DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Application Transfer
The application has been transferred to Patent Examiner Emily K. Johnson in Art Unit 1662. Any inconvenience to Applicant is regretted.
Restriction/Election
In response to the communication received on July 1st, 2026, from Agent Amy Dunstan, the election of markers M1 and M4 of claim 1; chromosomal regions on chromosome 3 and 4 of claim 2; SEQ ID NOs: 15 and 16 of claim 3; SEQ ID NOs: 13 and 15 or SEQ ID NOs: 14 and 16 of claim 4; SEQ ID NO: 3 of claim 5; SEQ ID NO: 15 of claim 6; and SEQ ID NO: 22 and 16 of claim 7 with traverse, is acknowledged (see end of pg. 3 into pg. 4 of Response to Election filed 07/01/2026).
Applicant contends that the technical feature common to all of claims 1-14 is “the specific combinations of Bv3 and Bv4 small ALS subunit alleles from best performing genotypes A through E that, together with the BvALS_W569” and that this technical feature is not disclosed or suggested by WO 2020064688. Applicant contends that the claimed ALS small subunit sequences are not unrelated species and that the requirement to elect between Bv3 and Bv4 is artificial. Lastly, Applicant contends that election between an amino acid sequence and its encoding nucleotide sequence is improper.
Applicant’s arguments have been carefully considered and were found persuasive. The requirement for an election of species is thus withdrawn.
Priority
Applicant’s claim for the benefit of a prior-filed application no. NZ779721 filed September 2nd, 2021, and application no. PCT/EP2022/073943 filed August 29th, 2022, under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Thus, the earliest possible priority for the instant application is September 2nd, 2021.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on May 14th, 2024, May 30th, 2024, August 29th, 2025, March 26th, 2026, were considered, initialed, and attached hereto. A signed copy of the list of references cited is included with this Office Action.
Status of Claims
Claims 1-14 filed April 18th, 2024, are pending and examined herein.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (see, for example ¶19). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The use of the term BLAST, which is a trade name or a mark used in commerce, has been noted in this application (see, for example ¶19). The term should be accompanied by the generic terminology; furthermore, the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM, or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claims 1-14 are objected to because of the following informalities:
Beta vulgaris as recited in claims 1-14 should be in italics.
Claim 6 should recite “comprising” instead of “comprises” in line 8.
Claims 6-7 should recite “an optimally fitted large subunit” instead of “optimally fitted subunit” in line 1.
Claim 7 should include “and” after part a. and recite “adapting” instead of “adapt” in line 8.
Claim 11 should recite “from” instead of “form” in line 5.
Claims 8-14 do not start with an article. Independent claims typically start with an indefinite article (i.e., a use of the hybrid Beta vulgaris plant according to claim 1…), while dependent claims start with the definite article “the” to refer back to an already introduced claim (i.e., “the method according to claim 12…”).
Appropriate correction is required.
Claim Interpretation
The term “Beta vulgaris growing areas” as recited in claims 9 and 12 is not defined in the instant specification and is interpreted to mean anywhere beets may grow, such as a field.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 8-11 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claims do not fall within at least one of the four categories of patent eligible subject matter. The claims recite uses of a plant or an ALS inhibitor herbicide.
Broadest Reasonable Interpretation
Claims 8-11 recite various uses of the plant of claim 1 or an herbicide. Since claims 8-11 do not recite a claim to a process, machine, manufacture or composition, claims 8-11 have not been interpreted to fall within one or the four patentable categories.
Step 1: Whether the claim is to a statutory category
Under Step 1 of the subject matter eligibility test for products and processes, it must be determined if the claim is to a process, machine, manufacture or a composition of matter. In the instant case, claims 8-11 are directed to “use of” claims. "Use" claims that do not purport to claim a process, machine, manufacture, or composition of matter fail to comply with 35 U.S.C. 101. In re Moreton, 288 F.2d 708, 709, 129 USPQ 227, 228 (CCPA 1961)("one cannot claim a new use per se, because it is not among the categories of patentable inventions specified in 35 U.S.C. § 101 "). Claims 8-11 recite no active method steps, further demonstrating that the claims are not to a process. The claims are therefore not directed to a statutory category.
Therefore, claims 8-11 are directed to subject matter that does not fall within at least one of the four categories of patent eligible subject matter and are, as a result, rejected under 35 U.S.C. 101.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-14 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 (and claims 2-5 and 8-14 depending therefrom) recites a small subunit of ALS which can be selected by identification with marker M1 and M4. The actual small subunit of ALS is not defined except in claims 3-7. ALS small subunits are naturally found in plants, as the Applicant discloses that the ALS holoenzyme consists of 4 catalytic and 4 regulatory subunits [¶9]. It is not clear that these markers would identify only the ALS sequences of the instant invention or any ALS sequence. Further, it is not clear if the Applicant simply identified and characterized Beta vulgaris ALS small subunits using methodology previously utilized and documented in Arabidopsis, according to the specification [pg. 62, ¶3-5], or if there are specific technical features that makes the ALS subunits novel. As plants naturally contain ALS small subunit sequences, it is not clear if the markers of the claims are merely intended to identify intrinsic sequences or if the ALS subunit sequences later claimed in claim 3-5 contain an additional feature that aids in the intended goal of the plant beyond the mutation of the large subunit to result in ALS inhibitor herbicide tolerance.
Regarding claim 1 (and claims 2-5 and 8-14 depending therefrom), the phrase "such as" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d). In the instant case, three subspecies of Beta vulgaris are typically recognized. Some of the most popular cultivar groups include: the sugar beet (used to produce table sugar), the root vegetable known as the beetroot or garden beet, the leaf vegetable known as chard or spinach beet or silverbeet, and mangelwurzel (a fodder crop)1.
The term “optimally fitted” in claims 6-7 is a relative term which renders the claim indefinite. The term “optimally fitted” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. A person of skill in the art may not know what physical dimensions or parameters satisfy “optimal.” It is additionally unclear how the plant can be optimally fitted with the large subunit. Is the subunit the best fit for enhancing catalytic activity of the large subunit as compared to a control? What parameters make it optimally fitted and how could one determine that the large subunit is an optimal fit?
Claims 8-11 are uses of the plant according to claim 1 for specific outcomes, such as the production of sugar. Although the claims recite a desired outcome, the claims merely recite a use without any active, positive steps delimiting how this use is actually practiced. It is not clear what the Applicant is claiming with the recitation of a use of the plant and no active steps to achieve the desired outcome, thus, these claims are indefinite.
Claims 8-11 recite use of a hybrid Beta vulgaris plant according to claim 1. It is not clear if Applicant intends to recite that the plant of claim 1 was crossed and a hybrid is used uniquely for claims 8-11 or if the original plant of claim 1 is identified as a hybrid plant. Claims 8-11 are interpreted as a use of the plant of claim 1.
Claims 9-14 are essentially directed to methods for controlling unwanted vegetation in Beta vulgaris growing areas. Controlling unwanted vegetation by applying an ALS inhibiting herbicide or a non-ALS inhibiting herbicide does not require the presence of the plant of claim 1. The presence of the tolerant plant does not add any limitation to the application of an herbicide or the removal of unwanted vegetation. It is not clear if the Applicant intends for the presence of the plants of claim 1 to provide some required structure or meaningful limitation to the claims.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Written Description
Claims 6-7 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 6-7 are broadly drawn to methods for producing a Beta vulgaris plant with an optimally fitted large subunit and one or more regulatory subunits of an ALS holoenzyme. Claim 7 further requires adapting the nucleotide sequence of an allele on chromosome 3 and/or the allele on chromosome 4 encoding a small subunit of ALS to obtain a specific nucleotide sequence.
The Applicant describes determining enzymatic activity of BvALS subunits from sugar beet [pg. 62, ¶1-2], combining the regulatory subunits with the purified catalytic subunit in vitro, and identifying combinations of catalytical and regulatory ALS subunits for robust enzyme performance [pg. 62, ¶3-4]. The Applicant describes identifying Beta vulgaris orthologues found in BLAST based on ALS regulatory subunits published in Arabidopsis thaliana [pg. 62, ¶5], and the genotypes with the best performance [pg. 63, ¶2].
The Applicant does not describe what an optimally fitted large subunit is. The Applicant does not describe any adaptation by genome-editing or directed mutation of an allele on chromosome 3 or 4 encoding a small subunit of ALS.
First, the Applicant describes that the genotypes with the best enzyme performance were genotypes A, B, C, D, and E with respective amino acid and nucleotide sequences for both BV3 and BV4 in ¶114. Paragraph 114 is a list of the amino acid sequences corresponding to specific genotypes. The Applicant has not claimed a specific combination representing a genotype. The Applicant has additionally not included the large subunit sequences in the genotype combinations stated as the best fitting combinations. Thus, the Applicant has not reduced to practice Beta vulgaris plants with an optimally fitted large subunit as there is no support in the specification. Additionally, this is not a term typically used regarding the large subunit. The prior art cannot remedy the deficiencies of the specification.
Second, the Applicant appears to have isolated and characterized Beta vulgaris ALS small subunit sequences in methodology previously known in Arabidopsis and does not provide any examples of adaptation of the nucleotide sequence of the allele on chromosome 3 or 4 encoding a small subunit of ALS to obtain one of the nucleotide sequences as listed in part b of claim 7. It is not clear what adaptation must occur if the sequences were identified within the sugar beet genome and may occur within the Beta vulgaris breeding pool without any inventive step. Additionally, the term “adapt, by genome-editing or directed mutation,” provides an extremely broad array of possible mutations. The Applicant has not provided any examples to reduce to practice a genetic modification of any sort or what improvement such a modification would contribute to the intended goal of ALS inhibiting herbicide tolerance.
Examiner notes that claims 1 and 11-14 are not currently rejected under 35 USC § 112(a) written description because, as written, the claims do not actually require that the Beta vulgaris plant is tolerant to another herbicide that does not belong to the class of ALS inhibitor herbicides. The claims merely require use of an ALS inhibitor herbicide and another non-ALS inhibitor herbicide.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-2, 5, and 7-14 are rejected under 35 U.S.C. 103 as being unpatentable over Rüdiger, H. et al. “Use of ALS Inhibitor Herbicides for Control of Unwanted Vegetation in ALS Inhibitor Herbicide Tolerant Beta vulgaris plants.” International Publication No. WO 2014090760 A1. Published 06/19/2014, in view of Dezfulian, M. et al. (2017). “Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development.” BMC Plant Biology. 17:71, Lee, Y. et al. (2001). “Identification of the Regulatory Subunit of Arabidopsis thaliana Acetohydroxyacid Synthase and Reconstitution with Its Catalytic Subunit.” Biochemistry. 40:6836-6844 (see, IDS filed 5/14/2024), and NCBI Reference Sequence: XM_010674185.2. PREDICTED: Beta vulgaris subsp. vulgaris acetolactate synthase small subunit 2, chloroplastic (LOC104889046), mRNA. Published 11/29/2016.
Claim 1 recites an acetolactate synthase (ALS) inhibitor-herbicide tolerant Beta vulgaris plant or seed, such as a sugar beet plant or seed, comprising an ALS holo-enzyme comprising a. a large subunit of ALS comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 1 and further comprising a leucine at a position corresponding to amino acid position 569 instead of the naturally occurring tryptophan; and b. a small subunit of ALS which can be selected by identification with marker M1 (comprising the nucleotide sequence of SEQ ID NO. 33).
Claim 2 recites the Beta vulgaris plant or seed of claim 1, wherein said small subunit of ALS is encoded by a chromosomal region located on chromosome 3, between a marker selected from marker M5 (comprising the nucleotide sequence of SEQ ID NO. 37), marker M6 (comprising the nucleotide sequence of SEQ ID NO. 38) or marker M7 (comprising the nucleotide sequence of SEQ ID NO. 39) and a marker selected from marker M11 (comprising the nucleotide sequence of SEQ ID NO. 43), marker M12 (comprising the nucleotide sequence of SEQ ID NO. 44) or marker M13 (comprising the nucleotide sequence of SEQ ID NO. 45) or wherein the small subunit of ALS is encoded by a chromosomal region located on chromosome 4, between a marker selected from marker M8 (comprising the nucleotide sequence of SEQ ID NO. 40), marker M9 (comprising the nucleotide sequence of SEQ ID NO. 41) or marker M10 (comprising the nucleotide sequence of SEQ ID NO. 42) and a marker selected from marker M14 (comprising the nucleotide sequence of SEQ ID NO. 46), marker M15 (comprising the nucleotide sequence of SEQ ID NO. 47) or marker M16 (comprising the nucleotide sequence of SEQ ID NO. 48).
Claim 5 recites the Beta vulgaris plant or seed of claim 1, wherein said large subunit of ALS comprises the amino acid sequence of SEQ ID NO. 3.
Claim 7 recites a method for producing a Beta vulgaris plant with optimally fitted large subunit and one or more regulatory subunits of an ALS holoenzyme comprising: a. providing a Beta vulgaris plant comprising an allele encoding a large subunit of ALS comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 1 and further comprising a leucine at a position corresponding to amino acid position 569 instead of the naturally occurring tryptophan, such as an amino acid sequence of SEQ ID NO. 3; b. adapt, by genome-editing or directed mutation, the nucleotide sequence of the allele on chromosome 3 and/or the allele on chromosome 4 encoding a small subunit of ALS to obtain a nucleotide sequence selected from the group of SEQ ID NO 22.
Claim 8 recites use of a hybrid Beta vulgaris plant according to claim l for the production of sugar.
Claim 9 recites use of one or more ALS inhibitor herbicide(s) for controlling unwanted vegetation in Beta vulgaris growing areas wherein the Beta vulgaris plants are hybrid Beta vulgaris plants according to claim 1.
Claim 10 recites use of one or more ALS inhibitor herbicide(s) according to claim 9, wherein the ALS inhibitor herbicide(s) comprises foramsulfuron.
Claim 11 recites use of one or more ALS inhibitor herbicide(s) according to claim 9 in combination with non-ALS inhibitor herbicides (i.e. herbicides showing a mode of action that is different to the inhibition of the ALS enzyme [acetohydroxyacid synthase; EC 2.2.1.6] group D herbicides), and wherein the non-ALS inhibitor herbicide(s) is/are selected form the group consisting of chloridazon.
Claim 12 recites a method for controlling unwanted vegetation in Beta vulgaris plant growing areas, characterized by: (a) the presence of Beta vulgaris plants according to claim l; (b) the application of one or more ALS inhibitor herbicide(s) alone or in combination with one or more herbicide(s) that do(es) not belong to the class of ALS inhibitor herbicides (non- ALS inhibitor herbicides), and (c) wherein the application of the respective herbicides as defined under (b) (i) takes place jointly or simultaneously, or (ii) takes place at different times and/or in a plurality of portions (sequential application), in pre-emergence applications followed by post-emergence applications or early post-emergence applications followed by medium or late post-emergence applications.
Claim 13 recites a method according to claim 12 for controlling unwanted vegetation, wherein the ALS inhibitor herbicide(s) comprise foramsulfuron.
Claim 14 recites a method according to claim 12, wherein the non-ALS inhibitor herbicide(s) are taken from the group consisting of chloridazon.
Regarding claim 1, Rüdiger teaches that a broad variety of ALS/AHAS inhibitor herbicides enable a farmer to control a wide range of weed species independent of their growth stages, but these highly efficient herbicides cannot be used in Beta vulgaris because Beta vulgaris, especially conventional sugar beet plants/commercial sugar beet varieties are highly susceptible against/affected by these ALS inhibitor herbicides [pg. 2, lns. 12-19]. Rüdiger teaches that a more reliable and more flexible way to obtain Beta vulgaris plants that stand against ALS inhibitor herbicide treatment is to generate mutants that are sufficiently tolerant to agronomically useful/necessary quantities of ALS inhibitor herbicides [pg. 2, lns. 25-28]. Modification of the ALS enzyme avoids the targeted shut down by ALS-inhibiting herbicides which starve the plant of essential amino acids, allowing for normal amino acid production and plant survival.
Rüdiger teaches the use of the ALS inhibitor herbicides for controlling unwanted vegetation in ALS inhibitor herbicide tolerant Beta vulgaris plants [Abstract]. Rüdiger teaches mutations in the ALS gene where the tryptophan at position 569 in the encoded ALS enzyme (corresponding to position 574 in the Arabidopsis thaliana ALS enzyme) is substituted by another amino acid (preferably by leucine) [pg. 16, lns. 24-31; claim 4]. Rüdiger teaches SEQ ID NO: 2, a Beta vulgaris sequence with 100% identity to SEQ ID NO: 1 of the instant application (i.e., a large subunit of ALS comprising an amino acid sequence of SEQ ID NO: 1, and further comprising a leucine at a position corresponding to amino acid position 569 instead of the naturally occurring tryptophan) (see, alignment below).
Query Match 100.0%; Score 3393; Length 664;
Best Local Similarity 100.0%;
Matches 660; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAATFTNPTFSPSSTPLTKTLKSQSSISSTLPFSTPPKTPTPLFHRPLQISSSQSHKSSA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAATFTNPTFSPSSTPLTKTLKSQSSISSTLPFSTPPKTPTPLFHRPLQISSSQSHKSSA 60
Qy 61 IKTQTQAPSSPAIEDSSFVSRFGPDEPRKGSDVLVEALEREGVTNVFAYPGGASMEIHQA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 IKTQTQAPSSPAIEDSSFVSRFGPDEPRKGSDVLVEALEREGVTNVFAYPGGASMEIHQA 120
Qy 121 LTRSKTIRNVLPRHEQGGVFAAEGYARATGKVGVCIATSGPGATNLVSGLADALLDSVPL 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LTRSKTIRNVLPRHEQGGVFAAEGYARATGKVGVCIATSGPGATNLVSGLADALLDSVPL 180
Qy 181 VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRIVKEAFFLANSGRPGP 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRIVKEAFFLANSGRPGP 240
Qy 241 VLIDLPKDIQQQLVVPDWDRPFKLGGYMSRLPKSKFSTNEVGLLEQIVRLMSESKKPVLY 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 VLIDLPKDIQQQLVVPDWDRPFKLGGYMSRLPKSKFSTNEVGLLEQIVRLMSESKKPVLY 300
Qy 301 VGGGCLNSSEELRRFVELTGIPVASTLMGLGSYPCNDELSLHMLGMHGTVYANYAVDKAD 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 VGGGCLNSSEELRRFVELTGIPVASTLMGLGSYPCNDELSLHMLGMHGTVYANYAVDKAD 360
Qy 361 LLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSICADVKLALRGMNKI 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSICADVKLALRGMNKI 420
Qy 421 LESRIGKLNLDFSKWREELGEQKKEFPLSFKTFGDAIPPQYAIQVLDELTNGNAIISTGV 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 LESRIGKLNLDFSKWREELGEQKKEFPLSFKTFGDAIPPQYAIQVLDELTNGNAIISTGV 480
Qy 481 GQHQMWAAQHYKYRNPRQWLTSGGLGAMGFGLPAAIGAAVARPDAVVVDIDGDGSFIMNV 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 GQHQMWAAQHYKYRNPRQWLTSGGLGAMGFGLPAAIGAAVARPDAVVVDIDGDGSFIMNV 540
Qy 541 QELATIRVENLPVKIMLLNNQHLGMVVQWEDRFYKANRAHTYLGNPSKSADIFPDMLKFA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 QELATIRVENLPVKIMLLNNQHLGMVVQWEDRFYKANRAHTYLGNPSKSADIFPDMLKFA 600
Qy 601 EACDIPSARVSNVADLRAAIQTMLDTPGPYLLDVIVPHQEHVLPMIPSGAGFKDTITEGD 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 EACDIPSARVSNVADLRAAIQTMLDTPGPYLLDVIVPHQEHVLPMIPSGAGFKDTITEGD 66
Rüdiger does not explicitly teach a small subunit of ALS which can be selected by identification with marker M1 or M4, however, small subunits of ALS are known in the art in a variety of plants. For example, Dezfulian teaches the regulatory processes governing ALS activity in Arabidopsis thaliana. Dezfulian teaches a set of interacting proteins, which are evolutionarily conserved orthologs of bacterial feedback-regulatory proteins and are implicated in ALS activity [Abstract]. Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. ALS is the first enzyme in the BCAA synthesis pathway. This has stimulated interest in the genetic manipulation of plant BCAA metabolism both for the generation of herbicide-tolerant crops [pg. 2, col. 1, ¶1].
Plant genomes commonly contain multiple genes known or predicted to encode ALS regulatory subunits, including two putative regulatory ALS subunit genes [pg. 2, col. 2, ¶1]. Dezfulian teaches identification of two ALS regulatory subunits AIP1 (At2g31810) and AIP3/VAT1 (At5g16290) with high deduced protein similarity to prokaryotic and eukaryotic regulatory ALS subunits [pg. 3, col. 1, ¶1]. Dezfulian teaches that both AIP1 and AIP3 regulatory subunits have acquired functional attributes independent of the ALS holoenzyme [pg. 10, col. 1, ¶2]. Given that both ALS regulatory subunits can bind BCAAs even in the absence of the ALS catalytic subunit, it is possible that AIP1 and AIP3 can contribute to binding and degradation of BCAAs in peroxisomes via a yet-to-be determined mechanism [pg. 10, col. 2, ¶1]. Further, catalytic activity of the holoenzyme in complex with AIP3 and BCAAs as cofactors exhibits a greater sensitivity to Val and Ile [pg. 10, col. 1, ¶1]. Thus, Dezfulian teaches that ALS regulatory subunits are found naturally in plants and perform their intended function of working with the catalytic subunit to enhance activity.
Rüdiger and Dezfulian do not explicitly teach the use of the main catalytic large subunit with the smaller regulatory subunit, however, Lee teaches that the combination of an ALS regulatory subunit of Arabidopsis thaliana with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine leucine, and isoleucine [Abstract]. Each regulatory subunit was individually able to activate the catalytic subunit and confer sensitivity to BCAA inhibition, though they were most effective as an entire regulatory subunit [pg. 6842, col. 1, ¶3]. This demonstrates that the regulatory subunits aid in enhancing catalytic activity.
Rüdiger, Dezfulian, and Lee do not explicitly teach identification of a small subunit of ALS with a marker comprising the nucleotide sequence of SEQ ID NO: 33, however, an NCBI BLAST search revealed NCBI Reference Sequence: XM_010674185.2, PREDICTED: Beta vulgaris subsp. vulgaris acetolactate synthase small subunit 2, chloroplastic (LOC104889046), mRNA (see, alignment below). As this sequence is annotated as a small subunit of ALS, one of ordinary skill in the art could have attempted to locate the known gene using a predicted sequence as the marker may be within the gene itself. This appears to be found naturally within the plant genome.
Alignment statistics for match #1
Score
Expect
Identities
Gaps
Strand
93.5 bits(50)
1e-15
50/50(100%)
0/50(0%)
Plus/Plus
Query 1 CATTTACCGTTTGCTGAGCGAGAGTTGATGTTGGTAAAGGTAGCTGTGAA 50
||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1390 CATTTACCGTTTGCTGAGCGAGAGTTGATGTTGGTAAAGGTAGCTGTGAA 1439
Given that Rüdiger teaches a catalytic subunit of ALS comprising the amino acid sequence of instant SEQ ID NO: 1 with demonstrated activity in an ALS inhibitor-tolerant Beta vulgaris; Dezfulian teaches identification of plant small regulatory subunits and suggests their function relative to herbicide tolerant plants; Lee teaches the combination of an ALS regulatory subunit and catalytic subunit for enhanced catalytic efficiency; and marker M1 was known in the art at the time of filing, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use the known mutation that confers herbicide tolerance to Beta vulgaris in combination with small regulatory subunits found in all plants that can enhance catalytic efficiency, thus overcoming the amino acid starvation effects of ALS inhibiting herbicides.
It would have been obvious to one of ordinary skill to isolate and characterize the ALS regulatory subunits as taught by Dezfulian in Arabidopsis. One would have reasonable expectation of success given that Dezfulian teaches that plant genomes commonly contain multiple genes known or predicted to encode ALS regulatory subunits and that they are evolutionarily conserved orthologs of the bacterial subunits implicated in ALS regulatory activity. One would be motivated to combine the mutated catalytic subunit and the inherent regulatory subunits of ALS in view of Lee’s teachings, specifically that the combination of the two regulatory subunits and the catalytic subunit enhanced catalytic efficiency. The mutation, as proven in the prior art, prevents the binding of the herbicide to the ALS enzyme, allowing for the normal production of essential amino acids. The regulatory subunits would aid in regular plant functioning with application of herbicides that would usually target the catalytic domain.
Further, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use a previously known sequence annotated as the gene of interest to identify the small subunit of ALS in Beta vulgaris. Alternatively, one would have come across such a marker or alternative markers that would effectively identify the small subunits of ALS given that they are found naturally in plants. One of ordinary skill would be capable of routinely validating the predicted sequence and would have been motivated to try the predicted sequence out of a finite number of Beta vulgaris sequences annotated as small subunits of ALS.
Regarding claim 2, although the sequences of markers M5, M6, M7, and M11 are not disclosed in the art, NCBI Reference Sequence: XM_010674185.2 is annotated as a small subunit of ALS, has 100% identity to marker M1, and is annotated as being on chromosome 3. Given the structure of the NCBI sequence and the claimed structure, NCBI Reference Sequence: XM_010674185.2 would additionally have the same features of marker M1, wherein said small subunit of ALS is encoded by a chromosomal region located on chromosome 3, between marker M5 and marker M11, absent evidence to the contrary.
Regarding claim 5, Rüdiger teaches Beta vulgaris acetolactate synthase variant (W569L), SEQ ID 4, sharing 100% identity with SEQ ID NO: 3 of the instant application (i.e., wherein said large subunit of ALS comprises the amino acid sequence of SEQ ID NO: 3) (see, alignment below).
Query Match 100.0%; Score 3386; Length 664;
Best Local Similarity 100.0%;
Matches 660; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAATFTNPTFSPSSTPLTKTLKSQSSISSTLPFSTPPKTPTPLFHRPLQISSSQSHKSSA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAATFTNPTFSPSSTPLTKTLKSQSSISSTLPFSTPPKTPTPLFHRPLQISSSQSHKSSA 60
Qy 61 IKTQTQAPSSPAIEDSSFVSRFGPDEPRKGSDVLVEALEREGVTNVFAYPGGASMEIHQA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 IKTQTQAPSSPAIEDSSFVSRFGPDEPRKGSDVLVEALEREGVTNVFAYPGGASMEIHQA 120
Qy 121 LTRSKTIRNVLPRHEQGGVFAAEGYARATGKVGVCIATSGPGATNLVSGLADALLDSVPL 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LTRSKTIRNVLPRHEQGGVFAAEGYARATGKVGVCIATSGPGATNLVSGLADALLDSVPL 180
Qy 181 VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRIVKEAFFLANSGRPGP 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRIVKEAFFLANSGRPGP 240
Qy 241 VLIDLPKDIQQQLVVPDWDRPFKLGGYMSRLPKSKFSTNEVGLLEQIVRLMSESKKPVLY 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 VLIDLPKDIQQQLVVPDWDRPFKLGGYMSRLPKSKFSTNEVGLLEQIVRLMSESKKPVLY 300
Qy 301 VGGGCLNSSEELRRFVELTGIPVASTLMGLGSYPCNDELSLHMLGMHGTVYANYAVDKAD 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 VGGGCLNSSEELRRFVELTGIPVASTLMGLGSYPCNDELSLHMLGMHGTVYANYAVDKAD 360
Qy 361 LLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSICADVKLALRGMNKI 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSICADVKLALRGMNKI 420
Qy 421 LESRIGKLNLDFSKWREELGEQKKEFPLSFKTFGDAIPPQYAIQVLDELTNGNAIISTGV 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 LESRIGKLNLDFSKWREELGEQKKEFPLSFKTFGDAIPPQYAIQVLDELTNGNAIISTGV 480
Qy 481 GQHQMWAAQHYKYRNPRQWLTSGGLGAMGFGLPAAIGAAVARPDAVVVDIDGDGSFIMNV 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 GQHQMWAAQHYKYRNPRQWLTSGGLGAMGFGLPAAIGAAVARPDAVVVDIDGDGSFIMNV 540
Qy 541 QELATIRVENLPVKIMLLNNQHLGMVVQLEDRFYKANRAHTYLGNPSKSADIFPDMLKFA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 QELATIRVENLPVKIMLLNNQHLGMVVQLEDRFYKANRAHTYLGNPSKSADIFPDMLKFA 600
Qy 601 EACDIPSARVSNVADLRAAIQTMLDTPGPYLLDVIVPHQEHVLPMIPSGAGFKDTITEGD 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 EACDIPSARVSNVADLRAAIQTMLDTPGPYLLDVIVPHQEHVLPMIPSGAGFKDTITEGD 660
Regarding claim 7, Rüdiger teaches SEQ ID NO: 2, a Beta vulgaris sequence with 100% identity to SEQ ID NO: 1 of the instant application (i.e., providing a Beta vulgaris plant comprising an allele encoding a large subunit of ALS comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1, and further comprising a leucine at a position corresponding to amino acid position 569 instead of the naturally occurring tryptophan) (see, alignment above). Rüdiger teaches Beta vulgaris acetolactate synthase variant (W569L), SEQ ID 4, sharing 100% identity with SEQ ID NO: 3 of the instant application (i.e., such as the amino acid sequence of SEQ ID NO: 3) (see, alignment above). Given the identical sequences, this is interpreted to read on an “optimally fitted large subunit”, as recited in claim 7, due to the structural similarity.
Dezfulian teaches screening to identify novel and existing protein interactors of ALS catalytic subunits of an ALS holoenzyme in Arabidopsis. Dezfulian teaches the identification of two ALS small regulatory subunits, AIP1 and AIP3 [Table 1], and teaches that both interact with the catalytic subunit [pg. 3, col. 1, ¶1]. Lee teaches in vitro reconstitution of A. thaliana ALS regulatory subunits to enhance activity of the catalytic subunit [pg. 6837, col. 1, ¶1].
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to perform the same screening for novel ALS subunits in Beta vulgaris, given the necessity for ALS herbicide tolerant plants, as taught by Rüdiger [pg. 1, lns. 15-21]. One would have reasonable expectation of success in identifying and characterizing the ALS subunits and using them in combination with the catalytic subunit to improve herbicide tolerance, given the enhanced activity of the catalytic subunit with the ALS small subunits, as taught by Lee. One would have narrowed down the identified proteins to the best fitting combinations of subunits based on the catalytic activity. It would be obvious to adapt the nucleotide sequence of the allele on chromosome 3 and/or the allele on chromosome 4 to reflect the optimal small subunit nucleotide sequence to enhance catalytic activity (i.e., adapt, by genome-editing or directed mutation, the nucleotide sequence of the allele on chromosome 3 and/or the allele on chromosome 4 encoding a small subunit of ALS). Additionally, sequences with high similarity to the claimed nucleotide sequences were predicted in the art at the time of filing and annotated as acetolactate synthase small subunits. For example, NCBI Reference Sequence: XM_010674185.2 has 99.38% identity to instant SEQ ID NO: 22 (see, alignment below) and is located on chromosome 3.
Alignment statistics for match #1
Score
Expect
Identities
Gaps
Strand
2649 bits(1434)
0.0
1452/1461(99%)
0/1461(0%)
Plus/Plus
Query 1 ATGGCGGCTGTGTCAACTCACCCATCAACGAGTTTAAACAGCTGTCTGAAAAATGAGCAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 286 ATGGCGGCTGTGTCAACTCACCCATCAACGAGTTTAAACAGCTGTCTGAAAAATGAGCAA 345
Query 61 ACCCAAAAATTTATCAAGTCCCATGCTTTCTTATTAAAACCTCAAATGTTGGGTTTTAAT 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 346 ACCCAAAAATTTATCAAGTCCCATGCTTTCTTATTAAAACCTCAAATGTTGGGTTTTAAT 405
Query 121 CCTAGAAAATGGAAGAATTTGGAGTTTGATAAACTGGTTGTATCTGCTAGCAATGTTGAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 406 CCTAGAAAATGGAAGAATTTGGAGTTTGATAAACTGGTTGTATCTGCTAGCAATGTTGAT 465
Query 181 CATCAGGGAAATGAGAGTAATTTACCCTTTAATGGCGTTTCTTCCTCTACTCGATCAAAG 240
|| ||||||||||||||||||||||||||||||||||| |||||||||||||||||||||
Sbjct 466 CAGCAGGGAAATGAGAGTAATTTACCCTTTAATGGCGTCTCTTCCTCTACTCGATCAAAG 525
Query 241 GCGATGCGGCATACTATATCAGTATTTGTTGGGGATGAAAGTGGAATGATAAATCGAATT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 526 GCGATGCGGCATACTATATCAGTATTTGTTGGGGATGAAAGTGGAATGATAAATCGAATT 585
Query 301 GCAGGGGTCTTTGCTAGAAGAGGTTATAATATTGAATCCCTTGCTGTTGGGTTGAACGAG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 586 GCAGGGGTCTTTGCTAGAAGAGGTTATAATATTGAATCCCTTGCTGTTGGGTTGAACGAG 645
Query 361 GACAAAGCTCTCTTCACCATTGTTGTCTCTGGAACTGATAAGGTGTTGCACCAAGTGATG 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 646 GACAAAGCTCTCTTCACCATTGTTGTCTCTGGAACTGATAAGGTGTTGCACCAAGTGATG 705
Query 421 GAACAACTTCAGAAGCTTTTGAATGTTTTAAAGGTTGAAGATATATCCAGGGAGCCTCAG 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 706 GAACAACTTCAGAAGCTTTTGAATGTTTTAAAGGTTGAAGATATATCCAGGGAGCCTCAG 765
Query 481 GTAGAACGTGAATTAATGCTTGTAAAAGTTGGAGCTGATCAAAGTAGCCGCGGTGAGTTA 540
||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||
Sbjct 766 GTAGAACGTGAATTAATGCTTGTAAAAGTTGGAGCTGATCAAAGTAGCCGCGGCGAGTTA 825
Query 541 ATGTGGTTGGTGGACATCTTCCGCGCCAAAATTGTGGACATATCTGAAGAGTATCTTACA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 826 ATGTGGTTGGTGGACATCTTCCGCGCCAAAATTGTGGACATATCTGAAGAGTATCTTACA 885
Query 601 GTAGAGGTCACTGGAGATCCAGGAAAGATGGTTGCTGTGCTAAGAAATCTAAGCAAGTTT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 886 GTAGAGGTCACTGGAGATCCAGGAAAGATGGTTGCTGTGCTAAGAAATCTAAGCAAGTTT 945
Query 661 GGTATCAGAGAGATTGCTCGAACTGGAAAGATTGCTCTAAGAAGAGAAAAGTTGGGTGAG 720
|||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||
Sbjct 946 GGTATCAGAGAGATTGCTCGAACTGGAAAGATTGCTCTAAGAAGGGAAAAGTTGGGTGAG 1005
Query 721 TCTGCTCCTTTCTGGCGTTTTTCAGCAGCTTCTTATCCTGACCTTAAAGAAGCTACGCTG 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1006 TCTGCTCCTTTCTGGCGTTTTTCAGCAGCTTCTTATCCTGACCTTAAAGAAGCTACGCTG 1065
Query 781 GTTGATGCCCTTTCTGAAGTTGCAAGACAAGCACCTGATACTAAATCATCAGATTTCTCC 840
|||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||
Sbjct 1066 GTTGATGCCCTTTCTGAAGTTGCAAGACAAGCACCTGATACTGGATCATCAGATTTCTCC 1125
Query 841 GTCGAGGGTGATGTTTATCCTGTGGAACCTTATGATGGTTTCGCAGTTCCTCAAGTCCTT 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1126 GTCGAGGGTGATGTTTATCCTGTGGAACCTTATGATGGTTTCGCAGTTCCTCAAGTCCTT 1185
Query 901 GATGCTCACTGGGGTGTCTTGAATGATGATACAAGTGGGTTCCAGTCACACACTCTTTCG 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1186 GATGCTCACTGGGGTGTCTTGAATGATGATACAAGTGGGTTCCAGTCACACACTCTTTCG 1245
Query 961 ATGCTTGTCAATGACAGACCCGGGGTCCTTAACTTTGTTACAGGGGCTTTTGCTCGAAGG 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1246 ATGCTTGTCAATGACAGACCCGGGGTCCTTAACTTTGTTACAGGGGCTTTTGCTCGAAGG 1305
Query 1021 GGTTATAATATTCAGAGTTTAGCTGTGGGTCATGCGGAAACTAAGGGTCTATCTCGCATC 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1306 GGTTATAATATTCAGAGTTTAGCTGTGGGTCATGCGGAAACTAAGGGTCTATCTCGCATC 1365
Query 1081 ACTACTGTTGTAACTGGTACAGATGAATCAATTAGCAAATTGGTGCAGCAAATTTATAAG 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1366 ACTACTGTTGTAACTGGTACAGATGAATCAATTAGCAAATTGGTGCAGCAAATTTATAAG 1425
Query 1141 CTGGTTGATATTCATGAGGTCAAGGATCTTACCCATTTACCGTTTGCTGAGCGAGAGTTG 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1426 CTGGTTGATATTCATGAGGTCAAGGATCTTACCCATTTACCGTTTGCTGAGCGAGAGTTG 1485
Query 1201 ATGTTGGTAAAGGTAGCTGTGAATACTGCTGCACGTCGTGAGGTCCTTGACATTGCCAGT 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1486 ATGTTGGTAAAGGTAGCTGTGAATACTGCTGCACGTCGTGAGGTCCTTGACATTGCCAGT 1545
Query 1261 ATTTTTCGAGCTAAAGCTGTTGATGTATCTGATCATACCATTACACTTGAGCTCACTGGA 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1546 ATTTTTCGAGCTAAAGCTGTTGATGTATCTGATCATACCATTACACTTGAGCTCACTGGA 1605
Query 1321 GATGTGAATAAGATGGTTGCACTCCAAAGATTGTTGGAACCCTATGGAATCTGCGAGGTT 1380
||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||
Sbjct 1606 GATGTGAATAAGATGGTTGCACTCCAAAGATTGTTGGAACCCTATGGAATCTGTGAGGTT 1665
Query 1381 GCACGAACAGGACGTGTGGCTTTGGCTAGAGAGTCAGGTGTGGACTCAAGATATCTTCGT 1440
|||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||
Sbjct 1666 GCACGAACAGGACGTGTGGCTTTGGCTAGAGAGTCAGGTGTGGATTCAAGATATCTTCGT 1725
Query 1441 GGATATTCCTTTCCTGTCTAA 1461
|||||||||||||| ||||||
Sbjct 1726 GGATATTCCTTTCCCGTCTAA 1746
Regarding claim 8, Rüdiger teaches that about 20 % of the world sugar production is based on sugar beet. Rüdiger teaches that reliable weed control measures, such as the modified Beta vulgaris, are imperative in these crop areas [pg. 1, lns. 15-21]. Given that a large proportion of the world’s sugar production is derived from sugar beets, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use the Beta vulgaris plant with ALS inhibitor herbicide resistance in the production of sugar because it would be able to resist ALS herbicides, as well as have reduced undesirable weed populations (i.e., use of a hybrid Beta vulgaris plant according to claim 1 for the production of sugar). Although Rüdiger does not specify that the plant is a hybrid plant as recited in the claim (see, rejection under 35 USC § 112(b)), Rüdiger teaches that the invention includes the Beta vulgaris mutants and their progeny, which would include hybrids.
Regarding claim 9, Rüdiger teaches the use of ALS inhibitor herbicides for controlling unwanted vegetation around Beta vulgaris plants with Beta vulgaris plants that are tolerant to such ALS inhibitor herbicides (i.e., use of one or more ALS inhibitor herbicides for controlling unwanted vegetation in Beta vulgaris growing areas, wherein the Beta vulgaris plants are hybrid Beta vulgaris plants according to claim 1) [Abstract]. It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use the combined features as detailed above to create a tolerant Beta vulgaris plant and apply ALS inhibitor herbicide around the plants. Rüdiger teaches that the invention includes the Beta vulgaris mutants and their progeny, which would include hybrids.
Regarding claim 10, Rüdiger teaches that the ALS inhibitor herbicides which are preferably used for control of unwanted vegetation in Beta vulgaris growing areas include foramsulfuron, and teaches that the Beta vulgaris plant comprising the amino acid difference at position 569 (tryptophan to leucine) is tolerant to such an ALS inhibitor herbicide (i.e., use of one or more ALS inhibitor herbicides according to claim 9, wherein the ALS inhibitor herbicide comprises foramsulfuron) [pg. 36, lns. 24-26].
Regarding claim 11, Rüdiger teaches the use of one or more ALS inhibitor herbicide(s) alone or in combination with one or more non-ALS inhibitor herbicide(s) for weed control in Beta vulgaris, including the use of chloridazon (i.e., use of one or more ALS inhibitor herbicides according to claim 9 in combination with non-ALS inhibitor herbicides, and wherein the non-ALS inhibitor herbicides are selected from the group consisting of chloridazon) [claim 6].
Regarding claim 12, Rüdiger teaches a method for controlling unwanted vegetation in Beta vulgaris plants growing area characterized by the presence of the plant comprising the tryptophan mutation at position 569, application of one or more ALS inhibitor herbicides alone or in combination with one or more herbicides that does not belong to the class of ALS inhibitor herbicides, and wherein the application of the respective herbicides takes place at the same time of at different times (i.e., a method for controlling unwanted vegetation in Beta vulgaris plant growing areas, characterized by: (a) the presence of Beta vulgaris plants according to claim 1; (b) the application of one or more ALS inhibitor herbicide(s) alone or in combination with one or more herbicide(s) that do(es) not belong to the class of ALS inhibitor herbicides (non- ALS inhibitor herbicides), and (c) wherein the application of the respective herbicides as defined under (b) (i) takes place jointly or simultaneously, or (ii) takes place at different times and/or in a plurality of portions (sequential application), in pre-emergence applications followed by post-emergence applications or early post-emergence applications followed by medium or late post-emergence applications) [claim 9]. It would have been prima facie obvious to one of ordinary skill in the art at the time of filing for the reasons detailed above in the rejection of the plant of claim 1 to create the plant of the instant application and use in the methodology of Rüdiger.
Regarding claim 13, Rüdiger further teaches the method for controlling unwanted vegetation in Beta vulgaris as detailed in the rejection of instant claim 12 and teaches that the ALS inhibitor herbicide comprises foramsulfuron (i.e., wherein the ALS inhibitor herbicide comprises foramsulfuron) [claim 4].
Regarding claim 14, Rüdiger teaches that the non-ALS inhibitor is selected from the group consisting of chloridazon (i.e., wherein the non-ALS inhibitor herbicide(s) are taken from the group consisting of chloridazon) [claim 6].
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Rüdiger, Dezfulian, XM_010674185.2, and Lee, as applied to claim 1 above, and further in view of NCBI Reference Sequence: XP_010673855.1. PREDICTED: acetolactate synthase small subunit 1, chloroplastic [Beta vulgaris subsp. vulgaris]. Published 11/29/2016.
Claim 3 recites the Beta vulgaris plant or seed of claim 1 or 2, wherein said small subunit of ALS comprises an amino acid sequence having at least 95%, or at least 98% sequence identity with an amino acid sequence selected from the group of SEQ ID NO. 15.
Regarding claim 3, Rüdiger teaches a catalytic subunit of ALS comprising the amino acid sequence of instant SEQ ID NO: 1 with demonstrated activity in an ALS inhibitor-tolerant Beta vulgaris; Dezfulian teaches identification of plant small regulatory subunits and suggests their function relative to herbicide tolerant plants; Lee teaches the combination of an ALS regulatory subunit and catalytic subunit for enhanced catalytic efficiency; and marker M1 was known in the art at the time of filing. Thus, as detailed above, Rüdiger, Dezfulian, XM_010674185.2, and Lee combined render obvious the plant of claim 1.
Rüdiger, Dezfulian, XM_010674185.2, and Lee do not explicitly teach wherein said small subunit of ALS comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 15 of the instant application, however, a BLAST search revealed NCBI Reference Sequence: XP_010673855.1 annotated as PREDICTED: acetolactate synthase small subunit 1, chloroplastic [Beta vulgaris subsp. vulgaris]. NCBI Reference Sequence: XP_010673855.1 shares 99% identity to instant SEQ ID NO: 15 (i.e., the Beta vulgaris plant or seed of claim 1, wherein said small subunit of ALS comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 15).
Given that mutations and modifications for ALS herbicide tolerant Beta vulgaris plants were well known at the time of filing, as was the influence of ALS subunits in other plants and the relation to herbicide tolerant plants, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to validate a predicted small subunit in Beta vulgaris or to isolate and characterize the small subunits as taught by Dezfulian. As the predicted sequence known in the art at the time of filing, one would have reasonable expectation of success and sufficient motivation to generate or identify the sequence to enhance the ALS herbicide tolerance of Beta vulgaris plants with a known mutation of the tryptophan at amino acid position 569, as taught by Rüdiger. The sequence would function as claimed given the high sequence similarity SEQ ID NO: 15 of the instant claim 3 and the annotation of the NCBI sequence.
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
972 bits(2514)
0.0
Compositional matrix adjust.
476/477(99%)
477/477(100%)
0/477(0%)
Query 1 MAAVSFNNNGGKIGTLCSKPEFVCGFLRKLDFGAQTTVMAKPMSKISKLKAMEVSSNTSV 60
MAAVSFNNNGGKIGTLCSKPEFVCGFLRKLDFGAQTTVMAKPMSKISKLKAMEVSSNTSV
Sbjct 1 MAAVSFNNNGGKIGTLCSKPEFVCGFLRKLDFGAQTTVMAKPMSKISKLKAMEVSSNTSV 60
Query 61 NAVPVSAHPKVMRHTISVFVGDESGIINRIAGVISRRGYNIESLAVGLNKDKALFTIVVC 120
NAVPVSAHPKVMRHTISVFVGDESGIINRIAGVISRRGYNIESLAVGLNKDKALFTIVVC
Sbjct 61 NAVPVSAHPKVMRHTISVFVGDESGIINRIAGVISRRGYNIESLAVGLNKDKALFTIVVC 120
Query 121 GTDKVLRQVMEQLNKLVSVLKVEDLSREPQVERELMLVKLNADANTHTEIKWLVEIFRAK 180
GTDKVLRQVMEQLNKLVSVLKVEDLSREPQVERELMLVKLNADANTHTEIKWLVEIFRAK
Sbjct 121 GTDKVLRQVMEQLNKLVSVLKVEDLSREPQVERELMLVKLNADANTHTEIKWLVEIFRAK 180
Query 181 IVDVSDSLVTVEVTGDPGKMAAVLRNFSKFGIKEVARTGKIALRRERMGETAPFWRFSAA 240
IVDVSDSLVTVEVTGDPGKMAAVLRNFSKFGIKEVARTGKIALRRERMGETAPFWRFSAA
Sbjct 181 IVDVSDSLVTVEVTGDPGKMAAVLRNFSKFGIKEVARTGKIALRRERMGETAPFWRFSAA 240
Query 241 SYPDLEEKAVNTLVESTKRSINGDPGSSSTGDVYPVEPYDNPMVNQVLDAHWGVLHDGDS 300
SYPDLEEKAVNTLVESTKRSINGDPGSSSTGDVYPVEPYDNPMVNQVLDAHWGVLHDGDS
Sbjct 241 SYPDLEEKAVNTLVESTKRSINGDPGSSSTGDVYPVEPYDNPMVNQVLDAHWGVLHDGDS 300
Query 301 SGLRSHTLSMLVNNVSGVLNTVTGVISRRGYNIQSLAVGPAEKEGLSRITTVVPGNDESI 360
SGLRSHTLSMLVNNVSGVLNTVTGVISRRGYNIQSLAVGPAEKEGLSRITTVVPGNDESI
Sbjct 301 SGLRSHTLSMLVNNVSGVLNTVTGVISRRGYNIQSLAVGPAEKEGLSRITTVVPGNDESI 360
Query 361 GKLVQQLSKLVDLHEIQDLTHLPFAERELMLIKVAANTSARRDVLDIANIFRAKAVDVSD 420
GKLVQQLSKLVDLHEIQDLTHLPFAERELMLIKVAANTSARRDVLDIANIFRAKAVDVSD
Sbjct 361 GKLVQQLSKLVDLHEIQDLTHLPFAERELMLIKVAANTSARRDVLDIANIFRAKAVDVSD 420
Query 421 HTITLQLSGDLDKMVALQRLLEPYGICEVARTGRVALSRESRVDSALLRGYALPLSE 477
HTITLQLSGDLDKMVALQRLLEPYGICEVARTGRVAL+RESRVDSALLRGYALPLSE
Sbjct 421 HTITLQLSGDLDKMVALQRLLEPYGICEVARTGRVALTRESRVDSALLRGYALPLSE 477
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Rüdiger, Dezfulian, XM_010674185.2, and Lee, as applied to claim 1 above, and further in view of GenBank: KMT15552.1. hypothetical protein BVRB_3g059040 [Beta vulgaris subsp. vulgaris]. Published 07/07/2015, NCBI Reference Sequence: XP_010673855.1. PREDICTED: acetolactate synthase small subunit 1, chloroplastic [Beta vulgaris subsp. vulgaris]. Published 11/29/2016.
Claim 4 recites the Beta vulgaris plant or seed of claim 1, comprising: a. an allele of a small subunit of ALS on chromosome 3 encoding an amino acid sequence having 98% sequence identity to the amino acid sequence of SEQ ID NO. 13 and further comprising an allele of a small subunit of ALS on chromosome 4 encoding an amino acid sequence having 98% sequence identity to the amino acid sequence of SEQ ID NO. 15.
Regarding claim 4, Rüdiger teaches a catalytic subunit of ALS comprising the amino acid sequence of instant SEQ ID NO: 1 with demonstrated activity in an ALS inhibitor-tolerant Beta vulgaris; Dezfulian teaches identification of plant small regulatory subunits and suggests their function relative to herbicide tolerant plants; Lee teaches the combination of an ALS regulatory subunit and catalytic subunit for enhanced catalytic efficiency; and marker M1 was known in the art at the time of filing. Thus, as detailed above, Rüdiger, Dezfulian, XM_010674185.2, and Lee combined render obvious the plant of claim 1.
Rüdiger, Dezfulian, XM_010674185.2, and Lee do not explicitly teach the plant of claim 1 wherein an allele of a small subunit of ALS on chromosome 3 encoding an amino acid sequence having 98% identity to SEQ ID NO: 13, however a BLAST search revealed several known sequences with high alignment, including GenBank: KMT15552.1, hypothetical protein BVRB_3g059040 [Beta vulgaris subsp. vulgaris], annotated as acetolactate synthase 3 regulatory subunit or acetohydroxyacid synthase small subunit sharing 100% alignment with SEQ ID NO: 13 (see, alignment below). Further, GenBank: KMT15552.1 is located on chromosome 3. Although this was a hypothetical sequence, it was one of a finite number of sequences annotated as Beta vulgaris an acetolactate synthase 3 regulatory subunit or acetohydroxyacid synthase small subunit with a predictable structure and location. It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use a sequence characterized in the prior art as an acetolactate synthase 3 regulatory subunit or acetohydroxyacid synthase small subunit to use in the plant of claim 1 given that Dezfulian teaches the significance of small subunits in a plant tolerant to ALS inhibiting herbicide. One would have reasonable expectation of success in trying given that it was annotated as an ALS small subunit.
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
997 bits(2578)
0.0
Compositional matrix adjust.
486/486(100%)
486/486(100%)
0/486(0%)
Query 1 MAAVSTHPSTSLNSCLKNEQTQKFIKSHAFLLKPQMLGFNPRKWKNLEFDKLVVSASNVD 60
MAAVSTHPSTSLNSCLKNEQTQKFIKSHAFLLKPQMLGFNPRKWKNLEFDKLVVSASNVD
Sbjct 1 MAAVSTHPSTSLNSCLKNEQTQKFIKSHAFLLKPQMLGFNPRKWKNLEFDKLVVSASNVD 60
Query 61 QQGNESNLPFNGVSSSTRSKAMRHTISVFVGDESGMINRIAGVFARRGYNIESLAVGLNE 120
QQGNESNLPFNGVSSSTRSKAMRHTISVFVGDESGMINRIAGVFARRGYNIESLAVGLNE
Sbjct 61 QQGNESNLPFNGVSSSTRSKAMRHTISVFVGDESGMINRIAGVFARRGYNIESLAVGLNE 120
Query 121 DKALFTIVVSGTDKVLHQVMEQLQKLLNVLKVEDISREPQVERELMLVKVGADQSSRGEL 180
DKALFTIVVSGTDKVLHQVMEQLQKLLNVLKVEDISREPQVERELMLVKVGADQSSRGEL
Sbjct 121 DKALFTIVVSGTDKVLHQVMEQLQKLLNVLKVEDISREPQVERELMLVKVGADQSSRGEL 180
Query 181 MWLVDIFRAKIVDISEEYLTVEVTGDPGKMVAVLRNLSKFGIREIARTGKIALRREKLGE 240
MWLVDIFRAKIVDISEEYLTVEVTGDPGKMVAVLRNLSKFGIREIARTGKIALRREKLGE
Sbjct 181 MWLVDIFRAKIVDISEEYLTVEVTGDPGKMVAVLRNLSKFGIREIARTGKIALRREKLGE 240
Query 241 SAPFWRFSAASYPDLKEATLVDALSEVARQAPDTGSSDFSVEGDVYPVEPYDGFAVPQVL 300
SAPFWRFSAASYPDLKEATLVDALSEVARQAPDTGSSDFSVEGDVYPVEPYDGFAVPQVL
Sbjct 241 SAPFWRFSAASYPDLKEATLVDALSEVARQAPDTGSSDFSVEGDVYPVEPYDGFAVPQVL 300
Query 301 DAHWGVLNDDTSGFQSHTLSMLVNDRPGVLNFVTGAFARRGYNIQSLAVGHAETKGLSRI 360
DAHWGVLNDDTSGFQSHTLSMLVNDRPGVLNFVTGAFARRGYNIQSLAVGHAETKGLSRI
Sbjct 301 DAHWGVLNDDTSGFQSHTLSMLVNDRPGVLNFVTGAFARRGYNIQSLAVGHAETKGLSRI 360
Query 361 TTVVTGTDESISKLVQQIYKLVDIHEVKDLTHLPFAERELMLVKVAVNTAARREVLDIAS 420
TTVVTGTDESISKLVQQIYKLVDIHEVKDLTHLPFAERELMLVKVAVNTAARREVLDIAS
Sbjct 361 TTVVTGTDESISKLVQQIYKLVDIHEVKDLTHLPFAERELMLVKVAVNTAARREVLDIAS 420
Query 421 IFRAKAVDVSDHTITLELTGDVNKMVALQRLLEPYGICEVARTGRVALARESGVDSRYLR 480
IFRAKAVDVSDHTITLELTGDVNKMVALQRLLEPYGICEVARTGRVALARESGVDSRYLR
Sbjct 421 IFRAKAVDVSDHTITLELTGDVNKMVALQRLLEPYGICEVARTGRVALARESGVDSRYLR 480
Query 481 GYSFPV 486
GYSFPV
Sbjct 481 GYSFPV 486
Rüdiger, Dezfulian, XM_010674185.2, and Lee also do not explicitly teach wherein said small subunit of ALS comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 15 of the instant application, however, a BLAST search revealed NCBI Reference Sequence: XP_010673855.1 annotated as PREDICTED: acetolactate synthase small subunit 1, chloroplastic [Beta vulgaris subsp. vulgaris]. NCBI Reference Sequence: XP_010673855.1 shares 99% identity to instant SEQ ID NO: 15 (i.e., the Beta vulgaris plant or seed of claim 1, wherein said small subunit of ALS comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 15) (see, sequence alignment below).
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
972 bits(2514)
0.0
Compositional matrix adjust.
476/477(99%)
477/477(100%)
0/477(0%)
Query 1 MAAVSFNNNGGKIGTLCSKPEFVCGFLRKLDFGAQTTVMAKPMSKISKLKAMEVSSNTSV 60
MAAVSFNNNGGKIGTLCSKPEFVCGFLRKLDFGAQTTVMAKPMSKISKLKAMEVSSNTSV
Sbjct 1 MAAVSFNNNGGKIGTLCSKPEFVCGFLRKLDFGAQTTVMAKPMSKISKLKAMEVSSNTSV 60
Query 61 NAVPVSAHPKVMRHTISVFVGDESGIINRIAGVISRRGYNIESLAVGLNKDKALFTIVVC 120
NAVPVSAHPKVMRHTISVFVGDESGIINRIAGVISRRGYNIESLAVGLNKDKALFTIVVC
Sbjct 61 NAVPVSAHPKVMRHTISVFVGDESGIINRIAGVISRRGYNIESLAVGLNKDKALFTIVVC 120
Query 121 GTDKVLRQVMEQLNKLVSVLKVEDLSREPQVERELMLVKLNADANTHTEIKWLVEIFRAK 180
GTDKVLRQVMEQLNKLVSVLKVEDLSREPQVERELMLVKLNADANTHTEIKWLVEIFRAK
Sbjct 121 GTDKVLRQVMEQLNKLVSVLKVEDLSREPQVERELMLVKLNADANTHTEIKWLVEIFRAK 180
Query 181 IVDVSDSLVTVEVTGDPGKMAAVLRNFSKFGIKEVARTGKIALRRERMGETAPFWRFSAA 240
IVDVSDSLVTVEVTGDPGKMAAVLRNFSKFGIKEVARTGKIALRRERMGETAPFWRFSAA
Sbjct 181 IVDVSDSLVTVEVTGDPGKMAAVLRNFSKFGIKEVARTGKIALRRERMGETAPFWRFSAA 240
Query 241 SYPDLEEKAVNTLVESTKRSINGDPGSSSTGDVYPVEPYDNPMVNQVLDAHWGVLHDGDS 300
SYPDLEEKAVNTLVESTKRSINGDPGSSSTGDVYPVEPYDNPMVNQVLDAHWGVLHDGDS
Sbjct 241 SYPDLEEKAVNTLVESTKRSINGDPGSSSTGDVYPVEPYDNPMVNQVLDAHWGVLHDGDS 300
Query 301 SGLRSHTLSMLVNNVSGVLNTVTGVISRRGYNIQSLAVGPAEKEGLSRITTVVPGNDESI 360
SGLRSHTLSMLVNNVSGVLNTVTGVISRRGYNIQSLAVGPAEKEGLSRITTVVPGNDESI
Sbjct 301 SGLRSHTLSMLVNNVSGVLNTVTGVISRRGYNIQSLAVGPAEKEGLSRITTVVPGNDESI 360
Query 361 GKLVQQLSKLVDLHEIQDLTHLPFAERELMLIKVAANTSARRDVLDIANIFRAKAVDVSD 420
GKLVQQLSKLVDLHEIQDLTHLPFAERELMLIKVAANTSARRDVLDIANIFRAKAVDVSD
Sbjct 361 GKLVQQLSKLVDLHEIQDLTHLPFAERELMLIKVAANTSARRDVLDIANIFRAKAVDVSD 420
Query 421 HTITLQLSGDLDKMVALQRLLEPYGICEVARTGRVALSRESRVDSALLRGYALPLSE 477
HTITLQLSGDLDKMVALQRLLEPYGICEVARTGRVAL+RESRVDSALLRGYALPLSE
Sbjct 421 HTITLQLSGDLDKMVALQRLLEPYGICEVARTGRVALTRESRVDSALLRGYALPLSE 477
Given that modifications for ALS herbicide tolerant Beta vulgaris plants were well known at the time of filing, as was the influence of ALS subunits in other plants and the relation to herbicide tolerant plants, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to validate a predicted small subunit in Beta vulgaris or to isolate and characterize the small subunits as taught by Dezfulian and result in structurally and functionally similar sequences. As the predicted sequence known in the art at the time of filing, one would have reasonable expectation of success and sufficient motivation to generate or identify the sequence to enhance the ALS herbicide tolerance of Beta vulgaris plants with a known mutation of the tryptophan at amino acid position 569, as taught by Rüdiger. One would be motivated to use both small subunit sequences in the Beta vulgaris plant given that Lee teaches that the catalytic efficiency increased with both small subunits functioning on the catalytic subunit in Arabidopsis. One would reasonably expect similar functionality given that Dezfulian teaches that ALS regulatory subunits are commonly known or predicted across plant genomes.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Rüdiger, H. et al. “Use of ALS Inhibitor Herbicides for Control of Unwanted Vegetation in ALS Inhibitor Herbicide Tolerant Beta vulgaris plants.” International Publication No. WO 2014090760 A1. Published 06/19/2014, in view of Dezfulian, M. et al. “Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development.” BMC Plant Biology. 17:71, Lee, Y. et al. (2001). “Identification of the Regulatory Subunit of Arabidopsis thaliana Acetohydroxyacid Synthase and Reconstitution with Its Catalytic Subunit.” Biochemistry. 40:6836-6844, and NCBI Reference Sequence: XP_010673855.1. PREDICTED: acetolactate synthase small subunit 1, chloroplastic [Beta vulgaris subsp. vulgaris]. Published 11/29/2016.
Claim 6 recites a method for producing a Beta vulgaris plant with optimally fitted large subunit and one or more regulatory subunits of an ALS holoenzyme comprising: a. crossing a Beta vulgaris plant comprising an allele encoding a large subunit of ALS comprising an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO. 1 and further comprising a leucine at a position corresponding to amino acid position 569 instead of the naturally occurring tryptophan, with a Beta vulgaris plant comprising at least one allele encoding a small subunit of ALS comprises an amino acid sequence having at least 95% sequence identity with an amino acid sequence selected from the group of SEQ ID NO. 15; and b. identifying progeny plants comprising said allele encoding said large subunit of ALS and said at least one allele encoding said regulatory subunit of ALS.
Regarding claim 6, Rüdiger teaches a method for producing a sugar beet mutant being resistant to one or more inhibitors of ALS [pg. 35, lns. 18-23]. Rüdiger teaches the use of the ALS inhibitor herbicides for controlling unwanted vegetation in ALS inhibitor herbicide tolerant Beta vulgaris plants [Abstract]. Rüdiger teaches mutations in the ALS gene where the tryptophan at position 569 in the encoded ALS enzyme (corresponding to position 574 in the Arabidopsis thaliana ALS enzyme) is substituted by another amino acid (preferably by leucine) [pg. 16, lns. 24-31; claim 4]. Rüdiger teaches SEQ ID NO: 2, a Beta vulgaris sequence with 100% identity to SEQ ID NO: 1 of the instant application (i.e., a large subunit of ALS comprising an amino acid sequence of SEQ ID NO: 1, and further comprising a leucine at a position corresponding to amino acid position 569 instead of the naturally occurring tryptophan) (see, alignment below). Rüdiger teaches the making, selection and propagation of the respective ALS inhibitor herbicide tolerant Beta vulgaris mutants and their progeny [pg. 41, lns. 2-5] and teaches the methods of other patents, incorporated by reference, such as a method for increasing the herbicide-resistance of a plant by: (a) crossing a first plant to a second plant, in which the first plant comprises an acetohydroxyacid synthase (AHAS) gene, in which the gene encodes a protein having a mutation at one or more amino acid positions; (b) screening a population resulting from the cross for increased AHAS herbicide-resistance; (c) selecting a member resulting from the cross having increased AHAS herbicide- resistance; and (d) producing seeds resulting from the cross2. In some embodiments, a hybrid seed is produced or plants are grown from seeds produced by any of the above methods (i.e., a method for producing a plant comprising: a. crossing a plant comprising a mutation; and b. identifying progeny plants).
Query Match 100.0%; Score 3393; Length 664;
Best Local Similarity 100.0%;
Matches 660; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAATFTNPTFSPSSTPLTKTLKSQSSISSTLPFSTPPKTPTPLFHRPLQISSSQSHKSSA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAATFTNPTFSPSSTPLTKTLKSQSSISSTLPFSTPPKTPTPLFHRPLQISSSQSHKSSA 60
Qy 61 IKTQTQAPSSPAIEDSSFVSRFGPDEPRKGSDVLVEALEREGVTNVFAYPGGASMEIHQA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 IKTQTQAPSSPAIEDSSFVSRFGPDEPRKGSDVLVEALEREGVTNVFAYPGGASMEIHQA 120
Qy 121 LTRSKTIRNVLPRHEQGGVFAAEGYARATGKVGVCIATSGPGATNLVSGLADALLDSVPL 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LTRSKTIRNVLPRHEQGGVFAAEGYARATGKVGVCIATSGPGATNLVSGLADALLDSVPL 180
Qy 181 VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRIVKEAFFLANSGRPGP 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRIVKEAFFLANSGRPGP 240
Qy 241 VLIDLPKDIQQQLVVPDWDRPFKLGGYMSRLPKSKFSTNEVGLLEQIVRLMSESKKPVLY 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 VLIDLPKDIQQQLVVPDWDRPFKLGGYMSRLPKSKFSTNEVGLLEQIVRLMSESKKPVLY 300
Qy 301 VGGGCLNSSEELRRFVELTGIPVASTLMGLGSYPCNDELSLHMLGMHGTVYANYAVDKAD 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 VGGGCLNSSEELRRFVELTGIPVASTLMGLGSYPCNDELSLHMLGMHGTVYANYAVDKAD 360
Qy 361 LLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSICADVKLALRGMNKI 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSICADVKLALRGMNKI 420
Qy 421 LESRIGKLNLDFSKWREELGEQKKEFPLSFKTFGDAIPPQYAIQVLDELTNGNAIISTGV 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 LESRIGKLNLDFSKWREELGEQKKEFPLSFKTFGDAIPPQYAIQVLDELTNGNAIISTGV 480
Qy 481 GQHQMWAAQHYKYRNPRQWLTSGGLGAMGFGLPAAIGAAVARPDAVVVDIDGDGSFIMNV 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 GQHQMWAAQHYKYRNPRQWLTSGGLGAMGFGLPAAIGAAVARPDAVVVDIDGDGSFIMNV 540
Qy 541 QELATIRVENLPVKIMLLNNQHLGMVVQWEDRFYKANRAHTYLGNPSKSADIFPDMLKFA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 QELATIRVENLPVKIMLLNNQHLGMVVQWEDRFYKANRAHTYLGNPSKSADIFPDMLKFA 600
Qy 601 EACDIPSARVSNVADLRAAIQTMLDTPGPYLLDVIVPHQEHVLPMIPSGAGFKDTITEGD 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 EACDIPSARVSNVADLRAAIQTMLDTPGPYLLDVIVPHQEHVLPMIPSGAGFKDTITEGD 66
Rüdiger does not explicitly teach one or more small subunit of ALS, however, small subunits of ALS are known in the art in a variety of plants. For example, Dezfulian teaches that the ALS holoenzyme is composed of distinct catalytic and regulatory subunits [pg. 2, col. 1, ¶3]. Dezfulian teaches a set of interacting proteins, which are evolutionarily conserved orthologs of bacterial feedback-regulatory proteins and are implicated in ALS activity [Abstract]. Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. ALS is the first enzyme in the BCAA synthesis pathway. This has stimulated interest in the genetic manipulation of plant BCAA metabolism both for the generation of herbicide-tolerant crops [pg. 2, col. 1, ¶1].
Plant genomes commonly contain multiple genes known or predicted to encode ALS regulatory subunits, including two putative regulatory ALS subunit genes [pg. 2, col. 2, ¶1]. Dezfulian teaches identification of two Arabidopsis ALS regulatory subunits AIP1 (At2g31810) and AIP3/VAT1 (At5g16290) with high deduced protein similarity to prokaryotic and eukaryotic regulatory ALS subunits [pg. 3, col. 1, ¶1]. Dezfulian teaches that both AIP1 and AIP3 regulatory subunits have acquired functional attributes independent of the ALS holoenzyme [pg. 10, col. 1, ¶2], and that catalytic activity of the holoenzyme in complex with AIP3 and BCAAs as cofactors exhibits a greater sensitivity to Val and Ile [pg. 10, col. 1, ¶1].
Rüdiger and Dezfulian do not explicitly teach the use of the main catalytic large subunit with the smaller regulatory subunit or that the subunit of ALS comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 15, however, Lee teaches that the combination of an ALS regulatory subunit of Arabidopsis thaliana with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine leucine, and isoleucine [Abstract]. Each regulatory subunit was individually able to activate the catalytic subunit and confer sensitivity to BCAA inhibition, though they were most effective as an entire regulatory subunit [pg. 6842, col. 1, ¶3]. This demonstrates that the plant’s native regulatory subunits aid in enhancing catalytic activity.
Rüdiger, Dezfulian, and Lee do not explicitly teach that the subunit of ALS comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 15, however, a BLAST search revealed NCBI Reference Sequence: XP_010673855.1 annotated as PREDICTED: acetolactate synthase small subunit 1, chloroplastic [Beta vulgaris subsp. vulgaris]. NCBI Reference Sequence: XP_010673855.1 shares 99% identity to instant SEQ ID NO: 15 (i.e., with a Beta vulgaris plant comprising at least one allele encoding a small subunit of ALS comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 15) (see, alignment below).
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
972 bits(2514)
0.0
Compositional matrix adjust.
476/477(99%)
477/477(100%)
0/477(0%)
Query 1 MAAVSFNNNGGKIGTLCSKPEFVCGFLRKLDFGAQTTVMAKPMSKISKLKAMEVSSNTSV 60
MAAVSFNNNGGKIGTLCSKPEFVCGFLRKLDFGAQTTVMAKPMSKISKLKAMEVSSNTSV
Sbjct 1 MAAVSFNNNGGKIGTLCSKPEFVCGFLRKLDFGAQTTVMAKPMSKISKLKAMEVSSNTSV 60
Query 61 NAVPVSAHPKVMRHTISVFVGDESGIINRIAGVISRRGYNIESLAVGLNKDKALFTIVVC 120
NAVPVSAHPKVMRHTISVFVGDESGIINRIAGVISRRGYNIESLAVGLNKDKALFTIVVC
Sbjct 61 NAVPVSAHPKVMRHTISVFVGDESGIINRIAGVISRRGYNIESLAVGLNKDKALFTIVVC 120
Query 121 GTDKVLRQVMEQLNKLVSVLKVEDLSREPQVERELMLVKLNADANTHTEIKWLVEIFRAK 180
GTDKVLRQVMEQLNKLVSVLKVEDLSREPQVERELMLVKLNADANTHTEIKWLVEIFRAK
Sbjct 121 GTDKVLRQVMEQLNKLVSVLKVEDLSREPQVERELMLVKLNADANTHTEIKWLVEIFRAK 180
Query 181 IVDVSDSLVTVEVTGDPGKMAAVLRNFSKFGIKEVARTGKIALRRERMGETAPFWRFSAA 240
IVDVSDSLVTVEVTGDPGKMAAVLRNFSKFGIKEVARTGKIALRRERMGETAPFWRFSAA
Sbjct 181 IVDVSDSLVTVEVTGDPGKMAAVLRNFSKFGIKEVARTGKIALRRERMGETAPFWRFSAA 240
Query 241 SYPDLEEKAVNTLVESTKRSINGDPGSSSTGDVYPVEPYDNPMVNQVLDAHWGVLHDGDS 300
SYPDLEEKAVNTLVESTKRSINGDPGSSSTGDVYPVEPYDNPMVNQVLDAHWGVLHDGDS
Sbjct 241 SYPDLEEKAVNTLVESTKRSINGDPGSSSTGDVYPVEPYDNPMVNQVLDAHWGVLHDGDS 300
Query 301 SGLRSHTLSMLVNNVSGVLNTVTGVISRRGYNIQSLAVGPAEKEGLSRITTVVPGNDESI 360
SGLRSHTLSMLVNNVSGVLNTVTGVISRRGYNIQSLAVGPAEKEGLSRITTVVPGNDESI
Sbjct 301 SGLRSHTLSMLVNNVSGVLNTVTGVISRRGYNIQSLAVGPAEKEGLSRITTVVPGNDESI 360
Query 361 GKLVQQLSKLVDLHEIQDLTHLPFAERELMLIKVAANTSARRDVLDIANIFRAKAVDVSD 420
GKLVQQLSKLVDLHEIQDLTHLPFAERELMLIKVAANTSARRDVLDIANIFRAKAVDVSD
Sbjct 361 GKLVQQLSKLVDLHEIQDLTHLPFAERELMLIKVAANTSARRDVLDIANIFRAKAVDVSD 420
Query 421 HTITLQLSGDLDKMVALQRLLEPYGICEVARTGRVALSRESRVDSALLRGYALPLSE 477
HTITLQLSGDLDKMVALQRLLEPYGICEVARTGRVAL+RESRVDSALLRGYALPLSE
Sbjct 421 HTITLQLSGDLDKMVALQRLLEPYGICEVARTGRVALTRESRVDSALLRGYALPLSE 477
Given that Rüdiger teaches a catalytic subunit of ALS comprising the amino acid sequence of instant SEQ ID NO: 1 with demonstrated activity in an ALS inhibitor-tolerant Beta vulgaris; Dezfulian teaches identification of plant small regulatory subunits and suggests their function relative to herbicide tolerant plants; Lee teaches the combination of an ALS regulatory subunit and catalytic subunit for enhanced catalytic efficiency; and predicted Beta vulgaris NCBI sequence was known in the art at the time of filing, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use the known mutation that confers herbicide tolerance to Beta vulgaris in combination with native small regulatory subunits that can enhance catalytic efficiency, thus overcoming the amino acid starvation effects of ALS inhibiting herbicides.
It would have been obvious to one of ordinary skill to isolate and characterize the ALS regulatory subunits as taught by Dezfulian in Arabidopsis. One would have reasonable expectation of success given that Dezfulian teaches that plant genomes commonly contain multiple genes known or predicted to encode ALS regulatory subunits and that they are evolutionarily conserved orthologs of the bacterial subunits implicated in ALS regulatory activity. One would be motivated to combine the mutated catalytic subunit and the regulatory subunits of ALS in view of Lee’s teachings, specifically that the combination of the two regulatory subunits and the catalytic subunit enhanced catalytic efficiency. The mutation at position 569, as proven in the prior art, prevents the binding of the herbicide to the ALS enzyme, allowing for the normal production of essential amino acids. The regulatory subunits would aid in regular plant functioning with application of herbicides that would usually target the catalytic domain.
Given that mutations and modifications for ALS herbicide tolerant Beta vulgaris plants were well known at the time of filing, as was the influence of ALS subunits in other plants, the relation to herbicide tolerant plants, and predicted acetolactate synthase small subunit sequences were previously annotated, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to validate a known predicted small subunit in Beta vulgaris or to isolate and characterize the small subunits as taught by Dezfulian. As the predicted sequence known in the art at the time of filing, one would have reasonable expectation of success and sufficient motivation to generate or identify the sequence to enhance the ALS herbicide tolerance of Beta vulgaris plants with a known mutation of the tryptophan at amino acid position 569, as taught by Rüdiger. Given the high sequence similarity of NCBI Reference Sequence: XP_010673855.1 to SEQ ID NO: 15 of the instant application, this is interpreted to read on an “optimally fitted large subunit”, as recited in claim 6, due to the structural similarity.
Conclusion
No claims allowed.
Contact Information
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1 Beta vulgaris. https://en.wikipedia.org/wiki/Beta_vulgaris.
2 Schopke, C. et al. (“Mutated Acetohydroxyacid Synthase Genes in Brassica”. WO 2009/046334. Published 10/03/2008) teaches methods for producing plants with ALS resistance and methods for producing progeny through crosses and selecting the progeny through screening the population for enhanced resistance [¶13] (incorporated herein by reference).
Prosecution Insights