and/or B is expressed and accumulated in the cell as a fusion protein containing the above-mentioned elements. Preferably, being expressed as such a fusion protein does not hinder the function of the protein of interest A and/or B. However, at restrictive higher temperatures, the degradation cassette is still expressed but the resulting fusion protein is rapidly degraded via poly-ubiquitination by the proteasome.
In a furthermore preferred embodiment of the present invention, the at least one cell or part of the multicellular organism into which the low-temperature N-terminal degradation cassette or vector is transfected or transformed, preferably stably transfected or stably transformed, and wherein the degradation cassette further comprises at least one nucleotide sequence encoding a protein of interest A, B or a combination thereof, comprise at least one functioning allele of an endogenous gene for the protein of interest.
In a preferred embodiment of the present invention, the multicellular organism, part or at least one cell thereof provided in step a) of the present process lacks a functional endogenous gene encoding the protein of interest A and/or B. Thus, the protein of interest A or B which is provided via the transfected or transformed degradation cassette supplements the protein A and/or B, thus allowing temperature-dependent expression of the protein of interest A and/or B.
In a preferred embodiment of the present invention, the protein of interest A is a protein to be produced in a transfected or transformed cell in a temperature-dependent manner and which is of commercial or research interest. In a preferred embodiment, the protein of interest A is a protein improving the nutritional value of a plant, conveying tolerance against biotic or abiotic stresses, an enzyme, a pharmaceutical or nutraceutical protein, a medicinal enzyme or a protein that can be used for diagnostic, industrial or technical purposes. Preferred examples for a protein of interest A are storage proteins, amidases, peroxidases, viral core antigens, antibodies, vaccines, hormones and growth factors or proteinaceous toxins such as lectins. In case the protein of interest A is an enzyme, as e.g. TEV, it can preferably also be used for intracellular cleavage of a substrate which is either naturally occurring in the cell or has been transfected or transformed into the cell as well, e.g. as a protein of interest C (see below). Preferred proteins of interest A are green fluorescent protein (GFP), β-glucuronidase (GUS), tobacco etch virus protease (TEV), phosphinothricin-N-acetyltransferase (PAT) which is able to provide herbicide resistance and cyclin-dependent kinase (CDKA;1) which is able to increase cell division competency.
In a preferred embodiment of the present invention, a process for producing a protein of interest is, thus, provided which process comprises producing a temperature-sensitive conditional mutant of a multicellular organism according to the present invention, subjecting the obtained conditional mutant to conditions appropriate for producing a protein of interest under permissive temperature and recovering the protein of interest.
In a preferred embodiment of the present invention, the protein of interest B is able to control the development of a reaction compartment of the multicellular organism. In this way, the multicellular organism obtained in step c) of the present process is able to develop the reaction compartment temperature-dependently. In particular, the protein of interest B is a protein which controls the development of a reaction compartment of the multicellular organism either positively or negatively. Thus, in a preferred embodiment the reaction compartment is developed by the mutant multicellular organism at permissive temperatures, which means that the protein of interest B is accumulated in the cells at permissive temperatures and the protein of interest B affects the development of the reaction compartment positively, that means the expressed protein of interest B allows the development of the reaction compartment. In this case, the multicellular organism preferably lacks a functional gene encoding the endogenous protein of interest B. In another preferred embodiment, the protein of interest affects the development of the reaction compartment negatively, e.g. in form of a repressor. In this case, the reaction compartment can only be developed by the mutant multicellular organism at restrictive temperatures when the protein of interest B is degraded. Thus, preferably, the multicellular organism is allowed to grow under permissive or restrictive temperatures depending on the nature of the protein of interest B for a period of time sufficient for development of the reaction compartment.
In the context of the present invention, the reaction compartment is a specific tissue, cell type or organ of the multicellular organism. Particularly preferred, the reaction compartment(s) are trichomes (leaf hairs), floral meristem cells, stomata (epidermal pores) or specific cell types of the root, seed or entire silique, such as root hairs, seed coat or endosperm. Thus, preferably, the protein of interest B is a protein able to control the development of the above-mentioned tissues, cell types or organs, in particular of trichomes or floral meristem cells of a suitable plant. For example, protein of interest B preferably is TTG1 (TRANSPARENT TESTA GLABRA 1), other members of the GLABRA (GL) family or related proteins involved in trichome development, or CO (CONSTANS) and related proteins involved in the development of floral meristem cells in Arabidopsis thaliana, wherein the plant is either a mutant or wildtype with respect to protein of interest B. Other examples are members of the APETALA (AP) family, LEAFY (LFY), AGAMOUS (AGA) or similar candidates involved in flower development, endosperm or fruit specific factors such as hordein family members and similar target proteins.
In general, to be a suitable candidate for protein of interest A or B the respective protein must on the one hand—at least transiently—capable to localize or be enabled to localize to cellular compartments where the N-end rule pathway is active such as the cytosol and the nucleus and on the other hand cause developmental phenotypes in loss-of-function or gain-of-function.
In a preferred embodiment of the present process, the at least one cell or part of the multicellular organism is transfected or transformed in step b) of the present process with a degradation cassette comprising a nucleotide sequence encoding at least the protein of interest B and simultaneously or in a further step d) with a vector comprising a nucleotide sequence encoding at least a protein of interest C which nucleotide sequence is under the control of a promotor specific for the reaction compartment, which development is controlled by the protein of interest B, and wherein in step c) or a further step e) a multicellular organism is obtained which is able to develop the reaction compartment and to express the protein of interest C in the reaction compartment temperature-dependently. Preferably, in case the reaction compartments are Arabidopsis thaliana trichomes promotors like ProTRIPTYCHON, ProGLABRA2 or ProCAPRICE can be used. Further, in case the reaction compartment is meristematic tissue of Arabidopsis thaliana promotors like ProCDKA;1 or ProWUSCHEL can be used.
Thus, advantageously, it is possible to create a conditional reaction compartment which is developed by the mutated multicellular organism at permissive or restrictive temperatures depending on the protein of interest B. Furthermore, by transfecting or transforming the multicellular organism also with a nucleotide sequence encoding a protein of interest C which itself is under the control of a promotor specific for the reaction compartment developed depending on the protein of interest B, the protein of interest C is only expressed in the cells of the reaction compartment although every cell of the mutant multicellular organism might comprise the vector encoding the protein of interest C. Preferably, the protein of interest C is an enzyme, an enzymatic cascade or part of an enzymatic cascade or at least one regulatory element thereof, such as for example one or more transcription factors, so that the protein of interest C is able to directly or indirectly generate a molecule, in particular a small molecule, or for instance a metabolite, of interest. Preferably, the molecule or metabolite generated is an anthocyanin. In this case, protein of interest C preferably is at least one transcription factor, more preferably two transcription factors, in particular DELILA and ROSEA1 in Arabidopsis thaliana. Successful generation of anthocyanin in the reaction compartment, preferably trichomes, is preferably indicated by violet coloring. Furthermore, the molecule or metabolite generated can be a protein. Thus, advantageously, the conditional reaction compartment(s) of the mutated multicellular organism can be used as bioreactors for the production of molecules, metabolites or proteins of interest, which can be regulated temperature-dependently. This is especially advantageous in case the produced molecules or proteins are toxic for the whole multicellular organism but not for certain parts thereof or are only toxic during specific phases of development of the multicellular organism. Preferred toxic proteins of interest C are Barnase, a highly toxic bacterial ribonuclease from Bacillus amyloliquifaciens, and Diphtheria toxin A, a potent inhibitor of protein biosynthesis. Toxicity of these proteins is lost at restrictive temperatures because the protein is degraded, whereas at permissive temperatures cells of the reaction compartment get specifically ablated.
In a preferred embodiment, the protein of interest C is a developmental regulator such as a transcription factor or a repressor. Furthermore, the protein of interest C can preferably be a member of one of the six main functional enzyme families comprising oxidoreductases (EC.1), transferases (EC.2), hydrolases (EC.3), lyases (EC.4), isomerases (EC.5), and ligases (EC.6). In particular, the protein of interest C is a member of one of the 24 sub-family classes of EC.1 (oxidoreductases), a member of one of the 10 sub-family classes of EC.2 (transferases), a member of one of the 13 sub-family classes of EC.3 (hydrolases), a member of one of the 8 sub-family classes of EC.4 (lyases), a member of one of the 6 sub-family classes of EC.5 (isomerases), or a member of one of the 6 sub-family classes of EC.6 (ligases). More preferably, it is a protease, a kinase, a glucosyltransferase, an oxygenase, a hydroxylase, a reductase, a tyrosinase, or a peroxidase.
In a preferred embodiment of the present invention, the molecule, in particular the metabolite, of interest is a phenylpropanoid, a flavonoid, a terpenoid, an alkaloid, a glycoside, a coumarin, more preferably a member of the betalains such as anthocyanin or anthocyanidin, carotinoid or betanin.
In a preferred embodiment of the present invention, the permissive temperature is below 24° C. and the restrictive temperature is at least 24° C. In particular, the permissive temperature is from 10° C. to 24° C., preferably from 10° to below 24° C. and preferably from 10° to 23° C., preferably from 10° C. to 22° C., preferably from 12° C. to 20° C., more preferably from 13° C. to 19° C., preferably from 13° C. to 16° C., in particular the permissive temperature is 13° C. or 14° C., whereas the restrictive temperature is from 24° C. to 37° C., preferably from 24° C. to 35° C., preferably from 25° C. to 33° C., preferably from 25° C. to 31° C., preferably from 25° C. to 30° C., preferably from 25° C. to 29° C., more preferably from 25° C. to 28° C., preferably from 26° C. to 29° C., in particular the restrictive temperature is 27° C. or 29° C. Most preferably, the restrictive temperature is determined depending on the specific protein of interest A or B used.
In another embodiment of the present invention, the permissive temperature is below 27° C. and the restrictive temperature is at least 27° C.
Advantageously, the amount and/or functionality, in particular enzymatic activity, of protein of interest A and/or B is tunable from the maximal amount and/or functionality of said proteins at permissive temperatures over lower amounts at semi-restrictive temperatures up to no or almost no protein amount and/or functionality, in particular enzymatic activity, at restrictive temperatures. For example, semi-restrictive temperatures are from 18° C. to 26° C., preferably from 19° C. to 25° C., in particular from 20 to 24° C.
In a preferred embodiment of the present invention, the multicellular organism is a plant, in particular a flowering plant or an animal, in particular an insect. Most preferably, the multicellular organism is a member of the mustard family (Brassicaceae), in particular Arabidopsis thaliana, a member of the nightshade family (Solanaceae) such as of the genus Nicotiana (tobacco), in particular Nicotiana benthamiana, or of the genus Solanum, in particular Solanum lycopersicum (tomato) or Solanum tuberosum (potato), or a member of the grass family (Poaceae), in particular barley (Hordeum vulgare) or maize (Zea mays), or Drosophila melanogaster.
In a preferred embodiment of the present invention, the degradation cassette used in the present process further comprises:
v) a nucleotide sequence encoding a linker, preferably comprising the amino acids histidine, glycine, serine, glycine and isoleucine, which nucleotide sequence is located 5′ of the nucleotide sequence encoding the temperature-sensitive DHFR protein identified in i) and 3′ of the codon for the destabilizing amino acid identified in ii).
Preferably, the linker consists of the amino acids histidine, glycine, serine, glycine and isoleucine, preferably the linker consists of five amino acids, namely histidine, glycine, serine, glycine and isoleucine, preferably in this order (HGSGI, SEQ ID no. 30). Advantageously, such a linker enhances molecular flexibility thereby supporting the temperature-sensitivity of the degradation cassette.
In a preferred embodiment of the present invention, the degradation cassette used in the present process further comprises:
vi) a nucleotide sequence encoding a triple hemagglutinin epitope which nucleotide sequence is located 3′ of the nucleotide sequence encoding the temperature-sensitive DHFR protein identified in i) and 5′ of the nucleotide sequence encoding the protein of interest A, B or the combination thereof identified in iv).
Advantageously, the triple hemagglutinin epitope can be used for immunodetection of the fusion protein expressed by the transfected or transformed cells.
The hemagglutinin epitope is preferably YPYDVPDYA (SEQ ID no. 31), more preferably GSYPYDVPDYA (SEQ ID no. 32).
Thus, in a preferred embodiment, the degradation cassette comprises from 5′ to 3′ nucleotide sequences encoding: the ubiquitin moiety (iii), the destabilizing amino acid (ii), preferably the linker (v), the temperature-sensitive DHFR protein (i), preferably the triple hemagglutinin epitope (vi) and preferably the protein of interest A and/or B (iv). At permissive temperatures, this degradation cassette is expressed in the transfected or transformed cells of the mutant multicellular organism and the protein of interest A and/or B is expressed and accumulated in the cell as a fusion protein containing at least i), ii) and iii) of the above-mentioned elements, preferably also iv), preferably also v) and most preferably all elements. Preferably, being expressed as such a fusion protein does not hinder the function of the protein of interest A and/or B. However, at restrictive higher temperatures, the degradation cassette is still expressed but the resulting fusion protein is rapidly degraded via poly-ubiquitination by the proteasome.
In a preferred embodiment, the temperature-sensitive conditional mutant of the multicellular organism obtained in step c) or further step e) of the present process contains the low-temperature degradation cassette transiently or stably integrated into its genome.
The present invention also relates to a low-temperature N-terminal degradation cassette as defined above.
In the context of the present invention the term ‘at least one amino acid substitution’ refers to preferably one amino acid substitution, in particular solely one amino acid substitution. In a further preferred embodiment, the term ‘at least one amino acid substitution’ refers to two amino acid substitutions, in particular solely two amino acid substitutions. In a further preferred embodiment, the term ‘at least one amino acid substitution’ refers to three amino acid substitutions, in particular solely three amino acid substitutions. In a further preferred embodiment, the term ‘at least one amino acid substitution’ refers to four amino acid substitutions, in particular solely four amino acid substitutions. In a further preferred embodiment, the term ‘at least one amino acid substitution’ refers to five amino acid substitutions, in particular solely five amino acid substitutions.
In a preferred embodiment of the present invention, the at least one amino acid substitution is at least one amino acid substitution, is at least two amino acid substitutions, is at least three amino acid substitutions, is at least four amino acid substitutions or is at least five amino acid substitutions.
In a preferred embodiment of the present invention, the maximum number of amino acid substitutions is two, three, four, five, six, seven, eight, nine, ten, eleven, twelve and, most preferably, thirteen.
In the context of the present invention, a degron is a specific sequence of amino acids in a protein that controls the metabolic stability of a protein and thus makes it short lived in vivo or in vitro. A degron sequence can for instance occur at either the N- or C-terminus and is called N-degron or C-degron, respectively. Further, a degron sequence can also be located within a protein sequence.
In the context of the present invention, specifically N-degrons are concerned, in particular temperature-sensitive N-degrons. Temperature-sensitive N-degrons take advantage of the N-end rule pathway of targeted protein degradation (abbreviated NERD) according to which the N-terminal amino acid of a protein determines its half-life and therefore its likelihood to be degraded (Varshaysky 1997, Genes Cells 2: 13-28). Accordingly, a destabilizing N-terminal residue decreases the in vivo or in vitro half-life of a protein. Thus, an N-degron is an amino-terminal (N-terminal) degradation signal (Johnson, Gonda and Varshaysky, 1990, Nature 346:287-91) which is recognized by and subjects the whole protein to NERD. NERD is part of the ubiquitin proteasome system (UPS) in yeast, animals and plants and of the protease-dependent proteolytic machinery in bacteria. It controls protein stability of cytosolic and nuclear proteins but also for proteins localized in the membrane. Besides the initiating destabilizing amino acid, N-degrons comprise several determinants in order to target a substrate, in particular a protein, to NERD. First, the N-degron must contain a destabilizing N-terminal amino acid that can be recognized by NERD E3 ubiquitin ligases (N-recognins). Second, another crucial factor is a certain flexibility and accessibility of the N-terminal amino acid enabling a proper recognition of the substrate. Third, the N-degron needs to contain at least one internal lysine residue in appropriate distance to the N-terminal amino acid that may serve as poly-ubiquitination site. Thus, in a preferred embodiment, an N-degron as referred to in the present invention, is a degradation signal that contains at least a destabilizing N-terminal amino acid and a temperature-sensitive DHFR, wherein the destabilizing N-terminal amino acid is located at the N-terminus of the DHFR and the DHFR provides the at least one internal lysine residue serving as poly-ubiquitination site.
To be able to engineer the destabilizing amino acid of choice at the N-terminus of the temperature-sensitive DHFR protein the ubiquitin-fusion technique (UFT; Baker et al., 1994, J Biol Chem 269: 25381-6; Varshaysky, 2005, Methods Enzymol 399: 777-99) is used. Thus, the ubiquitin (Ub) moiety serves as an initiating protein which allows engineering of the very N-terminal amino acid of an N-degron which is encoded at the Ub-N-degron junction. Ub is cotranslationally cleaved by deubiquitinating enzymes after its last amino acid (Gly76) and leaves the following residue as neo-N-terminus of the C-terminally fused N-degron. Thereby, UFT leads automatically to the required exposure of the desired destabilizing amino acid at the N-terminus of the N-degron.
In the context of the present invention, a reference to a protein or an N-degron which uses or takes advantage of the N-end rule pathway means that such a protein or N-degron has the property of being recognized by and subjected to NERD in a temperature dependent way.
In the context of the present invention, the term ‘protein of interest A or B’ means either one protein A or B or refers to more than one protein A or B, for instance, two, three, four, five, six, seven, eight, nine, ten, eleven or more proteins A or B.
In a particularly preferred embodiment, the term ‘protein of interest B’ may refer to two, three, four, five, six, seven, eight, nine, ten, eleven or more proteins of interest B, which are involved in the development of a reaction compartment.
In a furthermore preferred embodiment, the term ‘protein of interest C’ may refer to two, three, four, five, six, seven, eight, nine, ten, eleven or more proteins of interest C, which are an enzymatic cascade or a part thereof or regulatory elements thereof responsible for or involved in the regulation of an enzymatic activity or of an enzymatic cascade, for the production of a molecule, preferably metabolite, of interest.
In the context of the present invention 5′ is understood to mean upstream from another element within a DNA sequence, whereas 3′ is understood to mean downstream from the respective element.
In the context of the present invention, an ‘endogenous’ gene, allele or protein refers to a non-recombinant sequence of a multicellular organism as the sequence occurs in the respective multicellular organism, in particular wildtype form of the multicellular organism. The term ‘mutated’ refers to a sequence subjected to a genetic manipulation step, preferably carried out by human interaction.
A nucleotide or amino acid sequence is ‘heterologous or exogenous to’ an organism if it originates from a foreign species, or, if from the same species, is modified from its original form. ‘Recombinant’ refers to a human-altered, i.e. transgenic nucleotide or amino acid sequence. A ‘transgene’ is used as the term is understood in the art and refers to a, preferably heterologous, nucleic acid introduced into a cell by human molecular manipulation of the cell's genome, e.g. by molecular transformation. Thus, a ‘transgenic multicellular organism’ is a multicellular organism comprising a transgene, i.e. is a genetically-modified multicellular organism. The transgenic multicellular organism can be the initial multicellular organism into which the transgene was introduced as well as progeny thereof whose genome contains the transgene as well.
The term ‘nucleotide sequence encoding’ or ‘coding sequence’ refers to a nucleic acid which directs the expression of one or more specific protein(s). The nucleotide sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into the protein(s). The nucleotide sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences.
The term ‘gene’ refers to a coding nucleotide sequence and associated regulatory nucleotide sequences.
A ‘promoter’ is a DNA sequence initiating transcription of an associated DNA sequence, in particular being located upstream (5′) from the start of transcription and being involved in recognition and binding of the RNA-polymerase. Depending on the specific promoter region it may also include elements that act as regulators of gene expression such as activators, enhancers, and/or repressors.
The term ‘vector’ refers to a recombinant DNA construct which may be a plasmid, in particular a Ti-plasmid, a transfer-DNA, a virus, autonomously replicating sequence, an artificial chromosome, such as the bacterial artificial chromosome BAC, phage or other suitable nucleotide sequence. A vector may be linear or circular. A vector may be composed of a single or double stranded DNA or RNA.
The term ‘expression’ refers to the transcription and/or translation of an endogenous gene or a transgene in a multicellular organism.
‘Transfecting’ or ‘transforming’ refers to methods to transfer nucleic acid molecules, in particular DNA, into cells including, but not limited to, biolistic approaches such as particle bombardment, microinjection, permeabilizing the cell membrane with various physical, for instance electroporation, or chemical treatments, for instance polyethylene glycol or PEG, treatments; the fusion of protoplasts or Agrobacterium tumefaciens or rhizogenes mediated transformation. For the injection and electroporation of DNA in cells there are no specific requirements for the plasmids used.
In the context of the present invention, the term ‘comprising’ as used herein is understood as to have the meaning of ‘including’ or ‘containing’, which means that in addition to the explicitly mentioned element further elements are possibly present.
In a preferred embodiment of the present invention, the term ‘comprising’ as used herein is also understood to mean ‘consisting of’ thereby excluding the presence of other elements besides the explicitly mentioned element.
In a furthermore preferred embodiment, the term ‘comprising’ as used herein is also understood to mean ‘consisting essentially of’ thereby excluding the presence of other elements providing a significant contribution to the disclosed teaching besides the explicitly mentioned element.
Further preferred embodiments of the present invention are the subject-matter of the subclaims.
The invention will now be described in some more detail by way of the non-limiting examples and figures.
The sequence protocol shows:
SEQ ID no. 1: wildtype mouse DHFR (dihydrofolate reductase) protein.
SEQ ID no. 2: unspecific temperature-sensitive DHFR protein with all putatively substituted amino acids replaced by Xaa.
SEQ ID no. 3: specific temperature-sensitive DHFR protein with two amino acid substitutions (in the following also termed ‘K2’).
SEQ ID nos. 4 to 29: primers used for DNA construct design.
SEQ ID no. 30: a linker.
SEQ ID nos. 31 and 32: hemagglutinin epitope.
To find the underlying molecular cause for the enhanced temperature-sensitivity of the K2-DHFR T39A/E173D (SEQ ID No. 3), compared to the classical yeast DHFR P67L (K1) and a DHFR protein comprising all three substitutions (K3), we predicted and analysed the impact of the three relevant substitutions by molecular dynamics (MD) and constructed models based on the crystal structure. By calculating root mean standard deviation (RMSD), we found enhanced molecular flexibility in the protein structure of all three substitutions indicating that the mutations lead to increased thermolability and cause a higher intramolecular flexibility between the neighbouring amino acid residues and even within entire domains of the degron-DHFR. MD simulations revealed that none of the three tested DHFR variants undergoes unfolding as suggested previously. To further elucidate the molecular origin of increased thermolability in the variants K2 and K3, we modelled the three different point mutations used in the DHFR variants onto the wildtype structure of DHFR (PDB ID: 1U70). By investigating side chain conformations, we found that T39A does not essentially alter the structure of DHFR in the close vicinity of the point of mutation. Nevertheless, the MD simulations show that Lys69 becomes more flexible and possibly also accessible for ubiquitination. Testing the effect of E173D mutation resulted in a higher flexibility and accessibility of Lys174 which was surprisingly accompanied with conformational changes of the side chains of Arg29 and Lys33.
To assess the application spectrum of the lt-degron (low-temperature degradation cassette) of the present invention comprising the K2 variant of the DHFR protein (SEQ ID No. 3) in plants, we chose green fluorescent protein (GFP), β-glucuronidase (GUS), and tobacco etch virus protease (TEV) as additional target proteins to be tested when stably transformed in Arabidopsis. K2: GFP robustly followed the accumulation/depletion regime (
Histological stainings for K2: GUS revealed activity of the fusion protein in vivo at permissive and a significantly reduced activity at restrictive temperature. K2:GUS protein levels and activity were strongly decreased for individuals grown constitutively at restrictive temperature with transcript levels unchanged. In time-course experiments, K2: GUS protein levels increased and correlated with high enzyme activity after shift to permissive temperature and vice versa if shifted to restrictive conditions. We also identified the K2:GUS population with a temperature-dependent hydrolase activity which accumulates at permissive temperature by mass spectrometry. The active K2: GUS population accumulated at the restrictive temperature after the addition of proteasome inhibitor to the plants indicating the degradation of the fusion protein via the ubiquitin/proteasome system.
Next, we applied our low temperature approach to TEV as a target, which is widely used for cleaving proteins in vitro and in vivo. The protease can be expressed for site-specific protein cleavage in many different host organisms without causing adverse effects. In Arabidopsis, K2: TEV transcript levels were unaffected by temperature shifts but the fusion protein accumulated exclusively at permissive temperature.
To test the activity in vivo, we designed the artificial test substrate HA: GST-tev-GFP:His containing epitope-tagged glutathione S-transferase and GFP flanking an internal TEV recognition site and transformed Nicotiana benthamiana (tobacco) plants with both K2:TEV and HA:GST-tev-GFP:His. Here, K2: TEV was depleted from plants shifted to 29° C. and accumulated in individuals grown at 13° C. Consequently, K2:TEV conditionally cleaved the test substrate at permissive temperature. Therefore, a conditional TEV offers opportunities for downstreaming of recombinant proteins by intracellular cleavage. In addition to K2:TEV, also K2: GUS and a fusion of K2 with the agriculturally important herbicide resistance phosphinothricin-N-acetyltransferase (PAT) were also expressed in a conditional manner in tobacco (
To assess functionality of the N-terminal degradation cassette in multicellular organisms, we chose easily scorable, quantitative, and irreversible biological readouts after temperature shifts in vivo, like the development of Arabidopsis thaliana trichomes (leaf hairs) and flower induction (see Example 4). The present temperature-sensitive DHFR (K2) was initiated by phenylalanine, a strongly destabilizing residue in plants. As the first test system, we used the formation of Arabidopsis trichomes, which are a well-established model in cell and developmental biology, and an attractive target for plant metabolic engineering. WD40 protein TRANSPARENT TESTA GLABRA 1 (TTG1) is an essential regulator for trichome development, ttg1 mutants are devoid of trichomes but the mutant phenotype can be rescued by expressing TTG1 under control of the constitutive Cauliflower Mosaic Virus 35S promoter (Pro35S). In this evaluation system, the readout for a functional N-terminal degradation cassette is the number of trichomes formed per leaf after the shift from permissive to restrictive temperature. Under permissive conditions, the degradation cassette is non-functional, i.e. the POI (protein of interest) will accumulate and form trichomes, whereas under restrictive conditions, the degradation cassette is active and leads to the degradation of the fusion protein, and leaves will be devoid of trichomes.
Thus, we fused TTG1 to DHFR T39A/E173D (“K2”), which contains two destabilizing point mutations T39A/E173D. The expression of K2:TTG1 caused a conditional, i.e. a temperature-dependent restoration of trichomes in the ttg1 mutant background. In time-course experiments, decreasing and increasing levels of K2:TTG1 protein could be tracked as a consequence of temperature up- or down-shifts causing depletion versus accumulation (
The high degree of tunability of K2: TTG1 and the conditional induction of trichomes in the mutant background allowed us to perform previously impossible experiments, i.e. to dissect the temporal requirement of TTG1 function during trichome establishment and maturation in great detail (
As a second test system with an easily scorable biological readout, we chose wildtype Arabidopsis plants that conditionally overexpress the transcription factor CONSTANS (CO), a key regulator of flowering time. Overexpressing CO causes a rapid shift from the vegetative to reproductive growth mode and thereby an early flowering. Thus, at permissive temperatures, plants expressing a functional degron-CO fusion protein (CO-td) were expected to flower earlier than plants grown under the restrictive temperature. The readout is hence the days until flowering at permissive temperature in comparison to the wildtype. The lt-degron (low-temperature degradation cassette) system very efficiently worked as expression of K2:CO at the permissive temperature caused early floral induction. Here, the inducible formation of flower meristems occurred about 14 days earlier than in the wild-type when grown at the same permissive temperature (
Thus, conditionally stable CO-td, enabled us to control the onset of flowering and to generate floral meristem cells, i.e. specialized cell types as reaction compartments, on demand.
We then tested the It-degron in Drosophila melanogaster using K2::GFP and K2:TEV as model POIs. K2:GFP was destabilized after a shift from stabilizing 15° to 29° C. in embryonic Kc cells subjected to a temperature shift for 4 h (
Materials and Methods
Plants
Arabidopsis thaliana (L.) Heynh. plants were sown on soil and grown under standard long-day (16/8 hours light/dark) or short day ( 8/16 hours light/dark) greenhouse conditions between 18 and 25° C. Seeds were grown aseptically in vitro under long-day regime (16 h light, 8 h dark) on 0.5% Murashige & Skoog (MS; Duchefa Biochemicals, M0221) containing 1% (w/v) sucrose and 8 g/L phytoagar (Duchefa Biochemicals, P10031). Plants used in this study were all in the background of the Columbia-0 (Col-0; “wildtype” or “wt”) accession and either wild-type plants or T-DNA insertion mutants for TTG1 (ttg1, GABI_580A05, NASC stock ID: N455589). For SEMs, 5 to 7 days old plants were treated for the indicated time. First leaves were not yet visible at the time of the pulse experiments. Before protein isolation, transgenic plants were subjected to temperature shifts by removing the potted plants constantly grown at the indicated starting temperature and shifting them to the corresponding destination temperature. To achieve a rapid acclimatization, plants were immediately watered with water prewarmed or precooled to the final temperature.
DNA Work and Degron Construct Design
DNA cloning was performed following standard procedures using Escherichia coli strain DH5α. All fusions were flanked by Gateway attB1 and -2 sites and recombined by BP reactions into pDONR201 (Invitrogen). The three different N-degron cassettes K1 to K3 were based on a 5′ synthetic human ubiquitin (Ub) gene (Ecker et al., 1987, J Biol Chem 262: 14213-21) and an Ω leader sequence contained in pRTUB8, a derivative of pRTUB1 (Bachmair et al., 1990, EMBO J 9: 4543-9; Bachmair et al., 1993, Proc Natl Acad Sci USA 90: 418-21). The leader contains the 20 nucleotides upstream of the start codon of tobacco mosaic virus strain U1 (Gallie et al., 1987, Proc Natl Acad Sci USA 90: 418-21). A triple hemagglutinin tag (HAT) was amplified from pSKTag3SUM6 (kind gift of Andreas Bachmair). Construct K2 is derived from pJH10mut (pJH23), containing a mutated mouse DHFRT39A,E173D, which was isolated in the yeast mutagenesis screen. Ω leader and Ub were amplified with ND70 (ss12attB1UbcoreN—GGGGA CAAGT TTGTA CAAAA AAGCA GGCTT CCTCG AGCTG CAGAA TTACT ATTTA C) and ND78 (as2UbcoreCDHFRovlpN—GATGC AGTTC AATGG TCGAA CCATG ATTCC AGATC CGTGG AACCC ACCTC TAAGT CTTAA GACAA G), DHFRT39A,E173D from pJH10mut with ND79 (ss2DHFRcoreNUbovlpC—CTTAG AGGTG GGTTC CACGG ATCTG GAATC ATGGT TCGAC CATTG AACTG) and ND80 (as2DHFRovlpCHATcoreN—GTAGG ATCCC ATAGA ACCGTC TTTCT TCTCG T), and HAT with ND81 (ss2HATcoreNovlpDHFRC GAAAG ACGGT TCTAT GGGAT CCTAC CCATA CGAT) and e.g. ND75 (as1234HATcoreCovlpTTG—CTGAA TTATC CATAG CACCA GCACC AGCGT AATCT GGAAC GTCGT ATG) or N138 (asHATNotCO—CTCTC TTGTT TCAAC ATACC AGCGG CCGCA CCAGC GTAAT CTGGA ACGTC GTATG), depending on the fusion partner to follow. For further K2 constructs, an Entry clone containing construct K2:TTG1 was used as a template. A Gateway-compatible Entry clone pEN-L1-K2-L2 containing the K2 degron cassette from Ω leader to shortly after the HAT epitope was generated to construct a K2:POI fusions by Gateway LR reactions. The K2 cassette was amplified from the previously described K2:PAT using primers 21 (K2-Pos2_frw—GCTGC CGCCA TGGGA GGGGA CAAGT TTGTA CAA) and 22 (K2-Pos2_rw—GGGAC CACTT TGTAC AAGAA AGCTG GGTAG GCGCT GCCGC GCGGC A) containing the appropriate attB1/attB2 recombination sites for a Gateway BP reaction.
Reporter and Target Proteins in Expression Constructs
Arabidopsis TTG1 and CO wild-type cDNAs (AT5G24520 and AT5G15840) were amplified with ND76 (ss1234TTGcoreNovlpHATC—GTTCC AGATT ACGCT GGTGC TGGTG CTATG GATAA TTCAG CTCCA GAT) and ND77 (as1234TTGcoreC_attB2—GGGGA CCACT TTGTA CAAGA AAGCT GGGTC TCAAA CTCTA AGGAG CTGCA T; K2:TTG1, K3:TTG1) and subcloned into pDONR201 (Invitrogen). Entry clones were recombined in an LR reaction into the attR site-containing binary Gateway destination vector pLEELA (Jakoby et al., 2006, Plant Physiol 141: 1293-305), containing a double CaMV 35S promoter or pAM-PAT-GW-ProUBQ10. These backbones carry the bar gene from Streptomyces hygroscopicus that translates to phosphinothricin-N-acetyltransferase (PAT) as plant selection marker conferring resistance towards phosphinotricin (glufosinate ammonium). To generate K2:GFP, the K2 containing Entry clone pEN-L1-K2-L2, see above, was recombined with pAM-Kan-35S-GW-GFP. K2:GUS was assembled by amplifying K2 with ND70 and TR08 (asGUS-HAT—GGGGT TTCTA CAGGA CGTAA CATAG CACCA GCACC AGCGT AATCT GGAAC) from K2:TTG1, as well as by TROT (ssHAT-GUS—GTTCC AGATT ACGCT GGTGC TGGTG CTATG TTACG TCCTG TAGAA ACCCC) and TR06 (asattB2+GUS—GGGGA CCACT TTGTA CAAGA AAGCT GGGTC TTATT GTTTG CCTCC C) which amplifies GUS from pENTR-gus (uidA CDS in pDONR1). K2:GUS was fused using ND70 and TR06 and introduced into pAM-PAT GW ProUBQ10 by LR reaction. For K2:TEV, K2 was amplified from K2:TTG1 with primers 41 (td-fwd—GGGGA CAAGT TTGTA CAAAA AAGC) and 36 (L4-HArev—CGCTC ATGGG GTGAT GGTGA TGGTG ATGTT TCATA GCGTA ATCTG GAACG TCGTA TG). TEV was isolated from pCT190-6 (Taxis et al., 2009, Mol Syst Biol 5: 267) with primers 46 (LINKER2FORWARD—ATCAC CATCA CCCCA TGAGC GGCCT GGTGC CGCGC GGCAG CGCC) and 29 (TEVREVERSE/TEVrev—TTACC CTTGC GAGTA CACCA ATTCA). By this, a linker sequence (“L4”) was generated comprising both triple HA and hexahistidine tags. K2 and TEV were fused with primers 41 and 29. K2:TEV was cloned into pCRII-TOPO (Invitrogen) by TOPO cloning between the XhoI and SpeI sites and then cloned into pAM-PAT-MCS using EcoRI. K2 for K2:PAT was amplified from K2:TTG1 with ND70 and TR11 (asPAT-HAT—GCCGG GCGTC GTTCT GGGCT CATAG CACCA GCACC AGCGT AATCT GGAAC) and PAT from pLEELA with TR10 (ssHAT-PAT—GTTCC AGATT ACGCT GGTGC TGGTG CTATG AGCCC AGAAC GACGC CCGGC) and TR09 (asattB2+PAT—GGGGA CCACT TTGTA CAAGA AAGCT GGGTC TCAGA TTTCG GTGAC GGGCA GGACC GG). The fusion was accomplished with ND70 and TR09. K2:PAT was recombined into pJan33 (double CaMV 35S promoter fused to the first WRYKY33 intron, selectable marker NPTII).
Drosophila Constructs
K2 from pEN-L1-K2-L2 was introduced into pAWG (DGRC; Murphy, T. D., et al. Construction and application of a set of Gateway vectors for expression of tagged proteins in Drosophila, unpublished data) via a Gateway LR reaction. ProActin5c::K2:TEV for cell culture transfection and transformation of flies contains DHFRT39A,E173D, amplified from an Entry clone carrying K2:TTG1 using primers 36 and 37 (F-DHFR—TTCCA CGGAT CTGGA ATCAT GG; Phe-K2) or primers 36 and 38 (M-DHFR—ATGCA CGGAT CTGGA ATCAT GG; Met-K2). Instead of the synthetic plant-optimized Ub, a Ub sequence from S. cerevisiae (Bachmair et al., 1986, Science 234: 179-86) was used and amplified with primers 9 (Ub-fwd—CACCA TGCAG ATTTT CGTCA AGACT TTGAC) and 39 (F-DHFR-UBrev—CCATG ATTCC AGATC CGTGG AAACC ACCTC TTAGC CTTAG CAC; Phe-K2) or primers 9 and 40 (M-DHFR-UBrev—CCATG ATTCC AGATC CGTGC ATACC ACCTC TTAGC CTTAG CAC; Met-K2). The resulting fragments were fused to Ub:X-DHFRT39A,E173D with primers 9 and 36 (L4-HArev—CGCTC ATGGG GTGAT GGTGA TGGTG ATGTT TCATA GCGTA ATCTG GAACG TCGTA TG). TEV protease was amplified from pCT190-6 with primers 46 (LINKER2FORWARD—ATCAC CATCA CCCCA TGAGC GGCCT GGTGC CGCGC GGCAG CGCC) and 29 (TEVREVERSE/TEVrev—TTACC CTTGC GAGTA CACCA ATTCA) and K2 combined with TEV with primers 9 and 29. The construct was then subcloned in pCRII-TOPO as mentioned above. K2:TEV was prepared as an XhoI-SpeI fragment which was partially cut to leave the internal SpeI site unaffected. The fragment was ligated into pAW (DGRC).
Plant Transformation and Selection of Transformants
The resulting binary plant Expression vectors were retransformed into Agrobacterium tumefaciens GV3101-pMP90RK (C58C1 Rifr Gmr Kmr) (Koncz and Schell, 1986, Mol Gen Genet 204: 383-96) and transformed by a modified version of the floral dip method (Dissmeyer and Schnittger, 2011, Methods Mol Biol 779: 93-138). For transient transformation of tobacco, leaves of four-week-old plants were infiltrated with Agrobacteria, carrying binary plant expression vectors. Bacteria suspensions were infiltrated into the epidermis on the lower side of the tobacco leaf. To allow for efficient transformation and expression plants were kept for 48 h in the greenhouse, before applying temperature shift experiments by putting them into a growth cabinet at either permissive or restrictive growth conditions. For one data point, 15 leaf discs of 5 mm diameter were harvested from infiltrated areas and snap frozen in liquid nitrogen. Extraction was performed using RIPA buffer as mentioned above for Arabidopsis.
Drosophila Work
Drosophila melanogaster Kc (Echalier and Ohanessian, 1969, Methods Mol Biol 779: 93-138) or Schneider 2 (S2) cells (Schneider, 1972) Embryol Exp Morphol 27: 353-65) were grown at 24° C. in Schneider's medium (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were passaged into fresh media every 5 days in a 1:10 dilution. Transfection was carried out in a 12-well dish using the Effectene transfection kit (Qiagen) according to the manufacturer's protocol for adherent cells. Temperature shifts were carried out at 15° C. or 29° C. respectively. Vectors carrying Actin5c::Phe-K2:TEV (F-K2:TEV) or Actin5c::Met-K2:TEV (M-K2:TEV) were injected in ZH-attP-2A or ZH-attP-51D embryos for phiC31 recombinase transgenesis system64 using standard Drosophila transgenesis methods. Transformants were grown at 25° C. standard conditions. To analyse constructs activity, adult flies were shifted either at 18° C. (permissive temperature) or 29° C. (restrictive temperature). After 24 h, total protein extracts were obtained from whole flies homogenized in standard lysis buffer.
Protein Extraction and Western Blot Analysis
Tissue of interest (Arabidopsis: leafs or seedlings, tobacco: 20 leaf discs of 5 mm diameter of an infiltrated area) was collected in a standard 2 mL reaction tube containing three Nirosta stainless steel beads (3.175 mm; cat. no. 75306, Malmeier) snap frozen in liquid nitrogen and stored at −80° C. until use. Material was ground frozen using a bead mill (Retsch; 45 s, 30 Hz) in collection microtube blocks (adaptor set from TissueLyser II, 69984, Qiagen). For K2:TTG1 time-courses, per time-point, one leaf was ground in 200 μL extraction buffer (Tris-Cl 50 mM (pH 7.6), NaCl 150 mM, EDTA 5 mM, SDS 0.1% (w/v), β-mercaptoethanol 0.1% (w/v), EDTA-free Complete Protease Inhibitor Cocktail (Roche Diagnostics)). Alternatively, tissue was lysed using radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris-Cl (pH 8), 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 1 mM benzamidine hydrochloride, 15 mM Na4P2O7, and 1% Nonidet P-40 supplemented with EDTA-free Complete Protease Inhibitor cocktail (Roche Diagnostics) added freshly. Extraction of K2:TEV protease was done in 25 mM Tris-Cl (pH 7.5), 75 mM NaCl, 15 mM MgCl2, 15 mM EGTA pH 8.0, 0.1% (v/v) Tween 20, 0.1% (v/v) Triton X-100, 5 mM DTT and EDTA-free Complete Protease Inhibitor Cocktail (Roche Diagnostics) in a chilled cooling block at 4° C. and 800 rpm for 30 min. Insoluble cell debris was pelleted via centrifugation for 20 min at 4° C. and 20,000 g, the supernatant was transferred to a fresh tube. Drosophila cells were harvested via centrifugation at 4° C. for 5 min at 300 g. The pellet was washed once with ice-cold PBS and cells lysed using RIPA buffer containing 50 mM Tris-Cl pH 8, 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 1 mM benzamidine hydrochloride 15 mM Na4P2O7, and 1% Nonidet P-40 with EDTA-free Complete Protease Inhibitor cocktail (Roche Diagnostics). Lysis was carried out on a chilled thermo block at 4° C. and 800 rpm for 20 min. Insoluble cell debris was pelleted via centrifugation for 20 min at 4° C. and 20,000 g, the supernatant was transferred to a fresh tube. S. cerevisiae cultures were grown at 30° C. to logarithmic growth phase, and cycloheximide was added to the cultures at a final concentration of 100 mg/L. Strains carrying temperature-sensitive alleles were expanded at permissive 25° C. and incubated at restrictive 37° C. for 30 min before CHX addition. Yeast proteins were extracted from samples of exponentially growing suspension culture (5-50 mL; OD600=0.8-1.2) at the time points indicated by centrifugation and grinding with glass beads at 4° C. in 150-300 μL Native lysis buffer (50 mM Na-HEPES (pH 7.5); 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, containing EDTA-free Complete Protease Inhibitor Cocktail (Roche Diagnostics), 20 μM MG132, followed by a second centrifugation (10 min, 4° C., 20,000 g).
Quantification of Glucoronidase Activity
Proteins from two-week-old seedlings of K2:GUS plants were extracted in GUS extraction buffer (50 mM Na-phosphate, 10 mM EDTA, 0.1% (w/v) SDS, 0.1% (v/v) Triton X-100, and 10 mM β-mercaptoethanol freshly added) (Kim et al., 2006, Methods Mol Biol 323: 263-73). The GUS extraction buffer was complemented with freshly added EDTA-free protease inhibitor cocktail (Roche). The assay was carried out by mixing 196 μL assay solution containing 1 mM 4-methylumbelliferyl-β-D-glucuronide (4-MUG) with 4 μL of GUS protein extract in a white 96-well plate. Samples were measured for a total of 60 min with one data point taken every minute and normalized to a 4-MU standard. Activity was normalized and determined as pmol 4-MU per min and mg total protein.
RNA Work and RT-PCR
About 50 mg of plant material were used for RNA extraction using the RNeasy Plant Mini Kit (Qiagen). For first-strand cDNA synthesis, 500 ng of total RNA were used with an equimolar mixture of four oligo(dT) primers (CDSIII-NotIA, CDSIII-NotIC, CDSIII-NotIG, CDSIII-NotIT) containing 30 desoxythymidines and XbaI and NotI sites each and RevertAid H Minus Reverse Transcriptase (Thermo Scientific). 1 μL of cDNA was used for subsequent PCR analysis using self-made Taq DNA polymerase. For each sample, two reactions were carried out. One with generic degron specific primers (DHFR_frw and DHFR_rev) to test transcript levels of the transgene.
Microscopical Work
Scanning electron microscopy was done with a SUPRA 40VP (Carl Zeiss MicroImaging) equipped with a K1250X Cryogenic SEM Preparation System (EMITECH), a CPD 030 critical point dryer (Bal-Tec), and SC 7600 sputter coater (Polaron) at the on-campus microscopy core facility Zentrale Mikroskopie (CeMic) of the Max Planck Institute for Plant Breeding Research at Cologne. Light and confocal laser scanning microscopy (CLSM) was performed with an LSM710 system (Carl Zeiss MicroImaging). K2:GFP fluorescence was observed in root tips of plants that were aseptically grown for 2 weeks under long-day conditions at either constitutively restrictive (13° C.) or permissive (29° C.) temperatures. Photographs of in vitro cultures and histological stainings were taken with a stereo microscope (Stemi 2000-C, Carl Zeiss MicroImaging) equipped with a Zeiss CL 6000 LED illumination unit, and a video adapter 60 C including an AxioCam ERc 5s digital camera (Carl Zeiss MicroImaging).
| Number | Date | Country | Kind |
|---|---|---|---|
| 15156650.2 | Feb 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/053849 | 2/24/2016 | WO | 00 |
