Patent Grant
10570412
This application is a National Stage application of International Application No. PCT/EP2016/052019, filed Feb. 1, 2016, which claims priority to European Patent Application No. 15153820.4, filed on Feb. 4, 2015.
The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a text file. The name of the text file containing the Sequence Listing is “76603_Seqlisting.txt”, which was created on Aug. 1, 2017 and is 43,652 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.
The present invention relates to a method of increasing resistance against fungal pathogens, in particular soybean rust and/or Fusarium graminearum and/or Fusarium verticillioides, in plants, plant parts, and/or plant cells. This is achieved by increasing the content of scopoletin and/or a derivative thereof, in particular by increasing the expression of F6H1 in a plant, plant part and/or plant cell. This can also be achieved by application of a formulation or solution containing scopoletin and/or a derivative thereof.
Furthermore, the invention relates to recombinant expression vector constructs comprising a sequence that is identical or homologous to a sequence encoding F6H1 protein.
The cultivation of agricultural crop plants serves mainly for the production of foodstuffs for humans and animals. Monocultures in particular, which are the rule nowadays, are highly susceptible to an epidemic-like spreading of diseases. The result is markedly reduced yields. To date, the pathogenic organisms have been controlled mainly by using pesticides. Nowadays, the possibility of directly modifying the genetic disposition of a plant or pathogen is also open to man. Alternatively, natural occurring fungicides produced by the plants after fungal infection can be synthesized and applied to the plants.
Resistance generally describes the ability of a plant to prevent, or at least curtail the infestation and colonization by a harmful pathogen. Different mechanisms can be discerned in the naturally occurring resistance, with which the plants fend off colonization by phytopathogenic organisms (Schopfer and Brennicke (1999) Pflanzenphysiologie, Springer Verlag, Berlin-Heidelberg, Germany).
With regard to the race specific resistance, also called host resistance, a differentiation is made between compatible and incompatible interactions. In the compatible interaction, an interaction occurs between a virulent pathogen and a susceptible plant. The pathogen survives, and may build up reproduction structures, while the host is seriously hampered in development or dies off. An incompatible interaction occurs on the other hand when the pathogen infects the plant but is inhibited in its growth before or after weak development of symptoms (mostly by the presence of R genes of the NBS-LRR family, see below). In the latter case, the plant is resistant to the respective pathogen (Schopfer and Brennicke, vide supra). However, this type of resistance is mostly specific for a certain strain or pathogen.
In both compatible and incompatible interactions a defensive and specific reaction of the host to the pathogen occurs. In nature, however, this resistance is often overcome because of the rapid evolutionary development of new virulent races of the pathogens (Neu et al. (2003) American Cytopathol. Society, MPMI 16 No. 7: 626-633).
Most pathogens are plant-species specific. This means that a pathogen can induce a disease in a certain plant species, but not in other plant species (Heath (2002) Can. J. Plant Pathol. 24: 259-264). The resistance against a pathogen in certain plant species is called non-host resistance. The non-host resistance offers strong, broad, and permanent protection from phytopathogens. Genes providing non-host resistance provide the opportunity of a strong, broad and permanent protection against certain diseases in non-host plants. In particular, such a resistance works for different strains of the pathogen.
Fungi are distributed worldwide. Approximately 100 000 different fungal species are known to date. Thereof rusts are of great importance. They can have a complicated development cycle with up to five different spore stages (spermatium, aecidiospore, uredospore, teleutospore and basidiospore).
During the infection of plants by pathogenic fungi, different phases are usually observed. The first phases of the interaction between phytopathogenic fungi and their potential host plants are decisive for the colonization of the plant by the fungus. During the first stage of the infection, the spores become attached to the surface of the plants, germinate, and the fungus penetrates the plant. Fungi may penetrate the plant via existing ports such as stomata, lenticels, hydatodes and wounds, or else they penetrate the plant epidermis directly as the result of the mechanical force and with the aid of cell-wall-digesting enzymes. Specific infection structures are developed for penetration of the plant. To counteract plants have developed physical barriers, such as wax layers, and chemical compounds having antifungal effects to inhibit spore germination, hyphal growth or penetration.
The soybean rust Phakopsora pachyrhizi directly penetrates the plant epidermis. After crossing the epidermal cell, the fungus reaches the intercellular space of the mesophyll, where the fungus starts to spread through the leaves. To acquire nutrients the fungus penetrates mesophyll cells and develops haustoria inside the mesophyl cell. During the penetration process the plasmamembrane of the penetrated mesophyll cell stays intact.
Fusarium species are important plant pathogens that attacks a wide range of plant species including many important crops such as maize and wheat. They cause seed rots, seedling blights as well as root rots, stalk rots and ear rots. Pathogens of the genus Fusarium infect the plants via infected seeds, roots or silks or they penetrate the plant via wounds or natural openings and cracks. After a very short establishment phase the Fusarium fungi start to secrete mycotoxins such as trichothecenes, zearalenone and fusaric acid into the infected host tissues leading to cell death and maceration of the infected tissue. Nourishing from dead tissue the fungus then starts to spread through the infected plant leading to severe yield losses and decreases in quality of the harvested grain.
Biotrophic phytopathogenic fungi depend for their nutrition on the metabolism of living cells of the plants. This type of fungi belong to the group of biotrophic fungi, like many rust fungi, powdery mildew fungi or oomycete pathogens like the genus Phytophthora or Peronospora. Necrotrophic phytopathogenic fungi depend for their nutrition on dead cells of the plants, e.g. species from the genus Fusarium, Rhizoctonia or Mycospaerella. Soybean rust has occupied an intermediate position, since it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic. After the penetration, the fungus changes over to an obligatory-biotrophic lifestyle. The subgroup of the biotrophic fungal pathogens which follows essentially such an infection strategy are heminecrotrohic.
Scopoletin and scopolin are antimicrobial phenolic hydroxycumarins that accumulate in different plants upon infection with various pathogens such as fungi or bacteria or in response to insect feeding damage, mechanical injury, dehydration or various other abiotic stresses.
Scopoletin shows broad antimicrobial activity and can inhibit development and growth of various fungi or bacteria in vitro (Goy, P. A., Signer, H., Reist, R., Aichholz, R., Blum, W., Schmidt, E., and Kessmann, H. (1993). Accumulation of scopoletin is associated with the high disease resistance of the hybrid Nicotiana glutinosa×Nicotiana debneyi. Planta 41: 200-206; Tal, B. and Robeson, D. J. (1986b). The Metabolism of Sunflower Phytoalexins Ayapin and Scopoletin: Plant-Fungus Interactions. Plant Physiology 82: 167-172.).
Scopoletin and its glucoside scopolin originate from the phenylpropanoid pathway (
Key steps of scopletin/scopolin synthesis comprise ortho hydroxylation of feruloyl-CoA, trans/cis isomeration of the side chain, lactonization and—considering scopolin synthesis—glycosylation (Kai et al., 2008). In Arabidopsis it has recently been shown that scopoletin production depends on ortho hydroxylation of feruloyl-CoA by the Fe(II)- and 2-oxoglutarate-dependent dioxygenase F6H1 (At3g13610). E-Z isomerisation of the side chain and lactonization were found to occur spontaneously. (Kai et al., 2008).
In planta accumulating scopoletin can finally be glucosylated to produce scopolin. Several Arabidopsis glucosyltransferases (e.g. UGT71C1) (Lim, E.-K., Baldauf, S., Li, Y., Elias, L., Worrall, D., Spencer, S. P., Jackson, R. G., Taguchi, G., Ross, J., and Bowles, D. J. (2003). Evolution of substrate recognition across a multigene family of glycosyltransferases in Arabidopsis. Glycobiology 13: 139-45.) as well as two different tobacco glucosyltransferases (Togt1 and Togt2) (Fraissinet-Tachet, L., Baltz, R., Chong, J., Kauffmann, S., Fritig, B., and Saindrenan, P. (1998). Two tobacco genes induced by infection, elicitor and salicylic acid encode glucosyltransferases acting on phenylpropanoids and benzoic acid derivatives, including salicylic acid. FEBS letters 437: 319-23) have been identified that can catalyze glycosylation of scopoletin in vitro.
Scopolin is generally regarded a less potent antimicrobial agent than scopoletin. Following pathogen-induced mechanical injury or hypersensitive reactions (HR), decompartimentalization of scopolin containing cells might lead to the release of scopolin from vacuoles into the cytoplasm and subsequent hydrolysis of the glucose conjugate by β-glucosidases.
Scopoletin and its glucoside scopolin are widely distributed among the plant kingdom and have been detected in various plant organs of approximately 80 different plant families. Interestingly, scopoletin biosynthesis seems to be lost in several economically important crops (e.g. Glycine max, Zea mays, Triticum aestivum, Oryza sativa etc.), indicating that the ability to synthesize this antimicrobial substance might have been lost during breeding. However, this does not apply to sweet potato, tobacco, sunflower, cotton or cassava since scopoletin has been shown to accumulate in these crops in response to infection (summarized by Gnonlonfin, G. J. B., Sanni, A., and Brimer, L. (2012). Review Scopoletin—A Coumarin Phytoalexin with Medicinal Properties. Critical Reviews in Plant Sciences 31: 47-56).
Soybean rust has become increasingly important in recent times. The disease may be caused by the biotrophic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts infect a wide spectrum of leguminosic host plants. P. pachyrhizi, also referred to as Asian rust, is the more aggressive pathogen on soy (Glycine max), and is therefore, at least currently, of great importance for agriculture. P. pachyrhizi can be found in nearly all tropical and subtropical soy growing regions of the world. P. pachyrhizi is capable of infecting 31 species from 17 families of the Leguminosae under natural conditions and is capable of growing on further 60 species under controlled conditions (Sinclair et al. (eds.), Proceedings of the rust workshop (1995), National SoyaResearch Laboratory, Publication No. 1 (1996); Rytter J. L. et al., Plant Dis. 87, 818 (1984)). P. meibomiae has been found in the Caribbean Basin and in Puerto Rico, and has not caused substantial damage as yet.
P. pachyrhizi can currently be controlled in the field only by means of fungicides. Soy plants with resistance to the entire spectrum of the isolates are not available. When searching for resistant soybean accessions, six dominant R-genes of the NBS-LRR family, which mediate resistance of soy to P. pachyrhizi, were discovered. The resistance was lost rapidly, as P. pachyrhizi develops new virulent races.
Increasing resistance to Fusarium is one of the most important goals in maize breeding. Despite having a great natural diversity in interaction phenotypes with Fusarium species, resistance seems to be distributed over many weak QTLs with low heritability. Therefore only little progress was made in increasing resistance against Fusarium by breeding.
In recent years, fungal diseases, e.g. soybean rust and Fusarium graminearum have gained in importance as pest in agricultural production. There was therefore a demand in the prior art for developing methods to control fungi and to provide fungal resistant plants.
Much research has been performed on the field of powdery and downy mildew infecting the epidermal layer of plants. However, the problem to cope with soybean rust which infects the mesophyll or Fusarium fungi that infect inaccessible inner tissues remains unsolved.
The object of the present invention is inter alia to provide a method of increasing resistance against fungal pathogens, preferably against fungal pathogens of the family Phakopsoraceae, more preferably against fungal pathogens of the genus Phakopsora, most preferably against Phakopsora pachyrhizi (Sydow) and/or Phakopsora meibomiae (Arthur), also known as soybean rust.
A further object of the present invention is inter alia to provide a method of increasing resistance against fungal pathogens, preferably against fungal pathogens of the genus Fusarium, most preferably against Fusarium graminearum and/or Fusarium verticillioides.
Surprisingly, we found that fungal pathogens, in particular of the genus Phakopsora, for example soybean rust and/or of the genus Fusarium, for example Fusarium graminearum and/or Fusarium verticillioides, can be controlled by increased production or increased accumulation of scopoletin or derivatives thereof in a plant and by direct application of scopoletin or derivatives thereof to the plant.
Surprisingly, we found that fungal pathogens, in particular of the genus Phakopsora, for example soybean rust and of the genus Fusarium, for example Fusarium graminearum and/or Fusarium verticillioides, can be controlled by increased expression of the F6H1 protein, optionally in combination with one or more proteins selected from the group consisting of CCoAOMT1, ABCG37 and UGT71C1.
The present invention therefore provides a method of increasing resistance against fungal pathogens, preferably against fungal pathogens of the family Phakopsoraceae and/or Nectriaceae, more preferably against fungal pathogens of the genus Phakopsora and/or Fusarium, most preferably against Phakopsora pachyrhizi (Sydow), Phakopsora meibomiae (Arthur), Fusarium graminearum and/or Fusarium verticillioides in transgenic plants, plant parts, or transgenic plant cells by increasing the production and/or accumulation of scopoletin and/or derivatives thereof or by exogenous application of scopoletin and/or derivatives thereof to plants, plant parts, or plant cells.
A further object is to provide transgenic plants resistant against fungal pathogens, preferably of the family Phakopsoraceae and/or Nectriaceae, more preferably against fungal pathogens of the genus Phakopsora and/or Fusarium, most preferably against Phakopsora pachyrhizi (Sydow), Phakopsora meibomiae (Arthur), Fusarium graminearum and/or Fusarium verticillioides, a method for producing such plants as well as a recombinant vector construct useful for the above methods.
The present invention also refers to a recombinant vector construct and a transgenic plant, plant part, or plant cell comprising exogenous nucleic acids or fragment thereof which lead to enhanced production of scopoletin and/or derivatives thereof. Furthermore, a method for the production of a transgenic plant, plant part or plant cell using the nucleic acids of the present invention is claimed herein. In addition, the use of a nucleic acid or the recombinant vector of the present invention for the transformation of a plant, plant part, or plant cell is claimed herein.
The present invention also refers to method for applying a scopoletin and/or derivatives to a surface of a plant, plant part or plant cell as well as plant surface or plant part surface coated with scopoletin and/or derivatives.
The objects of the present invention, as outlined above, are achieved by the subject-matter of the main claims. Preferred embodiments of the invention are defined by the subject matter of the dependent claims.
(a) 3-O-methylation of the caffeate unit occurs mainly via CCoAOMT1 using caffeoyl CoA. Ortho-hydroxylation of feruloyl CoA is catalyzed by F6H1, followed by trans/cis isomerization of the side chain and lactonization to form scopoletin. C3H, p-coumarate 3-hydroxylase; 4CL, 4-coumarate:CoA ligase.
(b) An ionic mechanism of trans/cis isomerization of the side chain and lactonization is proposed for the thioester.
Untransformed (wildtype) N. benthamiana is not able to produce Scopoletin. Transient expression of the F6H1 enzyme (original sequence (F6H1,
Spores of Phakopsora pachyrhizi were germinated on glass slides in water containing 0 (grey dotted bar), 10 μM (vertically striped bar), 100 μM (diagonally striped bar), 500 μM (horizontally striped bar) and 1 mM (black bar) scopoletin. Morphological status of spores was determined microscopically 6 h after inoculation (see example 5a). Quantitative microscopic analysis showed that the germination and appressorium formation of Phakopsora pachyrhizi is strongly inhibited by the presence of scopoletin in a dose dependent manner.
Leaves of soybean plants were treated with 10 μM, 100 μM or 1 mM scopoletin in parallel with the inoculation with P. pachyrhizi (Co-application). Plants not treated with Scopoletin are marked as control (see example 6.2). The diseased leaf area was assessed according to
Quantitative analysis of the ratio of the infected leaf area showed that the diseased leaf area caused by Phakopsora pachyrhizi infection is strongly reduced in a dose dependent manner by co-application of scopoletin.
Primary leaves (grey dotted bars) or first trifoliate leaves (vertically striped bars) and second trifoliate leaves (diagonally striped bars) of soy plants were treated with 1 mM scopoletin either 6 h before inoculation with P. pachyrhizi (Pre-treatment) or in parallel with the inoculation with P. Pachyrhizi (Co-application). Plants not treated with scopoletin are marked “ASR-only”)(see example 6.2). The diseased leaf area was assessed according to
Quantitative analysis of the ratio of the infected leaf area showed that the diseased leaf area caused by Phakopsora pachyrhizi infection is strongly reduced by the either pre-treatment or co-application of 1 mM scopoletin on primary leaves (grey dotted bars) and first trifoliate leaves (vertically striped bars) and second trifoliate leaves (diagonally striped bars).
The presence of 1 mM scopoletin in the agar leads to a reduction of the Fusarium graminearum growth rate per day by 61% in comparison to Fusarium graminearum grown on PDA+methanol, indicating that scopoletin is also toxic against Fusarium graminearum.
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the examples included herein.
Definitions
Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. In addition to the definitions of terms provided herein, definitions of common terms in molecular biology may also be found in Rieger et al., 1991 Glossary of genetics: classical and molecular, 5th Ed., Berlin: Springer-Verlag; and in Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1998 Supplement).
It is to be understood that as used in the specification and in the claims, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a cell” can mean that at least one cell can be utilized. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.
Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al., 1989 Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I; Wu (Ed.) 1979 Meth Enzymol. 68; Wu et al., (Eds.) 1983 Meth. Enzymol. 100 and 101; Grossman and Moldave (Eds.) 1980 Meth. Enzymol. 65; Miller (Ed.) 1972 Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose, 1981 Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink, 1982 Practical Methods in Molecular Biology; Glover (Ed.) 1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender 1979 Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.
“Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and/or enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having the same, essentially the same biological activity or similar as the unmodified protein from which they are derived.
“Homologues” of a nucleic acid encompass nucleotides and/or polynucleotides having nucleic acid substitutions, deletions and/or insertions relative to the unmodified nucleic acid in question, wherein the protein coded by such nucleic acids has the same, essentially the same or similar biological activity as the unmodified protein coded by the unmodified nucleic acid from which they are derived. In particular, homologues of a nucleic acid may encompass substitutions on the basis of the degenerative amino acid code.
The terms “identity”, “homology” and “similarity” are used herein interchangeably. “Identity” or “homology” or “similarity” between two nucleic acids sequences or amino acid sequences refers in each case over at least 70%, at least 80% or at least 90% of the entire length of the respective F6H1, CCoAOMT, ABCG37 and/or UGT71C1 nucleic acid sequence or the respective F6H1, CCoAOMT, ABCG37 and/or UGT71C1 amino acid sequence, preferably over the entire length of the respective F6H1, CCoAOMT, ABCG37 and/or UGT71C1 nucleic acid sequence or the respective F6H1, CCoAOMT, ABCG37 and/or UGT71C1 amino acid sequence.
Preferably, “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over a particular region, determining the number of positions at which the identical base or amino acid occurs in both sequences in order to yield the number of matched positions, dividing the number of such positions by the total number of positions in the region being compared and multiplying the result by 100.
Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity or similarity or homology and performs a statistical analysis of the identity or similarity or homology between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity/homology/identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates similarity/homology/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1); 195-7).
The sequence identity may also be calculated by means of the Vector NTI Suite 7.1 program of the company Informax (USA) employing the Clustal Method (Higgins D G, Sharp P M. Fast and sensitive multiple sequence alignments on a microcomputer. Comput Appl. Biosci. 1989 April; 5(2):151-1) with the following settings:
Multiple Alignment Parameter:
Alternatively the identity may be determined according to Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment with the Clustal series of programs. (2003) Nucleic Acids Res 31 (13):3497-500, the web page: http://www.ebi.ac.uk/Tools/clustalw/index.html# and the following settings
Sequence identity between the nucleic acid or protein useful according to the present invention and the F6H1, CCoAOMT, ABCG37 and UGT71C1 nucleic acids and the F6H1, CCoAOMT, ABCG37 and UGT71C1 proteins may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide or protein sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group).
A “deletion” refers to removal of one or more amino acids from a protein or to the removal of one or more nucleic acids from DNA, ssRNA and/or dsRNA.
An “insertion” refers to one or more amino acid residues or nucleic acid residues being introduced into a predetermined site in a protein or the nucleic acid.
A “substitution” refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or beta-sheet structures).
On the nucleic acid level a substitution refers to a replacement of one or more nucleotides with other nucleotides within a nucleic acid, wherein the protein coded by the modified nucleic acid has essentially the same or a similar function. In particular homologues of a nucleic acid encompass substitutions on the basis of the degenerative amino acid code.
Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the protein and may range from 1 to 10 amino acids; insertions or deletion will usually be of the order of about 1 to 10 amino acid residues. The amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Taylor W. R. (1986) The classification of amino acid conservation J Theor Biol., 119:205-18 and Table 1 below).
Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation.
Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gene in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
The terms “encode” or “coding for” is used for the capability of a nucleic acid to contain the information for the amino acid sequence of a protein via the genetic code, i.e., the succession of codons each being a sequence of three nucleotides, which specify which amino acid will be added next during protein synthesis. The terms “encode” or “coding for” therefore includes all possible reading frames of a nucleic acid. Furthermore, the terms “encode” or “coding for” also applies to a nucleic acid, which coding sequence is interrupted by non-coding nucleic acid sequences, which are removed prior translation, e.g., a nucleic acid sequence comprising introns.
The term “domain” refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein.
Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.
The nucleic acids according to the present invention may comprise domains as defined herein below when analysed with the software tool InterProScan (version 4.8, (see Zdobnov E. M. and Apweiler R.; “InterProScan—an integration platform for the signature-recognition methods in InterPro.”; Bioinformatics, 2001, 17(9): 847-8; InterPro database, release 42 (Apr. 4, 2013)).
As used herein the terms “fungal-resistance”, “resistant to a fungus” and/or “fungal-resistant” mean reducing, preventing, or delaying an infection by fungi. Preferably fungal resistance is soybean rust-resistance and/or fusarium-resistance. The term “resistance” refers to fungal resistance. Resistance does not imply that the plant necessarily has 100% resistance to infection. In preferred embodiments, enhancing or increasing fungal resistance means that resistance in a resistant plant is greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% in comparison to a wild type plant. Preferably the wild type plant is a plant of a similar, more preferably identical, genotype as the plant having increased resistance to fungi, in particular soy-rust and or fusarium, but does not comprise an exogenous F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAOMT, ABCG37 and UGT71C1 nucleic acids. Preferably, the wildtype plant is not capable to produce more than 10 μM scopoletin and/or a derivative thereof, more preferably more than 5 μM scopoletin and/or a derivative thereof, most preferably the wildtype plant is not capable to produce scopoletin and/or a derivative thereof.
As used herein the terms “soybean rust-resistance”, “resistant to a soybean rust”, “soybean rust-resistant”, “rust-resistance”, “resistant to a rust”, or “rust-resistant” mean reducing or preventing or delaying an infection of a plant, plant part, or plant cell by Phacopsoracea, in particular Phakopsora, more particularly soybean rust or Asian Soybean Rust (ASR), more particularly Phakopsora pachyrhizi, Phakopsora meibomiae and/or Fusarium solani—also known as, as compared to a wild type plant, wild type plant part, or wild type plant cell.
As used herein the terms “fusarium-resistance”, “resistant to a fusarium”, or “fusarium-resistant” mean reducing or preventing or delaying an infection of a plant, plant part, or plant cell by Fusarium, in particular Fusarium graminearum, Fusarium sporotrichioides, Fusarium pseudograminearum, Fusarium culmorum, Fusarium poae, Fusarium verticillioides (Fusarium moniliforme), Fusarium subglutinans, Fusarium proliferatum, Fusarium fujikuroi), Fusarium avenaceum, Fusarium oxysporum, Fusarium virguliforme and/or Fusarium solani as compared to a wild type plant, wild type plant part, or wild type plant cell.
The level of fungal resistance of a plant can be determined in various ways, e.g. by scoring/measuring the infected leaf area or three-dimensional space in relation to the overall area or three-dimensional space. Another possibility to determine the level of resistance is to count the number of fusarium colonies on the plant or to measure the amount of spores produced by these colonies. Another way to resolve the degree of fungal infestation is to specifically measure the amount of fungal DNA by quantitative (q) PCR. Specific probes and primer sequences for most fungal pathogens are available in the literature (Frederick R D, Snyder C L, Peterson G L, et al. 2002 Polymerase chain reaction assays for the detection and discrimination of the rust pathogens Phakopsora pachyrhizi and P. meibomiae, Phytopathology 92(2) 217-227). (Nicolaisen M, Suproniene S, Nielsen L K, Lazzaro I, Spliid N H, Justesen A F. 2009 Real-time PCR for quantification of eleven individual Fusarium species in cereals. J Microbiol Methods. 2009 March; 76(3): 234-40.) Another way of evaluating fungal biomass is to biochemically determining the amount of fungal specific compounds, such as ergosterol or chitin (L. M. Reid, R. W. Nicol, T. Ouellet, M. Savard, J. D. Miller, J. C. Young, D. W. Stewart, and A. W. Schaafsma (1999) Interaction of Fusarium graminearum and F. moniliforme in Maize Ears: Disease Progress, Fungal Biomass, and Mycotoxin Accumulation Phytopathology 89(11) 1028-1037; CA Roberts, R R Marquardt, A A Frohlich, R L McGraw, R G Rotter, J C Henning (1991) Chemical and spectral quantification of mold in contaminated barley; Cereal Chemistry 68(3):272-275).
The term “hybridization” as used herein includes “any process by which a strand of nucleic acid molecule joins with a complementary strand through base pairing” (J. Coombs (1994) Dictionary of Biotechnology, Stockton Press, New York). Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acid molecules) is impacted by such factors as the degree of complementarity between the nucleic acid molecules, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acid molecules.
As used herein, the term “Tm” is used in reference to the “melting temperature.” The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acid molecules is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid molecule is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985). Other references include more sophisticated computations, which take structural as well as sequence characteristics into account for the calculation of Tm. Stringent conditions, are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
In particular, the term “stringency conditions” refers to conditions, wherein 100 contigous nucleotides or more, 150 contigous nucleotides or more, 200 contigous nucleotides or more or 250 contigous nucleotides or more which are a fragment or identical to the complementary nucleic acid molecule (DNA, RNA, ssDNA or ssRNA) hybridizes under conditions equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C. or 65° C., preferably at 65° C., with a specific nucleic acid molecule (DNA; RNA, ssDNA or ss RNA). Preferably, the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C. or 65° C., preferably 65° C., more preferably the hybridizing conditions are equivalent to hybridization in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C. or 65° C., preferably 65° C. Preferably, the complementary nucleotides hybridize with a fragment or the whole nucleic acids of exogenous F6H1, CCoAOMT, ABCG37 genes and UGT71C1, respectively. Alternatively, preferred hybridization conditions encompass hybridisation at 65° C. in 1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at 65° C. in 0.3×SSC or hybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50% formamide, followed by washing at 50° C. in 2×SSC. Further preferred hybridization conditions are 0.1% SDS, 0.1 SSD and 65° C.
The term “plant” is intended to encompass plants at any stage of maturity or development, as well as any tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context. Plant parts include, but are not limited to, plant cells, stems, roots, flowers, ovules, stamens, seeds, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, hairy root cultures, and/or the like. The present invention also includes seeds produced by the plants of the present invention. Preferably, the seeds comprise the exogenous F6H1 nucleic acid optionally in combination one or more nucleic acid selected from CCoAOMT, ABCG37 and UGT71C1 nucleic acids. In one embodiment, the seeds can develop into plants with increased resistance to fungal infection as compared to a wild-type variety of the plant seed. As used herein, a “plant cell” includes, but is not limited to, a protoplast, gamete producing cell, and a cell that regenerates into a whole plant. Tissue culture of various tissues of plants and regeneration of plants therefrom is well known in the art and is widely published.
Reference herein to an “endogenous” nucleic acid and/or protein refers to the nucleic acid and/or protein in question as found in a plant in its natural form (i.e., without there being any human intervention).
The term “exogenous” nucleic acid refers to a nucleic acid that has been introduced in a plant by means of genetechnology. An “exogenous” nucleic acid can either not occur in a plant in its natural form, be different from the nucleic acid in question as found in a plant in its natural form, or can be identical to a nucleic acid found in a plant in its natural form, but integrated not within their natural genetic environment. The corresponding meaning of “exogenous” is applied in the context of protein expression. For example, a transgenic plant containing a transgene, i.e., an exogenous nucleic acid, may, when compared to the expression of the endogenous gene, encounter a substantial increase of the expression of the respective gene or protein in total. A transgenic plant according to the present invention includes an exogenous F6H1 nucleic acid optionally in combination one or more exogenous nucleic acid(s) selected from CCoAOMT, ABCG37 and UGT71C1 nucleic acids integrated at any genetic loci and optionally the plant may also include the endogenous gene within the natural genetic background. Preferably the plant, plant part or plant cell does not include endogenous F6H1 nucleic acid optionally in combination with one or more endogenous nucleic acid(s) selected from CCoAOMT, ABCG37 and UGT71C1.
For the purposes of the invention, “recombinant” means with regard to, for example, a nucleic acid sequence, a nucleic acid molecule, an expression cassette or a vector construct comprising F6H1 nucleic acid optionally in combination with any one or more of CCoAOMT, ABCG37 and/or UGT71C1 nucleic acid(s), all those constructions brought about by man by genetechnological methods in which either
are not located in their natural genetic environment within the genome of the wildtype plant or have been modified by man by genetechnological methods. The modification may take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library or the combination with the natural promoter.
For instance, a naturally occurring expression cassette—for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a protein useful in the methods of the present invention, as defined above—becomes a recombinant expression cassette when this expression cassette is modified by man by non-natural, synthetic (“artificial”) methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350, WO 00/15815 or US200405323. Furthermore, a naturally occurring expression cassette—for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a protein useful in the methods of the present invention, as defined above—becomes a recombinant expression cassette when this expression cassette is not integrated in the natural genetic environment but in a different genetic environment.
The term “isolated nucleic acid” or “isolated protein” refers to a nucleic acid or protein that is not located in its natural environment, in particular its natural cellular environment. Thus, an isolated nucleic acid or isolated protein is essentially separated from other components of its natural environment. However, the skilled person in the art is aware that preparations of an isolated nucleic acid or an isolated protein can display a certain degree of impurity depending on the isolation procedure used. Methods for purifying nucleic acids and proteins are well known in the art. The isolated gene may be isolated from an organism or may be manmade, for example by chemical synthesis. In this regard, a recombinant nucleic acid may also be in an isolated form.
As used herein, the term “transgenic” refers to an organism, e.g., a plant, plant cell, callus, plant tissue, or plant part that exogenously contains the nucleic acid, recombinant construct, vector or expression cassette described herein or a part thereof which is preferably introduced by non-essentially biological processes, preferably by Agrobacteria transformation. The recombinant construct or a part thereof is stably integrated into a chromosome, so that it is passed on to successive generations by clonal propagation, vegetative propagation or sexual propagation. Preferred successive generations are transgenic too. Essentially biological processes may be crossing of plants and/or natural recombination.
Preferably, the nucleic acids according to the invention or used according to the invention comprise
A transgenic plant, plants cell or tissue for the purposes of the invention is thus understood as meaning that an exogenous F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAOMT, ABCG37 and UGT71C1 nucleic acids is integrated into the genome by means of genetechnology.
A recombinant construct, vector or expression cassette for the purposes of the invention comprises a F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAOMT, ABCG37 and UGT71C1 nucleic acids and is prepared by means of genetechnology.
A “wild type” plant, “wild type” plant part, or “wild type” plant cell means that said plant, plant part, or plant cell does not express exogenous F6H1, CCoAOMT, ABCG37 and UGT71C1 nucleic acids and exogenous F6H1, CCoAOMT, ABCG37 and UGT71C1 proteins. Preferably, the wildtype plant is not capable to produce more than 10 μM scopoletin and/or a derivative thereof, more preferably not more than 5 μM scopoletin and/or a derivative thereof and most preferably the wildtype plant is not capable to produce scopoletin and/or a derivative thereof. A derivative of scopoletin is e.g. scopolin. Preferably, the wildtype plant plant does not express endogenous F6H1, CCoAOMT, ABCG37 and/or UGT71C1 nucleic acids and endogenous F6H1, CCoAOMT, ABCG37 and/or UGT71C1 proteins.
Natural locus means the location on a specific chromosome and/or the location between certain genes and/or the same sequence background as in the original plant which is transformed.
Preferably, the transgenic plant, plant cell or tissue thereof expresses the F6H1 nucleic acids optionally in combination with one or more nucleic acids selected from the group consisting of CCoAOMT, ABCG37 and UGT71C1 nucleic acids. Preferably, the transgenic plant, plant cell or tissue thereof is transformed with recombinant vector constructs comprising F6H1 nucleic acids optionally in combination with one or more nucleic acids selected from the group consisting of CCoAOMT, ABCG37 and UGT71C1 nucleic acids described herein. F6H1, CCoAOMT, ABCG37 and/or UGT71C1 nucleic acids may be located on the same vector or different recombinant vectors.
The term “expression” or “gene expression” means the transcription of a specific gene or specific genes or specific genetic vector construct. The term “expression” or “gene expression” in particular means the transcription of a gene or genes or genetic vector construct into structural RNA (rRNA, tRNA), or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting RNA product. The term “expression” or “gene expression” can also include the translation of the mRNA and therewith the synthesis of the encoded protein, i.e., protein expression.
The term “increased expression” or “enhanced expression” or “overexpression” or “increase of content” as used herein means any form of expression that is additional to the original wild-type expression level. For the purposes of this invention, the original wild-type expression level might also be zero (absence of expression).
Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers, or RNAa (Li et al 2006, PNAS 103(46) 17337-42). Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the protein of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., WO9322443), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
The term “functional fragment” refers to any nucleic acid or protein which comprises merely a part of the fulllength nucleic acid or fulllength protein, respectively, but still provides the essentially same or similar function, e.g., increased fungal resistance and/or the same, essentially the same or similar biological activity when expressed in a plant. Preferably, the fragment comprises at least 70%, at least 80%, at least 90% at least 95%, at least 98%, at least 99% of the original sequence. Preferably, the functional fragment comprises contiguous nucleic acids or amino acids as in the original nucleic acid or original protein, respectively. In one embodiment the fragment of any of the F6H1, CCoAOMT, ABCG37 and/or UGT71C1 nucleic acids has an identity as defined above over a length of at least 70%, at least 75%, at least 90% of the nucleotides of the respective F6H1, CCoAOMT, ABCG37 and/or UGT71C1 nucleic acid.
The term “the same biological activity”, “essentially the same biologicla activity”, “similar biological activity” or increased biological activity preferably means leading to an increased production and/or accumulation compared to the wildtype plant, wild type plant part, or wild type plant cell of more than 0.1 μM, preferably more than 1 μM, preferably more than 2 μM, more preferably more than 5 μM, most preferably more than 10 μM scopoletin and/or a derivative thereof when F6H1 and optionally CCoAOMT, ABCG37 and/or UGT71C1 nucleic acids or fragments thereof are expressed in a plant.
The term “splice variant” as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons or parts thereof have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened. Thus, a splice variant can have one or more or even all introns removed or added or partially removed or partially added. According to this definition, a cDNA is considered as a splice variant of the respective intron-containing genomic sequence and vice versa. Such splice variants may be found in nature or may be manmade. Methods for predicting and isolating such splice variants are well known in the art (see for example Foissac and Schiex (2005) BMC Bioinformatics 6: 25).
The wildtype plant may express the respective endogenous F6H1, CCoAOMT, ABCG37 and/or UGT71C1 nucleic acids. As far as overexpression of exogenous F6H1, CCoAOMT, ABCG37 and/or UGT71C1 nucleic acids is concerned, for the purposes of this invention, the original wild-type expression level of the corresponding endogenous nucleic acids might also be zero (absence of expression).
With respect to a vector construct and/or the recombinant nucleic acid molecules, the term “operatively linked” is intended to mean that the nucleic acid to be expressed is linked to the regulatory sequence, including promoters, terminators, enhancers and/or other expression control elements (e.g. polyadenylation signals), in a manner which allows for expression of the nucleic acid (e.g. in a host plant cell when the vector is introduced into the host plant cell). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) and Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology, Eds. Glick and Thompson, Chapter 7, 89-108, CRC Press: Boca Raton, Fla., including the references therein. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of nucleic acid desired, and the like.
The term “introduction” or “transformation” as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The host genome includes the nucleic acid contained in the nucleus as well as the nucleic acid contained in the plastids, e.g., chloroplasts, and/or mitochondria. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
The term “terminator” encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3′ processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The terminator to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
Detailed Description
F6H1, CCoAOMT, ABCG37 and/or UGT71C1 nucleic acids
The F6H1 nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens, e.g., of the family Phacopsoraceae, for example soybean rust, or of the genus of Fusarium, in particular Fusarium graminearum and/or Fusarium verticillioides, is preferably a nucleic acid
consisting of or comprising a nucleic acid selected from the group consisting of:
The F6H1 nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens is for example a nucleic acid selected from SEQ ID No. 1, 9, 11, 13, 15, 17, 19 and 21. The F6H1 nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens is for example a nucleic acid encoding a F6H1 protein selected from SEQ ID No. 2, 10, 12, 14, 16, 18, 20 and 22.
The F6H1 protein may comprise a domain as defined in SEQ ID No. 63, and having least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the protein sequence represented by the respective sequence.
The CCoAOMT nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens, e.g., of the family Phacopsoraceae, for example soybean rust, or of the genus of Fusarium, in particular Fusarium graminearum and/or Fusarium verticillioides, is preferably a nucleic acid
consisting of or comprising a nucleic acid selected from the group consisting of:
The CCoAOMT nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens is for example a nucleic acid selected from SEQ ID No. 3, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47. The CCoAOMT nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens is for example a nucleic acid encoding a CCoAOMT protein selected from SEQ ID No. 4, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 48.
The CCoAOMT protein may comprise a domain as defined in SEQ ID No. 64, having least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the protein sequence represented by the respective sequence.
The ABCG37 nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens, e.g., of the family Phacopsoraceae, for example soybean rust, or of the genus of Fusarium, in particular Fusarium graminearum and/or Fusarium verticillioides, is preferably a nucleic acid
consisting of or comprising a nucleic acid selected from the group consisting of:
The ABCG37 nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens is for example a nucleic acid selected from SEQ ID No. 5, 49, 51 and 53. The ABCG37 nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens is for example a nucleic acid encoding a ABCG37 protein selected from SEQ ID No. 6, 50, 52 and 54.
The ABCG37 protein may comprise at least one domain selected from the group as defined in SEQ ID No. 65, 66, 67 and/or 68 having least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the protein sequence represented by the respective sequence.
The UGT71C1 nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens, e.g., of the family Phacopsoraceae, for example soybean rust, or of the genus of Fusarium, in particular Fusarium graminearum and/or Fusarium verticillioides, is preferably a nucleic acid
consisting of or comprising a nucleic acid selected from the group consisting of:
The UGT71C1 nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens is for example a nucleic acid selected from SEQ ID No. 55, 57, 59 and 61. The UGT71C1 nucleic acid to be overexpressed in order to achieve increased resistance to fungal pathogens is for example a nucleic acid encoding an ABCG37 protein selected from SEQ ID No. 56, 58, 60 and 62.
The UGT71C1 protein may comprise at least one domain selected from the group as defined in SEQ ID No. 69 and/or 70 having least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the protein sequence represented by the respective sequence.
Percentages of identity of a nucleic acid are indicated with reference to the entire nucleotide region given in a sequence specifically disclosed herein.
Preferably the portion of the F6H1 nucleic acid fragment is about 500-600, about 600-700, about 700-800, about 800-900, about 900-1000, or about 1000-1086 nucleotides, preferably consecutive nucleotides, preferably counted from the 5′ or 3′ end of the nucleic acid, in length, of the nucleic acid sequences given in SEQ ID NO: 1.
Preferably the portion of the CCoAOMT nucleic acid fragment is about 400-500 about 500-600, about 600-700, about 700-780, preferably consecutive nucleotides, preferably counted from the 5′ or 3′ end of the nucleic acid, in length, of the nucleic acid sequences given in SEQ ID NO: 3.
Preferably the portion of the ABCG37 nucleic acid fragment is about 2500-2600, about 2600-2700, about 2700-2800 about 2800-2900, about 2900-3000, about 3000-3100, about 3100-3200, about 3200-3300, about 3300-3400, about 3400-3500, about 3500-3600, about 3600-3700, about 3700-3800, about 3800-3900, about 3900-4000, about 4000-4100, about 4100-4200, or about 4300-4353 nucleotides, preferably consecutive nucleotides, preferably counted from the 5′ or 3′ end of the nucleic acid, in length, of the nucleic acid sequences given in SEQ ID NO: 5.
Preferably the portion of the UGT71C1 nucleic acid fragment is about 500-600, about 600-700, about 700-800 about 800-900, about 900-1000, about 1000-1100, about 1100-1200, about 1200-1300, about 1300-1400 or about 1400-1446 nucleotides, preferably consecutive nucleotides, preferably counted from the 5′ or 3′ end of the nucleic acid, in length, of the nucleic acid sequences given in SEQ ID NO: 7.
All the nucleic acid sequences mentioned herein (single-stranded and double-stranded DNA and RNA sequences, for example cDNA and mRNA) can be produced in a known way by chemical synthesis from the nucleotide building blocks, e.g. by fragment condensation of individual overlapping, complementary nucleic acid building blocks of the double helix. Chemical synthesis of oligonucleotides can, for example, be performed in a known way, by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press, New York, pages 896-897). The accumulation of synthetic oligonucleotides and filling of gaps by means of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning techniques are described in Sambrook et al. (1989), see below.
The F6H1, CCoAOMT, ABCG37 and/or UGT71C1 nucleic acids described herein are useful in the constructs, methods, plants, harvestable parts and products of the invention.
F6H1, CCoAOMT, ABCG37 and/or UGT71C1 Proteins
In one embodiment of the invention, the F6H1 protein is encoded by a nucleic acid comprising an exogenous nucleic acid having
In one embodiment of the invention, the CCoAOMT protein is encoded by a nucleic acid comprising an exogenous nucleic acid having
In one embodiment of the invention, the ABCG37 protein is encoded by a nucleic acid comprising an exogenous nucleic acid having
In one embodiment of the invention, the UGT71C1 protein is encoded by a nucleic acid comprising an exogenous nucleic acid having
Preferably, the F6H1 polypeptide comprises about 200-225, about 225-250, about 250-275, about 275-300, about 300-325, about 325-350, or about 350-362 amino acid residues, preferably consecutive amino acid residues, preferably counted from the N-terminus or C-terminus of the amino acid sequence, or up to the full length of any of the amino acid sequences encoded by the nucleic acid sequences set out in SEQ ID NO: 1.
Preferably, the CCoAOMT polypeptide comprises about 100-125, about 125-150, about 150-175, about 175-200, about 200-225, about 225-250, or about 250-260 amino acid residues, preferably consecutive amino acid residues, preferably counted from the N-terminus or C-terminus of the amino acid sequence, or up to the full length of any of the amino acid sequences encoded by the nucleic acid sequences set out in SEQ ID NO: 3.
Preferably, the ABCG37 polypeptide comprises about 1100-1125, about 1125-1150, about 1150-1175, about 1175-1200, about 1200-1225, about 1200-1225, about 1225-1250, about 1250-1275, about 1275-1300, about 1300-1325, about 1325-1350, about 1350-1375, about 1375-1400, about 1400-1425, or about 1425-1451 amino acid residues, preferably consecutive amino acid residues, preferably counted from the N-terminus or C-terminus of the amino acid sequence, or up to the full length of any of the amino acid sequences encoded by the nucleic acid sequences set out in SEQ ID NO: 5.
Preferably, the UGT71C1 polypeptide comprises about 225-250, about 250-275, about 275-300, about 300-325, about 325-350, about 350-375, about 375-400, about 400-425, about 425-450, about 450-475, or about 475-482 amino acid residues, preferably consecutive amino acid residues, preferably counted from the N-terminus or C-terminus of the amino acid sequence, or up to the full length of any of the amino acid sequences encoded by the nucleic acid sequences set out in SEQ ID NO: 7.
The F6H1, CCoAOMT, ABCG37 and/or UGT71C1 proteins described herein are useful in the constructs, methods, plants, harvestable parts and products of the invention.
Methods for Increasing Fungal Resistance
One embodiment of the present invention is a method according to the present invention for increasing fungal resistance in a plant, a plant part, or a plant cell, wherein the method comprises the step of increasing the production of scopoletin and/or a derivative thereof in the plant, plant part, or plant cell in comparison to a wild type plant, wild type plant part, or wild type plant cell. The derivative of the scopoletin may be the scopolin.
Scopoletin is defined by the structural formula:
Scopolin is defined by the structural formula:
One embodiment of the present invention is a method for increasing fungal resistance in a plant, a plant part, or a plant cell, wherein the method comprises increasing the expression and/or biological activity of a F6H1 protein in the plant, plant part, or plant cell in comparison to a wild type plant, wild type plant part, or wild type plant cell, wherein said F6H1 protein is encoded by as defined above. In a preferred embodiment said method further comprises increasing the expression and/or biological activity of at least one or more additional protein(s) selected from the group consisting of a CCoAOMT1 protein, a ABCG37 protein and a UGT71C1 protein in the plant, plant part, or plant cell in comparison to a wild type plant, wild type plant part, or wild type plant cell, wherein said CCoAOMT1 protein, a ABCG37 protein and a UGT71C1 protein are defined as above. Preferably, said method comprises increasing the productions and/or accumulation of scopoletin and/or a derivative thereof in a plant, plant part or plant cell.
One embodiment of the invention is a method for increasing fungal resistance, preferably resistance to Phacopsoracea and/or Fusarium, in a plant, plant part, or plant cell by increasing the expression and/or biological activity of a F6H1 protein, and optionally in combination with increasing the expression and/or biological activity of one or more of the protein(s) selected from the group consisting of CCoAOMT, ABCG37 and/or UGT71C1 protein(s) or a functional fragment, homologue thereof in comparison to wild-type plants, wild-type plant parts or wild-type plant cells. Preferably, the F6H1 protein is expressed from an exogenous nucleic acid. Preferably, F6H1 protein and one or more the proteins selected from the group consisting of CCoAOMT, ABCG37 and/or UGT71C1 protein(s), are expressed from an exogenous nucleic acid.
One embodiment of the invention is a method for increasing fungal resistance in a plant, a plant part, or a plant cell comprises
A preferred method according to the present invention comprises
optionally wherein the nucleic acid(s) which codes for a CCoAOMT1, ABCG37 and/or UGT71C1 protein(s) is expressed in an amount and for a period sufficient to generate or to increase fungal resistance in said plant.
Preferably the nucleic acid(s) encoding F6H1, CCoAOMT1, ABCG37 and/or UGT71C1 protein(s) are in functional linkage with a promoter. Preferably, the promoter is a constitutive, pathogen inducible, preferably fungal inducible, mesophyll-specific promoter and/or epidermis-specific promoter and/or stalk specific, ear or kernel specific promoter
Preferably, the production of scopoletin and/or a derivative thereof in the plant, plant part, or plant cell in comparison to a wild type plant, wild type plant part, or wild type plant cell is increased.
In preferred embodiments, the protein amount and/or biological activity of the F6H1 protein in the plant is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the F6H1 nucleic acid.
In preferred embodiments, the protein amount and/or biological activity of the CCoAOMT protein in the plant is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the CCoAOMT nucleic acid.
In preferred embodiments, the protein amount and/or biological activity of the ABCG37 protein in the plant is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the ABCG37 nucleic acid.
In preferred embodiments, the protein amount and/or biological activity of the UGT71C1 protein in the plant is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the UGT71C1 nucleic acid.
The exogenous nucleic acid encoding F6H1, CCoAOMT1, ABCG37 and/or UGT71C1 are located on the same or different expression cassettes. Preferably, one expression cassette comprises exogenous nucleic acid encoding F6H1 and optionally in combination with one or more exogenous nucleic acid encoding CCoAOMT1, ABCG37 and/or UGT71C1. Preferably, the expression cassette comprises exogenous nucleic acid encoding
In another embodiment the exogenous nucleic acid encoding
The fungal pathogens or fungus-like pathogens (such as, for example, Chromista) can belong to the group comprising Plasmodiophoramycota, Oomycota, Ascomycota, Chytridiomycetes, Zygomycetes, Basidiomycota or Deuteromycetes (Fungi imperfecti). Pathogens which may be mentioned by way of example, but not by limitation, are those detailed in Tables 2 and 3, and the diseases which are associated with them.
Puccinia recondita
P. striiformis
Erysiphe graminis/Blumeria graminis
Puccinia sorghi
Puccinia polysora
Cercospora nicotianae
Phakopsora pachyrhizi, P. meibomiae
Physopella pallescens, P. zeae =
Angiopsora zeae
Septoria (Stagonospora) nodorum
Septoria tritici
Fusarium spp.
Phytophthora infestans
Anthrocnose leaf
Colletotrichum graminicola (teleomorph:
Glomerella graminicola Politis);
Anthracnose stalk
Glomerella tucumanensis
Curvularia
Curvularia clavata, C. eragrostidis, =C.
maculans (teleomorph:
Cochliobolus eragrostidis),
Curvularia inaequalis, C. intermedia
Curvularia lunata (teleomorph:
Cochliobolus lunatus),
Curvularia pallescens (teleomorph:
Cochliobolus pallescens),
Curvularia senegalensis, C. tuberculata
Didymella leaf spot
Didymella exitalis
Diplodia leaf spot
Stenocarpella macrospora =
Diplodialeaf macrospora
Sclerophthora rayssiae var. zeae
Sclerophthora macrospora =
Sclerospora macrospora
Sclerospora graminicola
Alternaria alternata, Ascochyta maydis,
A. tritici, A. zeicola,
Bipolaris victoriae =
Helminthosporium victoriae (teleomorph:
Cochliobolus victoriae), C. sativus
H. sorokinianum = H. sativum),
Epicoccum nigrum, Exserohilum prolatum =
Drechslera prolata (teleomorph:
Setosphaeria prolata)
Graphium penicillioides,
Leptosphaeria maydis, Leptothyrium zeae,
Ophiosphaerella herpotricha, (anamorph:
Scolecosporiella sp.),
Paraphaeosphaeria michotii, Phoma sp.,
Septoria zeae, S. zeicola, S. zeina
Setosphaeria turcica (anamorph:
Exserohilum turcicum =
Helminthosporium turcicum)
Cochliobolus carbonum (anamorph:
Bipolaris zeicola =
Helminthosporium
Helminthosporium carbonum)
Phaeosphaeria
Phaeosphaeria maydis = Sphaerulina maydis
Rostratum leaf spot
Setosphaeria rostrata, (anamorph:
xserohilum rostratum =
Helminthosporium rostratum)
Peronosclerospora maydis =
Sclerospora maydis
Peronosclerospora philippinensis =
Sclerospora philippinensis
Sorghum downy
Peronosclerospora sorghi =
Sclerospora sorghi
Spontaneum downy
Peronosclerospora spontanea =
Sclerospora spontanea
Peronosclerospora sacchari =
Sclerospora sacchari
Sclerotium ear rot
Sclerotium rolfsii Sacc. (teleomorph:
Athelia rolfsii)
Bipolaris sorokiniana, B. zeicola =
Helminthosporium carbonum,
Diplodia maydis, Exserohilum pedicillatum,
Exserohilum turcicum =
Helminthosporium turcicum,
Fusarium avenaceum, F. culmorum,
F. moniliforme, Gibberella zeae
Macrophomina phaseolina, Penicillium spp.,
Phomopsis sp., Pythium spp.,
Rhizoctonia solani, R. zeae,
Sclerotium rolfsii, Spicaria sp.
Selenophoma leaf
Selenophoma sp.
Ascochyta ischaemi, Phyllosticta maydis
Gloeocercospora sorghi
Preferred fungal pathogens are of the order Pucciniales, in particular the family Phacopsoracea, in particular the genus Phakopsora, more particularly the species Phakopsora pachyrhizi and/or Phakopsora meibomiae—also known as soybean rust or Asian Soybean Rust (ASR) and/or preferred fungal pathogens are of the family Nectriaceae, in particular the genus Fusarium, in particular the species Fusarium graminearum, Fusarium sporotrichioides, Fusarium pseudograminearum, Fusarium culmorum, Fusarium poae, Fusarium verticillioides (Fusarium moniliforme), Fusarium subglutinans, Fusarium proliferatum, Fusarium fujikuroi), Fusarium avenaceum, Fusarium oxysporum, Fusarium virguliforme and/or Fusarium solani. Most preferred is fusarium graminearum and/or fusarium verticolloides.
F6H1, CCoAOMT1, ABCG37 and/or UGT71C1 expression constructs and vector constructs
One embodiment of the present invention is a recombinant vector construct comprising the nucleic acid encoding F6H1 protein as defined above operably linked with a promoter and a transcription termination sequence.
One embodiment of the present invention is a recombinant vector construct comprising the nucleic acid encoding CCoAOMT1 protein as defined above operably linked with a promoter and a transcription termination sequence.
One embodiment of the present invention is a recombinant vector construct comprising the nucleic acid encoding ABCG37 protein as defined above operably linked with a promoter and a transcription termination sequence.
One embodiment of the present invention is a recombinant vector construct comprising the nucleic acid encoding UGT71C1 protein as defined above operably linked with a promoter and a transcription termination sequence.
In one embodiment the nucleic acid encoding F6H1 protein, CCoAOMT1 protein, ABCG37 and/or UGT71C1 protein are located on the same recombinant vector construct. In another embodiment the nucleic acid encoding F6H1 protein, CCoAOMT1 protein and/or ABCG37 protein are located on different vector constructs. Preferably, one expression cassette comprises the exogenous nucleic acid(s) encoding F6H1 and optionally in combination with exogenous nucleic acids encoding one or more selected from the group of the exogenous nucleic acid(s) CCoAOMT1, ABCG37 and/or UGT71C1. Preferably, the recombinant vector construct comprises exogenous nucleic acid encoding.
Promoters according to the present invention may be constitutive, inducible, in particular pathogen-inducible, developmental stage-preferred, cell type-preferred, tissue-preferred or organ-preferred. Examples for suitable promoters and terminators are:
The PcUbi promoter regulates constitutive expression of the ubi4-2 gene (accession number X64345) of Petroselinum crispum (Kawalleck, P., Somssich, I. E., Feldbrügge, M., Hahlbrock, K., & Weisshaar, B. (1993). Polyubiquitin gene expression and structural properties of the ubi4-2 gene in Petroselinum crispum. Plant molecular biology, 21(4), 673-684. The p-Super promoter consists of three identical Octapine Synthase Enhancers followed by a MAS promoter (Lee et al., 2007 Plant Physiology Vol145 Issue 4 1294-1300). The p-Glyma14g06680 promoter has been identified in a screen for genes that are predominantly expressed in the leaf of soybean. The promoter regulates the expression of the gene Glyma14g06680, which is most likely a water channel protein (WO12127373) T-ocs and t-NOS terminators are both derived from Agrobacterium (Gielen, J., et al. “The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5.” The EMBO journal 3.4 (1984): 835. T-ocs is the terminator of the octopine synthase gene and t-NOS is the terminator of the nopaline synthase gene of Agrobacterium tumefaciens The StCATHD-pA is the terminator of the cathepsin D inhibitor gene from Solanum tuberosum (t-StCat) (Herbers et al. 1994)
One type of recombinant vector construct is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vector constructs are capable of autonomous replication in a host plant cell into which they are introduced. Other vector constructs are integrated into the genome of a host plant cell upon introduction into the host cell, and thereby are replicated along with the host genome. In particular the vector construct is capable of directing the expression of gene to which the vectors is operatively linked. However, the invention is intended to include such other forms of expression vector constructs, such as viral vectors (e.g., potato virus X, tobacco rattle virus, and/or Gemini virus), which serve equivalent functions.
Transgenic Organisms; Transgenic Plants, Plant Parts, and Plant Cells
A preferred embodiment is a transgenic plant, transgenic plant part, or transgenic plant cell overexpressing an exogenous F6H1 protein, optionally in combination with overexpressing one or more of CCoAOMT1 protein, ABCG37 protein and/or UGT71C1 protein encoded by a nucleic acid as defined above.
In preferred embodiments the biological activity of the F6H1 protein optional the biological activity of one or more of CCoAOMT1 protein, ABCG37 protein and/or UGT71C1 protein is increased in said transgenic plant, transgenic plant part, or transgenic plant cell.
In preferred embodiments, the protein amount of a F6H1 protein in the transgenic plant is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the F6H1 nucleic acid.
In preferred embodiments, the protein amount of a CCoAOMT1 protein in the transgenic plant is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the CCoAOMT1 nucleic acid.
In preferred embodiments, the protein amount of a ABCG37 protein in the transgenic plant is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the ABCG37 nucleic acid.
In preferred embodiments, the protein amount of a UGT71C1 protein in the transgenic plant is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the ABCG37 nucleic acid.
On preferred embodiments the amount of F6H1 protein in combination with CCoAOMT1 and/or ABCG37 and/or UGT71C1 in the transgenic plant is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more in comparison to a wild type plant that is not transformed with the respective nucleic acid(s).
More preferably, the transgenic plant, transgenic plant part, or transgenic plant cell according to the present invention has been obtained by transformation with one or more recombinant vector construct(s) described herein. In one embodiment a transgenic plant, transgenic plant part, or transgenic plant cell is transformed with one or more recombinant vector construct(s) as described, wherein the nucleic acid(s) encoding a F6H1 protein, and/or a CCoAOMT1 protein, and/or a ABCG37 protein and/or a UGT71C1 protein are located on the same recombinant vector construct or different vector constructs. Preferably, the recombinant vector construct comprises exogenous nucleic acid encoding F6H1 and CCoAOMT1, F6H1 and ABCG37, F6H1 and UGT71C1 or F6H1, CCoAOMT1, ABCG37 and UGT71C1 proteins.
A preferred embodiment comprises a transgenic plant, transgenic plant part, or transgenic plant cell overexpressing an exogenous F6H1 protein optionally in combination with one or more additional exogenous protein(s) selected from the group consisting of a CCoAOMT1 protein, an ABCG37 protein and an UGT71C1 protein, wherein the nucleic acid encodings the respective protein(s) is operably linked with a promoter and a transcription termination sequence.
Suitable methods for transforming or transfecting host cells including plant cells are well known in the art of plant biotechnology. Any method may be used to transform the recombinant expression vector into plant cells to yield the transgenic plants of the invention. General methods for transforming dicotyledonous plants are disclosed, for example, in U.S. Pat. Nos. 4,940,838; 5,464,763, and the like. Methods for transforming specific dicotyledonous plants, for example, cotton, are set forth in U.S. Pat. Nos. 5,004,863; 5,159,135; and 5,846,797. Soy transformation methods are set forth in U.S. Pat. Nos. 4,992,375; 5,416,011; 5,569,834; 5,824,877; 6,384,301 and in EP 0301749B1 may be used. Transformation methods may include direct and indirect methods of transformation. Suitable direct methods include polyethylene glycol induced DNA uptake, liposome-mediated transformation (U.S. Pat. No. 4,536,475), biolistic methods using the gene gun (Fromm M E et al., Bio/Technology. 8(9):833-9, 1990; Gordon-Kamm et al. Plant Cell 2:603, 1990), electroporation, incubation of dry embryos in DNA-comprising solution, and microinjection. In the case of these direct transformation methods, the plasmids used need not meet any particular requirements. Simple plasmids, such as those of the pUC series, pBR322, M13mp series, pACYC184 and the like can be used. If intact plants are to be regenerated from the transformed cells, an additional selectable marker gene is preferably located on the plasmid. The direct transformation techniques are equally suitable for dicotyledonous and monocotyledonous plants.
Transformation can also be carried out by bacterial infection by means of Agrobacterium (for example EP 0 116 718), viral infection by means of viral vectors (EP 0 067 553; U.S. Pat. No. 4,407,956; WO 95/34668; WO 93/03161) or by means of pollen (EP 0 270 356; WO 85/01856; U.S. Pat. No. 4,684,611). Agrobacterium based transformation techniques (especially for dicotyledonous plants) are well known in the art. The Agrobacterium strain (e.g., Agrobacterium tumefaciens or Agrobacterium rhizogenes) comprises a plasmid (Ti or Ri plasmid) and a T-DNA element which is transferred to the plant following infection with Agrobacterium. The T-DNA (transferred DNA) is integrated into the genome of the plant cell. The T-DNA may be localized on the Ri- or Ti-plasmid or is separately comprised in a so-called binary vector. Methods for the Agrobacterium-mediated transformation are described, for example, in Horsch R B et al. (1985) Science 225:1229. The Agrobacterium-mediated transformation is best suited to dicotyledonous plants but has also been adapted to monocotyledonous plants. The transformation of plants by Agrobacteria is described in, for example, White F F, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38; Jenes B et al. Techniques for Gene Transfer, Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S. D. Kung and R. Wu, Academic Press, 1993, pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225. Transformation may result in transient or stable transformation and expression. Although a nucleotide sequence of the present invention can be inserted into any plant and plant cell falling within these broad classes, it is particularly useful in crop plant cells.
The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S. D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer.
After transformation, plant cells or cell groupings may be selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above. The transformed plants may also be directly selected by screening for the presence of the F6H1, CCoAOMT1, ABCG37 and/or UGT71C1 protein nucleic acid(s).
Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques or crossed with appropriate tester lines to generate hybrids. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion). Preferably, constructs or vectors or expression cassettes are not present in the genome of the original plant or are present in the genome of the transgenic plant not at their natural locus of the genome of the original plant.
Preferably, the transgenic plant of the present invention or the plant obtained by the method of the present invention has increased resistance against fungal pathogens, preferably rust pathogens (i.e., fungal pathogens of the order Pucciniales), preferably against fungal pathogens of the family Phacopsoraceae, more preferably against fungal pathogens of the genus Phacopsora, most preferably against Phakopsora pachyrhizi and Phakopsora meibomiae, also known as soybean rust. Preferably, resistance against Phakopsora pachyrhizi and/or Phakopsora meibomiae is increased.
Preferably, the plant, plant part, or plant cell is a plant or derived from a plant selected from the group consisting of beans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches, groundnut, rice, wheat, barley, arabidopsis, lentil, banana, canola, cotton, potatoe, corn, sugar cane, alfalfa, and sugar beet.
In one embodiment of the present invention the plant is selected from the group consisting of beans, soy, pea, clover, kudzu, lucerne, lentils, lupins, vetches, and/or groundnut. Preferably, the plant is a legume, comprising plants of the genus Phaseolus (comprising French bean, dwarf bean, climbing bean (Phaseolus vulgaris), Lima bean (Phaseolus lunatus L.), Tepary bean (Phaseolus acutifolius A. Gray), runner bean (Phaseolus coccineus)); the genus Glycine (comprising Glycine soja, soybeans (Glycine max (L.) Merill)); pea (Pisum) (comprising shelling peas (Pisum sativum L. convar. sativum), also called smooth or round-seeded peas; marrowfat pea (Pisum sativum L. convar. medullare Alef. emend. C. O. Lehm), sugar pea (Pisum sativum L. convar. axiphium Alef emend. C. O. Lehm), also called snow pea, edible-podded pea or mangetout, (Pisum granda sneida L. convar. sneidulo p. shneiderium)); peanut (Arachis hypogaea), clover (Trifolium spec.), medick (Medicago), kudzu vine (Pueraria lobata), common lucerne, alfalfa (M. sativa L.), chickpea (Cicer), lentils (Lens) (Lens culinaris Medik.), lupins (Lupinus); vetches (Vicia), field bean, broad bean (Vicia faba), vetchling (Lathyrus) (comprising chickling pea (Lathyrus sativus), heath pea (Lathyrus tuberosus)); genus Vigna (comprising moth bean (Vigna aconitifolia (Jacq.) Maréchal), adzuki bean (Vigna angularis (Willd.) Ohwi & H. Ohashi), urd bean (Vigna mungo (L.) Hepper), mung bean (Vigna radiata (L.) R. Wilczek), bambara groundnut (Vigna subterrane (L.) Verdc.), rice bean (Vigna umbellata (Thunb.) Ohwi & H. Ohashi), Vigna vexillata (L.) A. Rich., Vigna unguiculata (L.) Walp., in the three subspecies asparagus bean, cowpea, catjang bean)); pigeonpea (Cajanus cajan (L.) Millsp.), the genus Macrotyloma (comprising geocarpa groundnut (Macrotyloma geocarpum (Harms) Maréchal & Baudet), horse bean (Macrotyloma uniflorum (Lam.) Verdc.); goa bean (Psophocarpus tetragonolobus (L.) DC.), African yam bean (Sphenostylis stenocarpa (Hochst. ex A. Rich.) Harms), Egyptian black bean, dolichos bean, lablab bean (Lablab purpureus (L.) Sweet), yam bean (Pachyrhizus), guar bean (Cyamopsis tetragonolobus (L.) Taub.); and/or the genus Canavalia (comprising jack bean (Canavalia ensiformis (L.) DC.), sword bean (Canavalia gladiata (Jacq.) DC.).
Further preferred is a plant selected from the group consisting of beans, soya, pea, clover, kudzu, lucerne, lentils, lupins, vetches, and groundnut. Most preferably, the plant, plant part, or plant cell is or is derived from soy and/or corn.
Preferably, the transgenic plant of the present invention or the plant obtained by the method of the present invention is a soybean plant and has increased resistance against fungal pathogens of the order Pucciniales (rust), preferably, of the family Phacopsoraceae, more preferably against fungal pathogens of the genus Phacopsora, most preferably against Phakopsora pachyrhizi and Phakopsora meibomiae, also known as soybean rust. Preferably, resistance against Phakopsora pachyrhizi and/or Phakopsora meibomiae is increased.
Preferably, the transgenic plant of the present invention or the plant obtained by the method of the present invention is a corn plant and has increased resistance against fungal pathogens of the family Nectriaceae, in particular the genus Fusarium, in particular the species Fusarium graminearum, Fusarium sporotrichioides, Fusarium pseudograminearum, Fusarium culmorum, Fusarium poae, Fusarium verticillioides (Fusarium moniliforme), Fusarium subglutinans, Fusarium proliferatum, Fusarium fujikuroi), Fusarium avenaceum, Fusarium oxysporum, Fusarium virguliforme and/or Fusarium solani. Most preferred is fusarium graminearum and/or fusarium verticolloides.
Methods for the Production of Transgenic Plants
One embodiment according to the present invention provides a method for the production of a transgenic plant, transgenic plant part, or transgenic plant cell having increased fungal resistance, comprising introducing
and further optionally introducing one or more nucleic acids selected from the group consisting of
into a plant, a plant part, or a plant cell,
wherein the exogenous nucleic acid encoding F6H1, CCoAMT1, ABCG37 and/or UGT71C1 protein are located on the same or different vector constructs,
generating a transgenic plant, transgenic plant part, or transgenic plant cell from the plant, plant part or plant cell; and expressing the protein(s) encoded by the recombinant vector construct(s).
In one embodiment, the present invention refers to a method for the production of a transgenic plant, transgenic plant part, or transgenic plant cell having increased fungal resistance, comprising
Preferably, said introducing and expressing does not comprise an essentially biological process.
Preferably, the method for the production of the transgenic plant, transgenic plant part, or transgenic plant cell further comprises the step of selecting a transgenic plant expressing F6H1 protein and one or more proteins selected from the group consisting of CCoAMT1, ABCG37 and/or UGT71C1 protein(s).
Preferably, the method for the production of the transgenic plant, transgenic plant part, or transgenic plant cell additionally comprises the step of harvesting the seeds of the transgenic plant and planting the seeds and growing the seeds to plants, wherein the grown plant(s) comprises a nucleic acid encoding F6H1 protein and one or more nucleic acids encoding proteins selected from the group consisting of CCoAMT1, ABCG37 and/or UGT71C1 protein(s) operably linked with a promoter and a transcription termination sequence.
Preferably, the step of harvesting the seeds of the transgenic plant and planting the seeds and growing the seeds to plants is repeated more than one time, preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times.
The transgenic plants may be selected by known methods as described above (e.g., by screening for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the F6H1, CCoAMT1, ABCG37 and/or UGT71C1 gene(s) or by directly screening for the FF6H1, CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid(s)).
Furthermore, the use of the exogenous F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid(s) or use of the recombinant vector construct comprising the F6H1 nucleic acid optionally in combination with one or more nucleic acid(s) selected from the group CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid(s) for the transformation of a plant, plant part, or plant cell to provide a fungal resistant plant, plant part, or plant cell is provided.
Harvestable Parts and Products
Harvestable parts of the transgenic plant according to the present invention are part of the invention. Preferably, the harvestable parts comprise the F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid(s) or F6H1 protein optionally in combination with one or more protein(s) selected from the group consisting of CCoAMT1, ABCG37 and UGT71C1 protein(s). The harvestable parts may be seeds, roots, leaves and/or flowers. Preferred parts of soy plants are soy beans. Preferred parts of corn plants are corn grains.
Products derived from a transgenic plant according to the present invention, parts thereof or harvestable parts thereof are part of the invention. A preferred product is oil, preferably, corn oil or soybean oil.
Preferred parts of soy plants are soy beans comprising the F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid(s) or F6H1 protein optionally in combination with one or more protein(s) selected from the group consisting of CCoAMT1, ABCG37 and UGT71C1 protein(s).
Preferred parts of corn plants are soy grains comprising the F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid(s) or F6H1 protein optionally in combination with one or more protein(s) selected from the group consisting of CCoAMT1, ABCG37 and UGT71C1 protein(s).
In a preferred embodiment a product is derived from the plant described above or from the harvestable part of the plant described above, wherein the product is preferably soybean oil and/or corn oil.
Preferably the soybean oil comprise the F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid(s) or F6H1 protein optionally in combination with one or more protein(s) selected from the group consisting of CCoAMT1, ABCG37 and UGT71C1 protein(s).
Preferably the corn oil comprises the F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid(s) or F6H1 protein optionally in combination with one or more protein(s) selected from the group consisting of CCoAMT1, ABCG37 and UGT71C1 protein(s).
Methods for Manufacturing a Product
In one embodiment the method for the production of a product comprises
In a further embodiment the method comprises the steps a) growing the plants of the invention, b) removing the harvestable parts as defined above from the plants and c) producing said product from or by the harvestable parts of the invention.
Preferably the products obtained by said method comprises an exogenous nucleic acid(s) and/or protein(s) according to the invention.
Method for the production of a product comprising
wherein the product comprise the F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid(s) or the proteins encoded by said nucleic acids.
The product may be produced at the site where the plant has been grown, the plants and/or parts thereof may be removed from the site where the plants have been grown to produce the product. Typically, the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant. The step of growing the plant may be performed only once each time the methods of the invention is performed, while allowing repeated times the steps of product production e.g. by repeated removal of harvestable parts of the plants of the invention and if necessary further processing of these parts to arrive at the product. It is also possible that the step of growing the plants of the invention is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts. Also, the steps of growing the plants and producing the product may be performed with an overlap in time, even simultaneously to a large extend or sequentially. Generally the plants are grown for some time before the product is produced.
In one embodiment the products produced by said methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fiber, cosmetic and/or pharmaceutical. Foodstuffs are regarded as compositions used for nutrition and/or for supplementing nutrition. Animal feedstuffs and animal feed supplements, in particular, are regarded as foodstuffs.
In another embodiment the inventive methods for the production are used to make agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.
It is possible that a plant product consists of one or more agricultural products to a large extent.
Methods for Breeding/Methods for Plant Improvement/Methods Plant Variety Production
The transgenic plants of the invention may be crossed with similar transgenic plants or with transgenic plants lacking the nucleic acids of the invention or with non-transgenic plants, using known methods of plant breeding, to prepare seeds. Further, the transgenic plant cells or plants of the present invention may comprise, and/or be crossed to another transgenic plant that comprises one or more exogenous nucleic acids, thus creating a “stack” of transgenes in the plant and/or its progeny. The seed is then planted to obtain a crossed fertile transgenic plant comprising the F6H1 nucleic acid optionally in combination with nucleic acids selected from the group consisting of CCoAMT1, ABCG37 and UGT71C1 nucleic acid(s). The crossed fertile transgenic plant may have the particular expression cassette inherited through a female parent or through a male parent. The second plant may be an inbred plant. The crossed fertile transgenic may be a hybrid. Also included within the present invention are seeds of any of these crossed fertile transgenic plants. The seeds of this invention can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plant lines comprising the exogenous nucleic acid.
Thus, one embodiment of the present invention is a method for breeding a fungal resistant plant comprising the steps of
Another preferred embodiment is a method for plant improvement comprising
The transgenic plants may be selected by known methods as described above (e.g., by screening for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the F6H1, CCoAMT1, ABCG37 and/or UGT71C1 gene or screening for the F6H1, CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid itself).
According to the present invention, the introduced F6H1 nucleic acid optionally in combination with one or more nucleic acids selected from the group consisting of CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid may be maintained in the plant cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes. Whether present in an extra-chromosomal non-replicating or replicating vector construct or a vector construct that is integrated into a chromosome, the exogenous F6H1, CCoAMT1, ABCG37 and/or UGT71C1 nucleic acid preferably resides in one or more a plant expression cassette. A plant expression cassette preferably contains regulatory sequences capable of driving gene expression in plant cells that are functional linked so that each sequence can fulfill its function, for example, termination of transcription by polyadenylation signals. Preferred polyadenylation signals are those originating from Agrobacterium tumefaciens t-DNA such as the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al., 1984, EMBO J. 3:835) or functional equivalents thereof, but also all other terminators functionally active in plants are suitable. As plant gene expression is very often not limited on transcriptional levels, a plant expression cassette preferably contains other functional linked sequences like translational enhancers such as the overdrive-sequence containing the 5′-untranslated leader sequence from tobacco mosaic virus increasing the polypeptide per RNA ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711). Examples of plant expression vectors include those detailed in: Becker, D. et al., 1992, New plant binary vectors with selectable markers located proximal to the left border, Plant Mol. Biol. 20:1195-1197; Bevan, M. W., 1984, Binary Agrobacterium vectors for plant transformation, Nucl. Acid. Res. 12:8711-8721; and Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung and R. Wu, Academic Press, 1993, S. 15-38.
A preferred method according to the invention is a method for applying a scopoletin and/or a derivative thereof to a surface of a plant, plant part or plant cell, wherein the resistance to a fungal pathogen of the plant, plant part or plant cell is increased by applying scopoletin and/or a derivative thereof to the surface of the plant, plant part or plant cell in comparison to a plant, plant part or plant cell to which surface scopoletin and/or a derivative has not been applied, wherein the plant is soy and/or corn.
In one embodiment according to the invention a plant surface or plant part surface is coated with scopoletin and/or a derivative thereof, wherein the plant is soy and/or corn.
In one embodiment according to the invention a plant, plant part or plant cell has a surface coated with scopoletin and/or a derivative thereof. wherein the plant is soy and/or corn.
The following examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the present invention.
The chemical synthesis of oligonucleotides can be affected, for example, in the known fashion using the phosphoamidite method (Voet, Voet, 2nd Edition, Wiley Press New York, pages 896-897). The cloning steps carried out for the purposes of the present invention such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking DNA fragments, transformation of E. coli cells, bacterial cultures, phage multiplication and sequence analysis of recombinant DNA, are carried out as described by Sambrook et al. Cold Spring Harbor Laboratory Press (1989), ISBN 0-87969-309-6. The sequencing of recombinant DNA molecules is carried out with an MWG-Licor laser fluorescence DNA sequencer following the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977).
To obtain cDNA, RNA was extracted from leaf tissue of Arabidopsis thaliana pen2 mutants that had been inoculated with P. pachyrhizi two days before harvest. cDNA was produced using RevertAid H minus reverse trancriptase (Thermo Scientific). All steps of cDNA preparation and purification were performed according as described in the manual.
The SEQ-ID 1-sequence (F6H1) was amplified from the cDNA by PCR as described in the protocol of the Phusion High-Fidelity DNA Polymerase (Thermo Scientific) hot-start, Pfu Ultra, Pfu Turbo or Herculase DNA polymerase (Stratagene). The composition for the protocol of the Pfu Ultra, Pfu Turbo or Herculase DNA polymerase was as follows: 1×PCR buffer, 0.2 mM of each dNTP, 100 ng cDNA of Arabidopsis thaliana (var Columbia-0), 50 pmol forward primer, 50 pmol reverse primer, 1 u Phusion hot-start, Pfu Ultra, Pfu Turbo or Herculase DNA polymerase.
The amplification cycles were as follows:
1 cycle of 30 seconds at 98° C., followed by 35 cycles of in each case 10 seconds at 98° C., 30 seconds at 62° C. and 40 seconds at 72° C., followed by 1 cycle of 10 minutes at 72° C., then 4° C.
The following primer sequences were used to specifically amplify the F6H1 full-length ORF for cloning purposes:
The amplified fragments were gel purified and cloned into the pDONR 207 entry vector (Invitrogen) using Gateway® cloning according to the manufacturer's instructions. Using this cloning technique the full-length F6H1 fragment is inserted in sense direction between the attL1 and attL2 recombination sites of the entry vector. To prepare an untagged F6H1 overexpression construct, a LR reaction (Gateway system, (Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturers protocol by using a pDONR207 vector containing the F6H1 fragment. As target a binary pB2GW7 (Ghent University, Belgium) vector was used, which is composed of: (1) a Spectinomycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a pBR322 origin of replication for stable maintenance in E. coli and (4) between the right and left border a bar selection gene under control of a pNos-promoter. The recombination reaction was transformed into competent E. coli (DH5alpha), mini-prepped and screened by specific restriction digestions. A positive clone from each the vector construct was sequenced and submitted to soy transformation (see
The amplified fragments were gel purified and to prepare a FLAG tagged F6H1 overexpression construct MultiSite Gateway® cloning was applied according to the manufacturer's manual. First, the Ω-FLAG sequence was PCR amplified from the vector pTA7002 (Shuqun Zhang, Columbia University, Missouri, USA) harboring the Arabidopsis thaliana MKK4 gene 5′ flanked by a tobacco mosaic virus Ω translational enhancer and a FLAG Tag sequence using following primers (attB primer extensions underlined):
GGGGACAAGTTTGTACAAAAAAGCAGGCTCATATTTTTACAACAATTAC
GGGGACAACTTTTGTATACAAAGTTGTCTTGTCATCGTCGTCCTTGT
Second, the F6H1 full length coding sequence was PCR amplified from pen2 cDNA prepared as described above. Following primer sequences carrying 5″attB5 and attB2 extensions were used for F6H1 amplification by PCR
GGGGACAACTTTGTATACAAAAGTTGCAATGGCTCCAACACTCTTGAC
GGGGACCACTTTGTACAAGAAAGCTGGGTATCAGATCTTGGCGTAATCG
PCR products were gel purified and the attB1-Ω-FLAG-attB5r sequence introduced into pDONR221 P1-P5r via gateway cloning (BP reaction). Analogously the attB5-F6H1-attB2 sequence was cloned into pDONR 221 P5-P2. Recombination reactions were transformed into competent E. coli (DH5alpha). Following plasmid extraction both vectors were used for LR recombination with the pB2GW7 destination vector. The resulting expression clone containing both sequences (Ω-FLAG and F6H1) was screened by specific restriction digestions and sequenced prior to transformation (
Transient transformation of N. benthamiana leaves was done according to a slightly modified protocol from Popescu et al. 2007 (Popescu, S. C., Popescu, G. V., Bachan, S., Zhang, Z., Seay, M., Gerstein, M., Snyder, M., and Dinesh-Kumar, S. P. (2007). Differential binding of calmodulin-related proteinsto their targets revealed through high-density Arabidopsis protein microarrays Proc Natl Acad Sci USA 104, 4730-4735.) A single Agrobacterium (strain AGL01) carrying a DNA construct of interest (see
Plant material was ground in liquid N2 and extracted for 24 h with 90% (v/v) methanol (1 ml per 0.5 g fresh material) supplemented with 4-methylumbelliferone as an internal standard. Extracts were centrifuged for 10 min at 15,000 g. The supernatants were concentrated in a speed vac and the dried residue resolved in 150 μl 100% methanol. Samples (20 μl injection volume) were subsequently subjected to reverse-phase high-performance liquid chromatography (HPLC) analysis on a Nucleosil C18 column (EC 150/4.6 Nucleosil 100-5 C18; Macherey-Nagel) with a gradient mobile phase built with 1% (v/v) formic acid in water (A) and 1% (v/v) formic acid in methanol (B), and a flow rate of 1.0 ml/min at RT. The gradient program started at 15% B for 2 min, then increased linearly to 21.5% for 18 min followed by a linear increase to 55% B between 20 and 40 min. The gradient then increased to 95% B for 5 min. This proportion was maintained for 10 min and then returned to initial conditions in 5 min. Scopoletin was detected with a fluorescence detector with an excitation wavelength of 345 nm and an emission wavelength of 460 nm and identified by comparison with the pure reference compound (Scopoletin, SIGMA-ALDRICH).
Total RNA was extracted from leaves of the described Arabidopsis mutants as described by Chomczynski and Sacchi (1987). 1 μg RNA was transcribed to cDNA using random primers (9-mers) and RevertAid™ reverse transcriptase (Fermentas) according to manufacturer's instructions. Accumulation of gene transcripts was quantified in an ABI7300 using SYBR green (Invitrogen) at the following conditions for RT-qPCR: 50° C. for 2 min, 95° C. for 10 min, 95° C. for 15 s, 60° C. for 1 min, 95° C. for 15 s, 60° C. for 1 min, and 95° C. for 15 s (the third and fourth steps were repeated 40 times).
All primers were designed according to standard criteria (Udvardi et al., 2008), off target search using Primer Blast tool at NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast/)). Expression of the genes was normalized to Actin2. Data were analyzed using the ABI 7300 software and the expression relative to actin was calculated according to Livak and Schmittgen (2001) with 2−(Ct F6H1-Ct Actin2).
Spores of Phakopsora pachyrhizi were resuspended in H2O supplemented with Tween-20 and 10 μM, 100 μM, 500 μM and 1 mM scopoletin. Spores of Phakopsora pachyrhizi resuspended in H2O supplemented with Tween-20 were used as control. All resuspended spores were transferred onto glass slides. After six hours incubation time the ASR spores were germinated and started to form appressoria.
The germination rate and appressoria formation rate was determined by quantitative microscopic analysis. Spores showing a visible germtube formation but no thickening of the germ tube tip were counted as “germinated”, whereas the presence of a thickened germ tube tip indicated the formation of an appressoria (
Application of scopoletin to ASR spores decreases the germination and appressoria formation in a dose dependent manner. At 1 mM concentration scopoletin completely abolishes spore germination in-vitro.
1 cm2 Agar plugs from 7 day old F. graminearum cultures grown on potato dextrose agar (PDA) at 24° C. were placed on fresh PDA plates supplemented with 1 mM scopoletin in methanol or equal volumes of methanol lacking scopoletin as control. Fungal spores were stained by spraying Uvitex2 solution (0.1% Uvitex 2B (Polyscience, Warrington, UK) solved in 0.1 M Tris-/HCl-buffer, pH 8.5). Fungal growth was measured daily using a fluorescence microscope to determine the average growth rate of the fungus. 100 spores were counted per sample (see
6.1 Arabidopsis
Arabidopsis seeds were sown on soil (type VM, Einheitserde Werkverband) and stratified at 4° C. for two days. Plants were grown at short day conditions (in a chamber at 8 h photoperiod, 120 μmol m-2 s-1 photon irradiance) 22° C., and 65% humidity. Five to six-week-old plants were inoculated with P. pachyrhizi as described below.
For pre-treatment experiments Arabidopsis plants were sprayed with—1 mM scopoletin (solved in H2O, 0.01% Tween-20); incubated for 6 h at short day conditions and subsequently inoculated with 1 mg/ml P. pachyrhizi uredospores. For co-treatment experiments spores of Phakopsora pachyrhizi were solved in 0.01% Tween-20 supplemented with 1 mM scopoletin.
Following inoculation plants were covered with moistened plastic domes to ensure high humidity and incubated at short day conditions (see above). 24 h later plastic domes were removed and plants incubated at the same conditions for another 24 h. Leaves were harvested 2 dpi and destained on tissue soaked with a saturated (2.5 g/ml) chloralhydrate solution. Germination and penetration on destained leaves was determined by quantitative microscopic analysis. Spores showing a visible germtube formation are assigned to the category “germinated” Pretreated as well as co-treated plants showed a drastically reduced formation of germinated spores, proving the toxic effect of scopoletin against soybean rust fungus (
6.2 Soybean
Soy seeds were sown on soil (type VM, Einheitserde Werkverband) and grown at short day conditions in a chamber (at 8 h photoperiod, 120 μmol m-2 s-1 photon irradiance) 22° C., and 65% humidity. Five to six-week-old plants were inoculated with P. pachyrhizi as described below.
For co-treatment experiments spores of Phakopsora pachyrhizi were solved in 0.01% Tween-20 supplemented with 10 μM, 100 μM, 500 μM and 1 mM scopoletin (
For pre-treatment experiments soy plants were sprayed with 1 mM scopoletin (solved in H2O, 0.01% Tween-20); incubated for 6 h and subsequently inoculated with 1 mg/ml P. pachyrhizi uredospores (
Following inoculation plants were covered with moistened plastic domes to ensure high humidity and incubated at short day conditions (see above). 24 h later plastic domes were removed and plants incubated at the same conditions for another 11 days. At 12 dpi the diseased leaf area was rated on primary leaves, first and second trifoliate leaves by using the program Assess2.0 (Lobet G., Draye X., Périlleux C. 2013 An online database for plant image analysis software tools, Plant Methods, vol. 9 (38)). The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves is considered as diseased leaf area.
Pretreated as well as co-treated plants showed a drastically reduced formation of infected leaf area (
The DNA sequence of the F6H1 (AT3G13610, SEQ ID No: 1), CCoAOMT1 (At4g34050, SEQ ID No: 3), ABCG37(PDR9; AT3G53480, SEQ ID No: 5) and UGT71C1 (SEQ ID No: 7) genes mentioned in this application were generated by DNA synthesis (Geneart, Regensburg, Germany).
The F6H1 DNA (as shown in SEQ ID No: 1) was synthesized in a way that a PacI restriction site is located in front of the start-ATG and a AscI restriction site downstream of the stop-codon. The synthesized DNA was digested using the restriction enzymes PacI and AscI (NEB Biolabs) and ligated in a PacI/AscI digested Gateway pENTRY-C vector (Invitrogen, Life Technologies, Carlsbad, Calif., USA) in a way that the full-length fragment is located in sense direction between the parsley ubiquitin promoter and the Agrobacterium tumefaciens derived octopine synthase terminator (t-OCS). The PcUbi promoter regulates constitutive expression of the ubi4-2 gene (accession number X64345) of Petroselinum crispum (Kawalleck et al. 1993 Plant Molecular Biology 21(4): 673-684).
To obtain the binary plant transformation vector, a triple LR reaction (Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturer's protocol by using an empty pENTRY-A vector, an empty pENTRY-C, and the PcUbi promoter::F6H1::OCS-terminator in the above described pENTRY-C vector. As target a binary pDEST vector was used which is composed of: (1) a Spectinomycin/Streptomycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a ColE1 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection under control of a AtAHASL-promoter (see
To obtain the F6H1-CCoAOMT1 double gene construct (
To obtain the binary plant transformation vector containing F6H1 and CCoAOMT1, a triple LR reaction (Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturer's protocol by using an empty pENTRY-A vector, the pSuper promoter::CCoAOMT1::nos-terminator in the above described pENTRY-B vector and the PcUbi promoter::F6H1::OCS-terminator in the above described pENTRY-C vector. As target a binary pDEST vector was used which is composed of: (1) a Spectinomycin/Streptomycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a ColE1 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection under control of a AtAHASL-promoter (see
To obtain the F6H1-UGT71C1 double gene construct (
To obtain the binary plant transformation vector containing F6H1 and UGT71C1, a triple LR reaction (Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturer's protocol by using an empty pENTRY-A vector, the pSuper promoter::UGT71C1::nos-terminator in the above described pENTRY-B vector and the PcUbi promoter::F6H1::OCS-terminator in the above described pENTRY-C vector. As target a binary pDEST vector was used which is composed of: (1) a Spectinomycin/Streptomycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a ColE1 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection under control of a AtAHASL-promoter (see
To obtain the F6H1-CCoAOMT1-ABCG37 (
The synthesized DNA was digested using the restriction enzymes PacI and AscI (NEB Biolabs) and ligated in a PacI/AscI digested Gateway pENTRY-A vector (Invitrogen, Life Technologies, Carlsbad, Calif., USA) in a way that the full-length fragment is located in sense direction between the pGlyma14g06680 promoter (see WO 2012/127373) and the Solanum tuberosum cathepsin D inhibitor (Herbers, Karin, Salomé Prat, and Lothar Willmitzer. “Functional analysis of a leucine aminopeptidase from Solanum tuberosum L.” Planta 194.2 (1994): 230-240.). The pGlyma14g06680 promoter mediates a medium strong constitutive expression in soybean.
To obtain the binary plant transformation vector containing F6H1, CCoAOMT1 and ABCG37, a triple LR reaction (Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturer's protocol by using the Glyma14g06680 promoter::ABCG37::cathepsin inhibitor terminator in the pENTRY-A vector, as described above, the pSuper promoter::CCoAOMT1::nos-terminator in the above described pENTRY-B vector and the PcUbi promoter::F6H1::OCS-terminator in the above described pENTRY-C vector.
As target a binary pDEST vector was used which is composed of: (1) a Spectinomycin/Streptomycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a ColE1 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection under control of a AtAHASL-promoter (see
The expression vector constructs (see example 2) is transformed into soy.
8.1 Sterilization and Germination of Soy Seeds
Virtually any seed of any soy variety can be employed in the method of the invention. A variety of soybean cultivar (including Jack, Williams 82, Jake, Stoddard, CD215 and Resnik) is appropriate for soy transformation. Soy seeds are sterilized in a chamber with a chlorine gas produced by adding 3.5 ml 12N HCl drop wise into 100 ml bleach (5.25% sodium hypochlorite) in a desiccator with a tightly fitting lid. After 24 to 48 hours in the chamber, seeds are removed and approximately 18 to 20 seeds are plated on solid GM medium with or without 5 μM 6-benzyl-aminopurine (BAP) in 100 mm Petri dishes. Seedlings without BAP are more elongated and roots develop especially secondary and lateral root formation. BAP strengthens the seedling by forming a shorter and stockier seedling.
Seven-day-old seedlings grown in the light (>100 μEinstein/m2s) at 25 degreeC are used for explant material for the three-explant types. At this time, the seed coat was split, and the epicotyl with the unifoliate leaves are grown to, at minimum, the length of the cotyledons.
The epicotyl should be at least 0.5 cm to avoid the cotyledonary-node tissue (since soycultivars and seed lots may vary in the developmental time a description of the germination stage is more accurate than a specific germination time).
For inoculation of entire seedlings, see Method A (example 8.3. and 8.3.2) or leaf explants see Method B (example 8.3.3).
For method C (see example 8.3.4), the hypocotyl and one and a half or part of both cotyledons are removed from each seedling. The seedlings are then placed on propagation media for 2 to 4 weeks. The seedlings produce several branched shoots to obtain explants from. The majority of the explants originated from the plantlet growing from the apical bud. These explants are preferably used as target tissue.
8.2—Growth and Preparation of Agrobacterium Culture
Agrobacterium cultures are prepared by streaking Agrobacterium (e.g., A. tumefaciens or A. rhizogenes) carrying the desired binary vector (e.g. H. Klee. R. Horsch and S. Rogers 1987 Agrobacterium-Mediated Plant Transformation and its further Applications to Plant Biology; Annual Review of Plant Physiology Vol. 38: 467-486) onto solid YEP growth medium YEP media: 10 g yeast extract. 10 g Bacto Peptone. 5 g NaCl. Adjust pH to 7.0, and bring final volume to 1 liter with H2O, for YEP agar plates add 20 g Agar, autoclave) and incubating at 25.degree C. until colonies appeared (about 2 days). Depending on the selectable marker genes present on the Ti or Ri plasmid, the binary vector, and the bacterial chromosomes, different selection compounds are be used for A. tumefaciens and A. rhizogenes selection in the YEP solid and liquid media. Various Agrobacterium strains can be used for the transformation method.
After approximately two days, a single colony (with a sterile toothpick) is picked and 50 ml of liquid YEP is inoculated with antibiotics and shaken at 175 rpm (25° C.) until an OD600 between 0.8-1.0 is reached (approximately 2 d). Working glycerol stocks (15%) for transformation are prepared and one-ml of Agrobacterium stock aliquoted into 1.5 ml Eppendorf tubes then stored at −80° C.
The day before explant inoculation, 200 ml of YEP are inoculated with 5 μl to 3 ml of working Agrobacterium stock in a 500 ml Erlenmeyer flask. The flask is shaken overnight at 25° C. until the OD600 is between 0.8 and 1.0. Before preparing the soy explants, the Agrobacteria ARE pelleted by centrifugation for 10 min at 5,500×g at 20° C. The pellet Is resuspended in liquid CCM to the desired density (OD600 0.5-0.8) and placed at room temperature at least 30 min before use.
8.3—Explant Preparation and Co-Cultivation (Inoculation)
8.3.1 Method A: Explant Preparation on the Day of Transformation.
Seedlings at this time had elongated epicotyls from at least 0.5 cm but generally between 0.5 and 2 cm. Elongated epicotyls up to 4 cm in length are successfully employed. Explants are then prepared with: i) with or without some roots, ii) with a partial, one or both cotyledons, all preformed leaves are removed including apical meristem, and the node located at the first set of leaves is injured with several cuts using a sharp scalpel.
This cutting at the node not only induces Agrobacterium infection but also distributes the axillary meristem cells and damaged pre-formed shoots. After wounding and preparation, the explants are set aside in a Petri dish and subsequently co-cultivated with the liquid CCM/Agrobacterium mixture for 30 minutes. The explants are then removed from the liquid medium and plated on top of a sterile filter paper on 15×100 mm Petri plates with solid co-cultivation medium. The wounded target tissues are placed such that they are in direct contact with the medium.
8.3.2 Modified Method A: Epicotyl Explant Preparation
Soyepicotyl segments prepared from 4 to 8 d old seedlings are used as explants for regeneration and transformation. Seeds of soya cv. L00106CN, 93-41131 and Jack are germinated in 1/10 MS salts or a similar composition medium with or without cytokinins for 4 to 8 d. Epicotyl explants are prepared by removing the cotyledonary node and stem node from the stem section. The epicotyl is cut into 2 to 5 segments. Especially preferred are segments attached to the primary or higher node comprising axillary meristematic tissue.
The explants are used for Agrobacterium infection. Agrobacterium AGL1 harboring a plasmid with the gene of interest (GOI) and the AHAS, bar or dsdA selectable marker gene is cultured in LB medium with appropriate antibiotics overnight, harvested and resuspended in a inoculation medium with acetosyringone. Freshly prepared epicotyl segments are soaked in the Agrobacterium suspension for 30 to 60 min and then the explants were blotted dry on sterile filter papers. The inoculated explants are then cultured on a co-culture medium with L-cysteine and TTD and other chemicals such as acetosyringone for increasing T-DNA delivery for 2 to 4 d. The infected epicotyl explants are then placed on a shoot induction medium with selection agents such as imazapyr (for AHAS gene), glufosinate (for bar gene), or D-serine (for dsdA gene). The regenerated shoots are subcultured on elongation medium with the selective agent.
For regeneration of transgenic plants the segments are then cultured on a medium with cytokinins such as BAP, TDZ and/or Kinetin for shoot induction. After 4 to 8 weeks, the cultured tissues are transferred to a medium with lower concentration of cytokinin for shoot elongation. Elongated shoots are transferred to a medium with auxin for rooting and plant development. Multiple shoots are regenerated.
Many stable transformed sectors showing strong cDNA expression are recovered. Soybean plants are regenerated from epicotyl explants. Efficient T-DNA delivery and stable transformed sectors are demonstrated.
8.3.3 Method B: Leaf Explants
For the preparation of the leaf explant the cotyledon is removed from the hypocotyl. The cotyledons are separated from one another and the epicotyl is removed. The primary leaves, which consist of the lamina, the petiole, and the stipules, are removed from the epicotyl by carefully cutting at the base of the stipules such that the axillary meristems are included on the explant. To wound the explant as well as to stimulate de novo shoot formation, any pre-formed shoots are removed and the area between the stipules was cut with a sharp scalpel 3 to 5 times.
The explants are either completely immersed or the wounded petiole end dipped into the Agrobacterium suspension immediately after explant preparation. After inoculation, the explants are blotted onto sterile filter paper to remove excess Agrobacterium culture and place explants with the wounded side in contact with a round 7 cm Whatman paper overlaying the solid CCM medium (see above). This filter paper prevents A. tumefaciens overgrowth on the soy-explants. Wrap five plates with Parafilm™ “M” (American National Can, Chicago, Ill., USA) and incubate for three to five days in the dark or light at 25° C.
8.3.4 Method C: Propagated Axillary Meristem
For the preparation of the propagated axillary meristem explant propagated 3-4 week-old plantlets are used. Axillary meristem explants can be pre-pared from the first to the fourth node. An average of three to four explants could be obtained from each seedling. The explants are prepared from plantlets by cutting 0.5 to 1.0 cm below the axillary node on the internode and removing the petiole and leaf from the explant. The tip where the axillary meristems lie is cut with a scalpel to induce de novo shoot growth and allow access of target cells to the Agrobacterium. Therefore, a 0.5 cm explant included the stem and a bud.
Once cut, the explants are immediately placed in the Agrobacterium suspension for 20 to 30 minutes. After inoculation, the explants are blotted onto sterile filter paper to remove excess Agrobacterium culture then placed almost completely immersed in solid CCM or on top of a round 7 cm filter paper overlaying the solid CCM, depending on the Agrobacterium strain. This filter paper prevents Agrobacterium overgrowth on the soy-explants. Plates are wrapped with Parafilm™ “M” (American National Can, Chicago, Ill., USA) and incubated for two to three days in the dark at 25° C.
8.4—Shoot Induction
After 3 to 5 days co-cultivation in the dark at 25° C., the explants are rinsed in liquid SIM medium (to remove excess Agrobacterium) (SIM, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soy using primary-node explants from seedlings In Vitro Cell. Dev. Biol.—Plant (2007) 43:536-549; to remove excess Agrobacterium) or Modwash medium (1×B5 major salts, 1×B5 minor salts, 1×MSIII iron, 3% Sucrose, 1×B5 vitamins, 30 mM MES, 350 mg/L Timentin pH 5.6, WO 2005/121345) and blotted dry on sterile filter paper (to prevent damage especially on the lamina) before placing on the solid SIM medium. The approximately 5 explants (Method A) or 10 to 20 (Methods B and C) explants are placed such that the target tissue was in direct contact with the medium. During the first 2 weeks, the explants could be cultured with or without selective medium. Preferably, explants are transferred onto SIM without selection for one week.
For leaf explants (Method B), the explant should be placed into the medium such that it is perpendicular to the surface of the medium with the petiole imbedded into the medium and the lamina out of the medium.
For propagated axillary meristem (Method C), the explant is placed into the medium such that it is parallel to the surface of the medium (basipetal) with the explant partially embedded into the medium.
Wrap plates with Scotch 394 venting tape (3M, St. Paul, Minn., USA) are placed in a growth chamber for two weeks with a temperature averaging 25.degree. C. under 18 h light/6 h dark cycle at 70-100 μE/m2s. The explants remains on the SIM medium with or without selection until de novo shoot growth occurred at the target area (e.g., axillary meristems at the first node above the epicotyl). Transfers to fresh medium can occur during this time. Explants are transferred from the SIM with or without selection to SIM with selection after about one week. At this time, there is considerable de novo shoot development at the base of the petiole of the leaf explants in a variety of SIM (Method B), at the primary node for seedling explants (Method A), and at the axillary nodes of propagated explants (Method C).
Preferably, all shoots formed before transformation are removed up to 2 weeks after co-cultivation to stimulate new growth from the meristems. This helped to reduce chimerism in the primary transformant and increase amplification of transgenic meristematic cells. During this time the explant may or may not be cut into smaller pieces (i.e. detaching the node from the explant by cutting the epicotyl).
8.5—Shoot Elongation
After 2 to 4 weeks (or until a mass of shoots is formed) on SIM medium (preferably with selection), the explants are transferred to SEM medium (shoot elongation medium, see Olhoft et al 2007 A novel Agrobacterium rhizogenes-mediated transformation method of soy using primary-node explants from seedlings. In Vitro Cell. Dev. Biol.—Plant (2007) 43:536-549) that stimulates shoot elongation of the shoot primordia. This medium may or may not contain a selection compound.
After every 2 to 3 weeks, the explants are transferred to fresh SEM medium (preferably containing selection) after carefully removing dead tissue. The explants should hold together and not fragment into pieces and retain somewhat healthy. The explants are continued to be transferred until the explant dies or shoots elongate. Elongated shoots >3 cm are removed and placed into RM medium for about 1 week (Methods A and B), or about 2 to 4 weeks depending on the cultivar (Method C) at which time roots began to form. In the case of explants with roots, they are transferred directly into soil. Rooted shoots are transferred to soil and hardened in a growth chamber for 2 to 3 weeks before transferring to the greenhouse. Regenerated plants obtained using this method are fertile and produced on average 500 seeds per plant.
After 5 days of co-cultivation with Agrobacterium tumefaciens transient expression of the gene of interest (GOI) is widespread on the seedling axillary meristem explants especially in the regions wounding during explant preparation (Method A). Explants are placed into shoot induction medium without selection to see how the primary-node responds to shoot induction and regeneration. Thus far, greater than 70% of the explants were formed new shoots at this region. Expression of the GOI is stable after 14 days on SIM, implying integration of the T-DNA into the soybean genome. In addition, preliminary experiments results in the formation of cDNA expressing shoots forming after 3 weeks on SIM.
For Method C, the average regeneration time of a soybean plantlet using the propagated axillary meristem protocol is 14 weeks from explant inoculation. Therefore, this method has a quick regeneration time that leads to fertile, healthy soybean plants.
9.1. Growth of Plants
10 T1 soy plants per event are potted and grown for 3-4 weeks in the Phytochamber (16 h-day-und 8 h-night-Rhythm at a temperature of 16° and 22° C. und a humidity of 75%) till the first 2 trifoliate leaves were fully expanded.
9.2 Inoculation
The plants are inoculated with spores of P. pachyrhizi.
In order to obtain appropriate spore material for the inoculation, soybean leaves which are infected with rust 15-20 days ago, are taken 2-3 days before the inoculation and transferred to agar plates (1% agar in H2O). The leaves are placed with their upper side onto the agar, which allowed the fungus to grow through the tissue and to produce very young spores. For the inoculation solution, the spores are knocked off the leaves and are added to a Tween-H2O solution. The counting of spores is performed under a light microscope by means of a Thoma counting chamber. For the inoculation of the plants, the spore suspension is added into a compressed-air operated spray flask and applied uniformly onto the plants or the leaves until the leaf surface is well moisturized. For macroscopic assays a spore density of 1-5×105 spores/ml is used. For the microscopy, a density of >5×105 spores/ml is used. The inoculated plants are placed for 24 hours in a greenhouse chamber with an average of 22° C. and >90% of air humidity. The following cultivation is performed in a chamber with an average of 25° C. and 70% of air humidity.
For the evaluation of the pathogen development, the inoculated leaves of plants are stained with aniline blue 48 hours after infection.
The aniline blue staining serves for the detection of fluorescent substances. During the defense reactions in host interactions and non-host interactions, substances such as phenols, callose or lignin accumulate or are produced and are incorporated at the cell wall either locally in papillae or in the whole cell (hypersensitive reaction, HR). Complexes are formed in association with aniline blue, which lead e.g. in the case of callose to yellow fluorescence. The leaf material is transferred to falcon tubes or dishes containing destaining solution II (ethanol/acetic acid 6/1) and is incubated in a water bath at 90° C. for 10-15 minutes. The destaining solution II is removed immediately thereafter, and the leaves are washed 2× with water. For the staining, the leaves are incubated for 1.5-2 hours in staining solution II (0.05% aniline blue=methyl blue, 0.067 M di-potassium hydrogen phosphate) and analyzed by microscopy immediately thereafter.
The different interaction types are evaluated (counted) by microscopy. An Olympus UV microscope BX61 (incident light) and a UV Longpath filter (excitation: 375/15, Beam splitter: 405 LP) are used. After aniline blue staining, the spores appeared blue under UV light. The papillae can be recognized beneath the fungal appressorium by a green/yellow staining. The hypersensitive reaction (HR) is characterized by a whole cell fluorescence
The progression of the soybean rust disease is scored by the estimation of the diseased area (area which was covered by sporulating uredinia) on the backside (abaxial side) of the leaf. Additionally the yellowing of the leaf is taken into account. (for scheme see
At all 50 T1 soybean plants per construct are inoculated with spores of Phakopsora pachyrhizi. The macroscopic disease symptoms of soy against P. pachyrhizi of the inoculated soybean plants are scored 14 days after inoculation.
The average of the percentage of the leaf area showing fungal colonies or strong yellowing/browning on all leaves is considered as diseased leaf area. At all 50 soybean T1 plants per construct (expression checked by RT-PCR) are evaluated in parallel to non-transgenic control plants. Non-transgenic soy plants grown in parallel to the transgenic plants are used as controls.
The expression of the F6H1 gene will lead to enhanced resistance of corn against Phakopsora pachyrhizi.
The DNA sequence of the F6H1 (AT3G13610), CCoAOMT1 (At4g34050) and ABCG37 (PDR9; AT3G53480) genes mentioned in this application were generated by DNA synthesis (Geneart, Regensburg, Germany).
The F6H1 DNA (as shown in SEQ ID No: 1) was synthesized in a way that a PacI restriction site is located in front of the start-ATG and a AscI restriction site downstream of the stop-codon. The synthesized DNA was digested using the restriction enzymes PacI and AscI (NEB Biolabs) and ligated in a PacI/AscI digested Gateway pENTRY-C vector (Invitrogen, Life Technologies, Carlsbad, Calif., USA) in a way that the full-length fragment is located in sense direction between the maize ubiquitin promoter and the Agrobacterium tumefaciens derived octopine synthase terminator (t-OCS).
To obtain the binary plant transformation vector, a triple LR reaction (Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturer's protocol by using an empty pENTRY-A vector, an empty pENTRY-C, and the ZmUbi promoter::F6H1::OCS-terminator in the above described pENTRY-C vector. As target a binary pDEST vector was used which is composed of: (1) a Spectinomycin/Streptomycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a ColE1 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection (Z. mays acetohydroxyacid synthase (AHAS108) gene) under control of a Maize AHASL2 promoter. The recombination reaction was transformed into E. coli (DH5alpha), mini-prepped and screened by specific restriction digestions. A positive clone from the vector construct was sequenced and submitted to corn transformation.
To obtain the F6H1-CCoAOMT1 double gene construct the CCoAOMT1 DNA (as shown in SEQ ID No: 3) was synthesized in a way that a PacI restriction site is located in front of the start-ATG and a AscI restriction site downstream of the stop-codon. The synthesized DNA was digested using the restriction enzymes PacI and AscI (NEB Biolabs) and ligated in a PacI/AscI digested Gateway pENTRY-B vector (Invitrogen, Life Technologies, Carlsbad, Calif., USA) in a way that the full-length fragment is located in sense direction between the pSuper promoter (Lee et al., 2007 Plant Physiology Vol 145 Issue 4 1294-1300) and the Agrobacterium tumefaciens derived nopaline synthase terminator (t-nos). The Super promoter consists of three identical Octapine Synthase Enhancers followed by a MAS promoter (Lee et al., 2007 Plant Physiology Vol 145 Issue 4 1294-1300).
To obtain the binary plant transformation vector containing F6H1 and CCoAOMT1, a triple LR reaction (Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturer's protocol by using an empty pENTRY-A vector, the pSuper promoter::CCoAOMT1::nos-terminator in the above described pENTRY-B vector and the PcUbi promoter::F6H1::OCS-terminator in the above described pENTRY-C vector. As target a binary pDEST vector was used which is composed of: (1) a Spectinomycin/Streptomycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a ColE1 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection (Z. mays acetohydroxyacid synthase (AHAS108) gene) under control of a Maize AHASL2 promoter. The recombination reaction was transformed into E. coli (DH5alpha), mini-prepped and screened by specific restriction digestions. A positive clone from each vector construct was sequenced and submitted soy transformation.
To obtain the F6H1-UGT71C1 double gene construct the UGT71C1 DNA (as shown in SEQ ID No: 7) was synthesized in a way that a PacI restriction site is located in front of the start-ATG and a AscI restriction site downstream of the stop-codon. The synthesized DNA was digested using the restriction enzymes PacI and AscI (NEB Biolabs) and ligated in a PacI/AscI digested Gateway pENTRY-B vector (Invitrogen, Life Technologies, Carlsbad, Calif., USA) in a way that the full-length fragment is located in sense direction between the pSuper promoter (Lee et al., 2007 Plant Physiology Vol 145 Issue 4 1294-1300) and the Agrobacterium tumefaciens derived nopaline synthase terminator (t-nos). The Super promoter consists of three identical Octapine Synthase Enhancers followed by a MAS promoter (Lee et al., 2007 Plant Physiology Vol 145 Issue 4 1294-1300).
To obtain the binary plant transformation vector containing F6H1 and UGT71C1, a triple LR reaction (Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturer's protocol by using an empty pENTRY-A vector, the pSuper promoter::UGT71C1::nos-terminator in the above described pENTRY-B vector and the ZmUbi promoter::F6H1::OCS-terminator in the above described pENTRY-C vector. As target a binary pDEST vector was used which is composed of: (1) a Spectinomycin/Streptomycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a ColE1 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection (Z. mays acetohydroxyacid synthase (AHAS108) gene) under control of a Maize AHASL2 promoter. The recombination reaction was transformed into E. coli (DH5alpha), mini-prepped and screened by specific restriction digestions. A positive clone from the vector construct was sequenced and submitted to corn transformation.
To obtain the F6H1-CCoAOMT1-ABCG37 triple gene construct the ABCG37 DNA (as shown in SEQ ID No: 5) was synthesized in a way that a PacI restriction site is located in front of the start-ATG and a AscI restriction site downstream of the stop-codon. The synthesized DNA was digested using the restriction enzymes PacI and AscI (NEB Biolabs) and ligated in a PacI/AscI digested Gateway pENTRY-A vector (Invitrogen, Life Technologies, Carlsbad, Calif., USA) in a way that the full-length fragment is located in sense direction between the ScBV promoter (Bouhida, Mohammed, B. E. Lockhart, and Neil E. Olszewski. “An analysis of the complete sequence of a sugarcane bacilliform virus genome infectious to banana and rice.” The Journal of general virology 74 (1993): 15-22.) and the Solanum tuberosum cathepsin D inhibitor (Herbers, Karin, Salomé Prat, and Lothar Willmitzer. “Functional analysis of a leucine aminopeptidase from Solanum tuberosum L.” Planta 194.2 (1994): 230-240.). The ScBV promoter mediates a medium strong constitutive expression in corn.
To obtain the binary plant transformation vector containing F6H1, CCoAOMT1 and ABCG37, a triple LR reaction (Gateway system, Invitrogen, Life Technologies, Carlsbad, Calif., USA) was performed according to manufacturer's protocol by using the ScBV promoter::ABCG37::cathepsin inhibitor terminator in the pENTRY-A vector, as described above, the pSuper promoter::CCoAOMT1::nos-terminator in the above described pENTRY-B vector and the ZmUbi promoter::F6H1::OCS-terminator in the above described pENTRY-C vector.
As target a binary pDEST vector was used which is composed of: (1) a Spectinomycin/Streptomycin resistance cassette for bacterial selection (2) a pVS1 origin for replication in Agrobacteria (3) a ColE1 origin of replication for stable maintenance in E. coli and (4) between the right and left border an AHAS selection (Z. mays acetohydroxyacid synthase (AHAS108) gene) under control of a Maize AHASL2 promoter. The recombination reaction was transformed into E. coli (DH5alpha), mini-prepped and screened by specific restriction digestions. A positive clone from the vector construct was sequenced and submitted to corn transformation.
Agrobacterium cells harboring a plasmid containing the gene of interest (see above) and the mutated maize AHAS gene were grown in YP medium supplemented with appropriate antibiotics for 1-2 days. One loop of Agrobacterium cells was collected and suspended in 1.8 ml M-LS-002 medium (LS-inf). The cultures were incubated while shaking at 1,200 rpm for 5 min-3 hrs. Corn cobs were harvested at 8-11 days after pollination. The cobs were sterilized in 20% Clorox solution for 5 min, followed by spraying with 70% Ethanol and then thoroughly rinsed with sterile water. Immature embryos 0.8-2.0 mm in size were dissected into the tube containing Agrobacterium cells in LS-inf solution.
The constructs were transformed into immature embryos by a protocol modified from Japan Tobacco Agrobacterium mediated plant transformation method (U.S. Pat. Nos. 5,591,616; 5,731,179; 6,653,529; and U.S. Patent Application Publication No. 2009/0249514). Two types of plasmid vectors were used for transformation. One type had only one T-DNA border on each of left and right side of the border, and selectable marker gene and gene of interest were between the left and right T-DNA borders. The other type was so called “two T-DNA constructs” as described in Japan Tobacco U.S. Pat. No. 5,731,179. In the two DNA constructs, the selectable marker gene was located between one set of T-DNA borders and the gene of interest was included in between the second set of T-DNA borders. Either plasmid vector can be used. The plasmid vector was electroporated into Agrobacterium.
Agrobacterium infection of the embryos was carried out by inverting the tube several times. The mixture was poured onto a filter paper disk on the surface of a plate containing co-cultivation medium (M-LS-011). The liquid agro-solution was removed and the embryos were checked under a microscope and placed scutellum side up. Embryos were cultured in the dark at 22° C. for 2-4 days, and transferred to M-MS-101 medium without selection and incubated for four to seven days. Embryos were then transferred to M-LS-202 medium containing 0.75 μM imazethapyr and grown for three weeks at 27° C. to select for transformed callus cells.
Plant regeneration was initiated by transferring resistant calli to M-LS-504 medium supplemented with 0.75 μM imazethapyr and growing under light at 26° C. for two to three weeks. Regenerated shoots were then transferred to a rooting box with M-MS-618 medium (0.5 μM imazethapyr). Plantlets with roots were transferred to soil-less potting mixture and grown in a growth chamber for a week, then transplanted to larger pots and maintained in a greenhouse until maturity.
Transgenic maize plant production is also described, for example, in U.S. Pat. Nos. 5,591,616 and 6,653,529; U.S. Patent Application Publication No. 2009/0249514; and WO/2006136596, each of which are hereby incorporated by reference in their entirety.
Transformation of maize may be made using Agrobacterium transformation, as described in U.S. Pat. Nos. 5,591,616; 5,731,179; U.S. Patent Application Publication No. 2002/0104132, and the like. Transformation of maize (Zea mays L.) can also be performed with a modification of the method described by Ishida et al. (Nature Biotech., 1996, 14:745-750). The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation (Fromm et al., Biotech, 1990, 8:833), but other genotypes can be used successfully as well. Ears are harvested from corn plants at approximately 11 days after pollination (DAP) when the length of immature embryos is about 1 to 1.2 mm. Immature embryos are co-cultivated with Agrobacterium tumefaciens that carry “super binary” vectors and transgenic plants are recovered through organogenesis. The super binary vector system is described in WO 94/00977 and WO 95/06722. Vectors are constructed as described. Various selection marker genes are used including the maize gene encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. No. 6,025,541). Similarly, various promoters are used to regulate the trait gene to provide constitutive, developmental, inducible, tissue or environmental regulation of gene transcription. Excised embryos can be used and can be grown on callus induction medium, then maize regeneration medium, containing imidazolinone as a selection agent. The Petri dishes are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the imidazolinone herbicides and which are PCR positive for the transgenes.
Transgenic maize plants expressing the F6H1 DNA alone or in combination with CCoAOMT1, ABCG37 or UGT71C1 (as described above) are grown in greenhouse or phyto-chamber under standard growing conditions in a controlled environment (20-25° C., 60-90% humidity).
Shortly after the transgenic maize plants enter the reproductive phase they are inoculated near the base of the stalk using a fungal suspension of spores (105 spores in PBS solution) of Fusarium ssp. or Colletotrichum graminicola. Plants are incubated for 2-4 weeks at 20-25° C. and 60-90% humidity.
For scoring the stalk rot disease, stalks are split and the progression of the disease is scored by observation of the characteristic brown to black color of the fungus as it grows up the stalk. Disease ratings are conducted by assigning a visual score. Per experiment the diseased leaf area of more than 10 transgenic plants (and wild-type plants as control) is scored. For analysis the average of the diseased leaf area of the non-transgenic mother plant is set to 100% to calculate the relative diseased leaf area of the transgenic lines
The expression of the F6H1 gene will lead to enhanced resistance of corn against Fusarium ssp. and Colletotrichum graminicola.
The effect on resistance of Scopoletin accumulation in leaves was evaluated. To achieve accumulation of Scopoeltin in leaves a F6H1 overexpression construct generated. The F6H1 overexpression construct (
The 5 best working independent events were selected for further analysis. As trait efficacy is varying depending on the T-DNA insertion site, the average of those 5 independent events is seen as a good measure to estimate the overall effect of F6H1 overexpression.
At all 5 transgenic plants were cultivated per event. Additionally 11 non-transgenic wild type soybean plants were grown in parallel as controls. Presence of the construct was confirmed by qPCR, and Scopoletin accumulation was confirmed by presence of fluorescence (
Three weeks old plants (V1 stage) were inoculated with spores of Phakopsora pachyrhizi as described in example 9.
The progression of the soybean rust disease was scored 14 days after infection by visual rating of the diseased leaf area. Diseased leaf area is defined as area showing fungal colonies or strong yellowing/browning. The relative diseased area in percent is defined as diseased leaf area divided by overall leaf area (for scheme see
Evaluation of Scopoletin accumulating plants was done in parallel to the evaluation of the non-transformed wildtype controls. The average of the diseased leaf area for soybean plants transformed with the F6H1 overexpression construct (
Expression of F6H1 (construct 1,
This data clearly indicates that the in-planta accumulation of Scopoletin leads to a lower disease of transgenic plants compared to non-transgenic wild type controls. So, the expression of F6H1 in soybean significantly (p<0.05) increases the resistance of soy against soybean rust.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15153820 | Feb 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/052019 | 2/1/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/124515 | 8/11/2016 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6281414 | Foley | Aug 2001 | B1 |
| 7888553 | Dixon | Feb 2011 | B2 |
| 20050108791 | Edgerton | May 2005 | A1 |
| 20070214517 | Alexandrov et al. | Sep 2007 | A1 |
| 20070256192 | Herbers | Nov 2007 | A1 |
| 20090100536 | Adams et al. | Apr 2009 | A1 |
| 20160068856 | Schultheiss et al. | Mar 2016 | A1 |
| 20180010144 | Schultheiss et al. | Jan 2018 | A1 |
| Number | Date | Country |
|---|---|---|
| WO-0159140 | Aug 2001 | WO |
| WO-2008034648 | Mar 2008 | WO |
| WO-2011140329 | Nov 2011 | WO |
| WO-2012127373 | Sep 2012 | WO |
| Entry |
|---|
| Prats, E., et al. “Constitutive coumarin accumulation on sunflower leaf surface prevents rust germ tube growth and appressorium differentiation.” Crop science 47.3 (2007): 1119-1124 (Year: 2007). |
| Azelmat, Jabrane, et al. “Synthesis and evaluation of antibacterial and anti-inflammatory properties of naturally occurring coumarins.” Phytochemistry Letters 13 (2015): 399-405. (Year: 2015). |
| Cai, Xueting, et al. “Synthesis and biological evaluation of scopoletin derivatives.” Bioorganic & medicinal chemistry 21.1 (2013): 84-92. (Year: 2013). |
| Darla, Mark Manidhar, et al. “Synthesis and bio-evaluation of novel 7-hydroxy coumarin derivatives via Knoevenagel reaction.” Research on Chemical Intermediates 41.2 (2015): 1115-1133. (Year: 2015). |
| Shen, Qiong, et al. “Hydroxycoumarin derivatives: Novel and potent α-glucosidase inhibitors.” Journal of medicinal chemistry 53.23 (2010): 8252-8259. (Year: 2010). |
| Kontogiorgis, Christos A., and Dimitra J. Hadjipavlou-Litina. “Synthesis and antiinflammatory activity of coumarin derivatives.” Journal of medicinal chemistry 48.20 (2005): 6400-6408. (Year: 2005). |
| Witaicenis, Aline, et al. “Antioxidant and intestinal anti-inflammatory effects of plant-derived coumarin derivatives.” Phytomedicine 21.3 (2014): 240-246. (Year: 2014). |
| McArthur, John B., and Xi Chen. “Glycosyltransferase engineering for carbohydrate synthesis.” Biochemical Society Transactions 44.1 (2016): 129-142. (Year: 2016). |
| Lin, Yuheng, et al. “Combinatorial biosynthesis of plant-specific coumarins in bacteria.” Metabolic engineering 18 (2013): 69-77. (Year: 2013). |
| Kai, Kosuke, et al. “Accumulation of coumarins in Arabidopsis thaliana.” Phytochemistry 67.4 (2006): 379-386. (Year: 2006). |
| Peterson, Joseph K., et al. “Biological activities and contents of scopolin and scopoletin in sweetpotato clones.” HortScience 38.6 (2003): 1129-1133 (Year: 2003). |
| Sun, Huanhuan, et al. “Scopoletin is a phytoalexin against Alternaria alternata in wild tobacco dependent on jasmonate signalling.” Journal of experimental botany 65.15 (2014): 4305-4315. (Year: 2014). |
| Cai, Xueting, et al. “Synthesis and biological evaluation of scopoletin derivatives.” Bioorganic & medicinal chemistry 21.1 (2013): 84-92. (Year: 2013). |
| Darla, Mark Manidhar, et al. “Synthesis and bio-evaluation of novel 7-hydroxy coumarin derivatives via Knoevenagel reaction.” Research on Chemical Intermediates 41.2 (2015): 1115-1133. (Year: 2015). |
| Khunnawutmanotham, Nisachon, et al. “Synthesis and anti-acetylcholinesterase activity of scopoletin derivatives.” Bioorganic chemistry 65 (2016): 137-145. (Year: 2016). |
| Shen, Qiong, et al. “Hydroxycoumarin derivatives: Novel and potent α-glucosidase inhibitors.” Journal of medicinal chemistry 53.23 (2010): 8252-8259. (Year: 2010). |
| Kontogiorgis, Christos A., and Dimitra J. Hadjipavlou-Litina. “Synthesis and antiinflammatory activity of coumarin derivatives.” Journal of medicinal chemistry 48.20 (2005): 6400-6408. (Year: 2005). |
| Witaicenis, Aline, et al. “Antioxidant and intestinal anti-inflammatory effects of plant-derived coumarin derivatives.” Phytomedicine 21.3 (2014): 240-246. (Year: 2014). |
| Sun, Xinxiao, et al. “Structural insights into substrate specificity of Feruloyl-CoA 6′-Hydroxylase from Arabidopsis thaliana.” Scientific reports 5 (2015): 10355. (Year: 2015). |
| McArthur, John B., and Xi Chen. “Glycosyltransferase engineering for carbohydrate synthesis.” Biochemical Society Transactions 44.1 (2016): 129-142. (Year: 2016). |
| Levee, V., and A. Seguin. “Inducible expression of the heterologous PAL2 promoter from bean in white pine (Pinus strobus) transgenic cells.” Tree physiology 21.10 (2001): 665-672. (Year: 2001). |
| Nicole Beate Schmid: “Identification and characterization of Arabidopsis genes involved in tolerance to Fe deficiency mediated chlorosis”, Dissertation zur Erlangung des Doktorgrades der Naturmwissenschaften, May 28, 2014 (May 28, 2014), XP002740757, submitted in IDS (Year: 2014). |
| Jeandet, Philippe, et al. “Modulation of phytoalexin biosynthesis in engineered plants for disease resistance.” International journal of molecular sciences 14.7 (2013): 14136-14170. (Year: 2013). |
| Sanzani, Simona M., Leonardo Schena, and Antonio Ippolito. “Effectiveness of phenolic compounds against citrus green mould.” Molecules 19.8 (2014): 12500-12508. (Year: 2014). |
| Carpinella, Maria C., Carlos G. Ferrayoli, and Sara M. Palacios. “Antifungal synergistic effect of scopoletin, a hydroxycoumarin isolated from Melia azedarach L. fruits.” Journal of agricultural and food chemistry 53.8 (2005): 2922-2927. (Year: 2005). |
| Lin, Yuheng, et al. “Combinatorial biosynthesis of plant-specific coumarins in bacteria.” Metabolic engineering 18 (2013): 69-77. (Year: 2013). |
| Kai, Kosuke, et al. “Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl CoA by a 2-oxoglutarate-dependent dioxygenase in Arabidopsis thaliana.” The Plant Journal 55.6 (2008): 989-999. (Year: 2008). |
| Ahl Goy et al., Accumulation of scopoletin is associated with the high disease resistance of the hybrid Nicotiana glutinosa x Nicotiana debneyi, Planta, 191:200-6 (1993). |
| Database Geneseq [Online], “Amino Acid Sequence SEQ ID 18550”, XP002740752, Retrieved from EBI accession No. GSP:AWJ45345, Database accession No. AWJ45345, Oct. 29, 2009. |
| Database Geneseq [Online], “Arabidopsis Thaliana Amino Acid Sequence SEQ 1D 81044”, XP002740753, retrieved from EBI accession No. GSP:ARM81672, Database accession No. ARM81672. |
| Database Geneseq [Online], “Arabidopsis Thaliana Glucosyltransferase (GTase) A961 Protein”, XP002740756, retrieved from EBI accession No. GSP:AAE07511, Database accession No. AAE07511 (Jun. 15, 2007). |
| Database Geneseq [Online], “Arabidopsis Thaliana Protein, SEQ 1D 1605.”, XP002740755, retrieved from EBI accession No. GSP:AWW66938, Database accession No. AWW66938, Nov. 12, 2009. |
| Database Geneseq [Online], “Plant Biomass-Modulating DNA Encoded Protein SEQ :78.”, XP002740754, retrieved from EBI accession No. GSP:AZP53437, Database accession No. AZP53437, Jan. 5, 2012. |
| European Search Report for EP Patent Application No. 15153820.4, Completed on Jun. 15, 2015, 5 pages. |
| Fourcroy et al., Involvement of the ABCG37 transporter in secretion of scopoletin and derivatives by Arabidopsis roots in response to iron deficiency, New Phytol., 201(1):155-67 (2014). |
| Fraissinet-Tachet et al., Two tobacco genes induced by infection, elicitor and salicylic acid encode glucosyltransferases acting on phenylpropanoids and benzoic acid derivatives, including salicylic acid, FEBS Lett., 437(3):319-23 (1998). |
| Gielen et al., The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5, EMBO J., 3(4):835-46 (1984). |
| Gnonlonfin et al., Review scopoletin—a coumarin phytoalexin with medicinal properties, Crit. Rev. Plant Sci., 31:1, 47-56 (2012). |
| Herbers et al., Functional analysis of a leucine aminopeptidase from Solanum tuberosum L, Planta, 194(2):230-40 (1994). |
| International Application No. PCT/EP2016/052019, International Search Report and Written Opinion, dated Mar. 29, 2016. |
| Kai et al., Scopoletin is biosynthesized via ortho-hydroxylation of feruloyl CoA by a 2-oxoglutarate-dependent dioxygenase in Arabidopsis thaliana, Plant J., 55(6):989-99 (2008). |
| Kawalleck et al., Polyubiquitin gene expression and structural properties of the ubi4-2 gene in Petroselinum crispum, Plant Mol. Biol., 21(4):673-84 (1993). |
| Lee et al., Novel plant transformation vectors containing the superpromoter, Plant Physiol., 145(4):1294-300 (2007). |
| Lim et al., Evolution of substrate recognition across a multigene family of glycosyltransferases in Arabidopsis, Glycobiology, 13(3):139-45 (2003). |
| Schmid, “Identification and Characterization of Arabidopsis Genes Involved in Tolerance to Fe Deficiency-Mediated Chlorosis”, Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften der Naturwissenschaftlichen Faukultät I—Biowissenschaften—der Martin-Luther-Universität Halle-Wittenberg (Apr. 8, 1983). |
| Shimizu, 2-Oxoglutarate-dependent dioxygenases in the biosynthesis of simple coumarins, Front Plant Sci., 5:549 (2014). |
| Sun et al., Scopoletin is a phytoalexin against Alternaria alternata in wild tobacco dependent on jasmonate signalling, J. Exp. Bot., 65(15):4305-15 (2014). |
| Tal et al., The metabolism of sunflower phytoalexins ayapin and scopoletin: plant-fungus interactions, Plant Physiol., 81(1):167-72 (1986). |
| Number | Date | Country | |
|---|---|---|---|
| 20180010144 A1 | Jan 2018 | US |
