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 .
Priority
Acknowledgment This present application is a U.S. National Stage Application of International Application No. PCT/US2023/035047, filed 10/12/2023, which claims priority from U.S. Provisional Patent Application No. 63/415,863, filed 10/13/2022.
The effective filing date is 10/13/2022.
Status of the Claims
Amendments dated 02/25/2026 are entered
Claims 1 are pending and are examined herein.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 04/11/2025 Is acknowledged and is being considered by the examiner.
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Specification
The specification is objected to for the following reason(s):
The use of the trademark BLAST® (e.g., at page 15 and 16) has been noted in this application. The Basic Local Alignment Search Tool (BLAST)® is a registered trademark of the National Library of Medicine. The registered trademark symbol ® should be included following the trademarks wherever they appear, and the trademarks should be accompanied by the generic terminology.
Tables 1, 2, and 3 are referenced in the Specification but are not provided.
Claim Objections
Claims 1-12 are objected to for the following informalities:
Lack of consistency. Is it “Scz”, “SCZ”, “SISCZ”, “SCHIZORIZA”,or “SLSCHIZORIZA”? Various terms have been used in the claims and in the specification.
Claim 9 is further objected to for the following informalities:
Should recite “A plant having increased” instead of “A plant having increased having increased”
Claim Interpretations
Regarding claims 1-12, specification para 0029 defines that “the term ‘SlEXO transcription factor’ or ‘SlEXO polypeptide’ refers to a polypeptide encoded by a tomato C2H2 transcription factor gene available under accession number Solyc09g011120 (see, also NCBI GENE ID: 101261848 zinc finger protein 10 (Solanum lycopersicum)), or a homolog, or ortholog, thereof in another plant.” This locus or gene encodes the polypeptide depicted in NCBI Accession XP_019071045.1.
Regarding claims 1-12, specification para 0030 defines that “the term ‘SCZ transcription factor’ or ‘SCZ polypeptide’ refers to a polypeptide encoded by a plant SCHIZORIZA heat shock transcription factor gene, or homolog, or ortholog, thereof in another plant. An illustrative polypeptide sequence is available under accession number Solyc04g078770 (synonym SGN-U603561).” This locus or gene encodes the polypeptide depicted in NCBI Accession XP_004238144.1.
Regarding claims 9-12, specification para 0027, recites that “’increased drought tolerance’ of a plant refers to any measurable improvement in a physiological or physical characteristic, such as yield, as measured relative to a reference or control plant when grown under drought conditions.”
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.
Indefiniteness
Claim 10 is 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. The claim is recited below with emphasis and annotations for reference within this text:
10. The plant of claim 9, wherein the plant of (a) [i] is mutated or engineered to reduce or knockout expression of, or [ii] expresses an siRNA or antisense polynucleotide to reduce expression of, the SlEXO transcription factor, or [iii] expresses a modified endonuclease such as Cas9 targeted to reduce expression of the SlEXO transcription factor and/or the Scz transcription factor.
The claim recites the word “or” multiple times such that it is unclear what the alternative limitations are. The claim is drawn to the plant of claim 9 and further limited to the plant of claim9(a). According to claim 9, the plant of claim 9 (a) is “mutated or engineered to reduce or knockout expression of a gene encoding a SlEXO zinc finger transcription factor.” Does the recitation of “wherein the plant of (a) is mutated or engineered to…” in claim 10 mean that the plant of claim 9(a) is further mutated or engineered or is it just the mutation or engineering that was recited in claim 9? If it is the latter, then claim 10 would be subject to a rejection under 35 U.S.C. 112(d) as not further limiting preceding claim 9.
Are the alternative limitations [i], [ii], or [iii]? If so limitation [i] is incomplete as is lacks a target gene. Or is the target gene the SlEXO transcription factor?
If so, the alternative limitations might instead be [i] or [ii] targeting the SlEXO transcription factor, or [iii] targeting either the SlEXO transcription factor or the Scz transcription factor or both?
Claim 10 is further rejected under 35 U.S.C. 112(b). The phrase "such as" recited therein renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. Description of examples or preferences is properly set forth in the specification rather than the claims. If stated in the claims, examples and preferences may lead to confusion over the intended scope of a claim. See MPEP § 2173.05(d).
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.
Written Description
Claims 1-12 are rejected under 35 U.S.C. 112(a) 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. All dependent claims are included in these rejections unless they contain a limitation that overcomes the deficiencies of the parent claim from which they depend. Details are listed below.
The Federal Circuit has clarified the written description requirement. The court stated that a written description of an invention "requires a precise definition, such as by structure, formula, [or] chemical name, of the claimed subject matter sufficient to distinguish it from other materials". University of California v. Eli Lilly and Co., 119 F.3d 1559, 1568; 43 USPQ2d 1398, 1406 (Fed. Cir. 1997). The court also concluded that "naming a type of material generally known to exist, in the absence of knowledge as to what that material consists of, is not description of that material". Id. Further, the court held that to adequately describe a claimed genus, Patent Owner must describe a representative number of the species of the claimed genus, and that one of skill in the art should be able to "visualize or recognize the identity of the members of the genus".
Applicant broadly claims a method of generating a plant having enhanced lignification of the root exodermis by genetically modifying a gene encoding a SlEXO zinc finger transcription factor or a homolog or ortholog thereof and a gene encoding a heat shock transcription factor B4 Scz transcription factor or a homolog or ortholog thereof to reduce or knockout their expression, wherein the plant has enhanced lignification of root exodermis compared to a counterpart control plant, wherein genetically modifying comprises: gene editing, specifically CRISPR/Cas gene editing, of the gene encoding the SlEXO zinc finger transcription factor or a homolog or ortholog thereof and/or a gene encoding a heat shock transcription factor B4 Scz transcription factor or a homolog or ortholog thereof; or introducing an inhibitory RNA that targets a polynucleotide encoding the SlEXO zinc finger transcription factor or a homolog or ortholog thereof and/or a gene encoding a heat shock transcription factor B4 Scz transcription factor or a homolog or ortholog thereof, and wherein the polypeptide encoded by the gene encoding SlEXO has at least 75% identity to SEQ ID NO:1 and/or the polypeptide encoded by the Scz gene has at least 75% identity to SEQ ID NO: 3.
In a method of generating a plant having enhanced lignification of the root exodermis, Applicant has reduced to practice this invention only in Solanum lycopersicum plants(Specification, para 0105), yet claims the invention in all possible plants, which include plants yet to be discovered. It is unclear if genetically modifying a Solanum lycopersicum EXO polypeptide and/or a Solanum lycopersicum SCHIZORIZA heat shock transcription factor genes or orthologs or homologs expressed in a cactus or in a Sequoia tree to reduce their expression would also generate enhanced lignification of their root exodermis, for example.
Even though claims 7 and 12 are limited to the recitation(s) of tomato plants, they are still rejected due to their recitation(s) of all possible homologs and orthologs of SlEXO.
It appears that with the exception of the full length sequence of the SlEXO zinc finger transcription factor polypeptide (SEQ ID NO: 1) and the full length sequence of the heat shock transcription factor B4 Scz transcription factor polypeptide(SEQ ID NO: 1), Applicant has not reduced to practice the claimed method wherein the polypeptide encoded by the gene encoding SlEXO only has at least 75% identity to SEQ ID NO: 1 and/or polypeptide encoded by the gene encoding Scz only has at least 75% identity to SEQ ID NO: 3, in a method of generating a plant having enhanced lignification of the root exodermis.
There does not appear to be adequate support in the specification that the claimed invention will work in all possible plants. Also, reducing the polypeptide sequence identity to only 75% may not work, especially for applicants’ SlEXO. “The C2H2-type zinc finger protein (C2H2-ZFP) family represents one of the largest and most diverse groups of TFs in plants,” teaches Qu (Qu Y, Feng Y, Xia P. C2H2-type zinc finger proteins: Contributing regulators of plant tolerance to abiotic stress. Plant Sci. 2026 Apr;365:113023. doi: 10.1016/j.plantsci.2026.113023. Epub 2026 Feb 4. PMID: 41651224.)(page 1, col 2, last para). In a 2026 review of C2H2 zinc finger proteins, Qu additionally teaches that “within the overall [transcriptional] network, distinct C2H2-ZFP complexes form species- or stress-specific regulatory modules, reflecting functional specialization and integration” (page 9, col 2, para 4). Qu concludes that “C2H2-type zinc finger proteins exhibit multilevel regulatory properties in stress response. On the one hand, different family members may enhance plant stress tolerance through similar regulatory mechanisms (e.g. ABA signalling pathway); on the other hand, the same protein may be involved in multiple stress responses, showing functional pleiotropy…Furthermore, the contextual factors that determine whether a given C2H2-ZFP acts as a transcriptional activator or repressor under specific stress conditions remain poorly understood. Dynamic regulatory layers such as post-translational modifications, protein–protein interactions, and cellular redox states likely confer such functional plasticity, yet these aspects require deeper exploration” (conclusion, para 2). Because the claimed genus of sequences is so vast and the C2H2-type zinc finger protein families’ roles are not fully understood, the applicant must disclose sufficient findings relating the parts of sequence to the protein’s function beyond the single complete open reading frame sequence described in SEQ ID NO:1. It is noted that this rejection is to SlEXO. In contrast, the SCZ protein appears to be more conserved across plants, so the rejection does not pertain to it.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 9-10 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Mittler (Mittler R, et al. Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress. FEBS Lett. 2006 Dec 11;580(28-29):6537-42. doi: 10.1016/j.febslet.2006.11.002. Epub 2006 Nov 9. PMID: 17112521; PMCID: PMC1773020., published 2006). This rejection is made to the extent that the ZAT10 protein is being interpreted as an ortholog of SlEXO. See claim interpretation above.
Mittler teaches in the abstract that, in Arabidopsis [i.e., a plant], “knockout and RNAi mutants [i.e., claim 9 recitation of “mutated or engineered to reduce or knockout expression”, claim 10 recitation of “expresses an siRNA or antisense polynucleotide to reduce expression”] of Zat10 [i.e., SlEXO zinc finger transcription factor or ortholog] were also more tolerant to osmotic and salinity stress [i.e., having increased having increased drought and salt tolerance. “[Mittler’s] results suggest that Zat10 plays a key role as both a positive and a negative regulator of plant defenses.”
Thus, Mittler anticipates the claimed inventions.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1 and 4-12 are rejected under 35 U.S.C. 103 as being unpatentable over Yu (U.S. Patent Application Publication No. 20180163221A1, assigned to Academia Sinica, titled ‘Transgenic plants expressing type 2c protein phosphatase abscisic acid (pp2caba) proteins and uses thereof’, published 2018, Cited in IDS) in view of Hove (ten Hove CA, et al. SCHIZORIZA encodes a nuclear factor regulating asymmetry of stem cell divisions in the Arabidopsis root. Curr Biol. 2010 Mar 9;20(5):452-7. doi: 10.1016/j.cub.2010.01.018. Epub 2010 Feb 18. PMID: 20171102., published 2010), NCBI Accession XP_004238144.1 (PREDICTED: heat stress transcription factor B-4 [Solanum lycopersicum], published 2016), Lin (U.S. Patent Application Publication No. 20120102588A1, assigned to Shanghai Institutes for Biological Sciences SIBS of CAS, titled ‘Rice zinc finger protein transcription factor dst and use thereof for regulating drought and salt tolerance’, published 2012, Cited in IDS), Dinkins (Dinkins R, et al. Ectopic expression of an Arabidopsis single zinc finger gene in tobacco results in dwarf plants. Plant Cell Physiol. 2002 Jul;43(7):743-50. doi: 10.1093/pcp/pcf086. PMID: 12154136., published 2002), and NCBI Accession XP_019071045.1 (zinc finger protein 10 [Solanum lycopersicum], published 2018, Cited in IDS).
Claims 1 and 5 are drawn to a method of generating a plant having enhanced lignification of the root exodermis, the method comprising (a) genetically modifying a gene encoding a SlEXO zinc finger transcription factor (i.e., a polypeptide encoded by a tomato C2H2 transcription factor gene available under accession number Solyc09g011120 (see, also NCBI GENE ID: 101261848 zinc finger protein 10 (Solanum lycopersicum)), or a homolog, or ortholog, thereof in another plant) to reduce or knockout expression of said a zinc finger transcription factor; and (b) genetically modifying a gene encoding a heat shock transcription factor B4 Scz transcription factor (i.e., a polypeptide encoded by a plant SCHIZORIZA heat shock transcription factor gene, or homolog, or ortholog, thereof in another plant) to reduce or knockout expression of the Scz transcription factor; wherein the plant has enhanced lignification of the root exodermis compared to a counterpart control plant wherein the polypeptide encoded by the gene encoding SlEXO has at least 75% identity to SEQ ID NO:1 and/or the polypeptide encoded by the Scz gene has at least 75% identity to SEQ ID NO:3.
Claim 9 is drawn to a plant having increased drought and/or salt tolerance, compared to a counterpart control plant, wherein the plant is mutated or engineered to reduce or knockout expression of a gene encoding a SlEXO zinc finger transcription factor and/or a gene encoding a heat shock transcription factor B4 Scz transcription factor.
This rejection is made on the basis that rice C2H2 zinc finger transcription factor Lin is interpreted as being an ortholog of SlEXO zinc finger transcription factor of the instant application; see claim interpretation above.
Regarding claims 1, 5, and 9, Yu teaches that “ABA treatments mimic low water potentials or osmotic stress in dehydrated soils , with low concentrations inducing root system (lateral branching] for increasing root surface for more efficient water uptake, while high concentrations arrest root branching but promote mechanical strength and allow continued PR growth to facilitate water uptake from deep soil” where PP2CABA is induced in a dose-dependent manner by ABA (para 0014, 0123, and 0127).
Yu then teaches “The development of two protective sheaths, endodermis and exodermis, [which] play important roles in basic root function and protection against stresses such as drought, … and salinity” where “Cell walls of these two tissues are …connected by Casparian strips that are mainly made of lignin… The endodermis Casparian strip blocks the passive flow of materials such as water and solutes into the stele, and the exodermis Casparian strip… filters ions from the soil solution, and prevents root drying under limited water supply in soil” (emphasis provided; para 0005).
Yu also teaches a method “of producing the transgenic plants” where “ABA [abscisic acid] and PP2CABA enhanced the accumulation of lignin and suberin in endodermal and exodermal cell layers in roots”(c.f., claim 1: A method of generating a plant having enhanced lignification of the root exodermis)(para 0016 and 0123), and that “The method may comprise: (a) transforming a plant cell with a nucleic acid operably linked to a promoter to obtain a recombinant plant cell expressing a PP2CABA protein, wherein the nucleic acid encodes the PP2CABA protein; and (b) growing the recombinant plant cell obtained in (a) to generate the transgenic plant” ((c.f., instant claim 1 which recites “the method comprising genetically modifying a gene…” in parts (a) and (b)) (para 0016).
Yu also teaches that “the transgenic plant disclosed…exhibits a lower lateral roots (LR) to primary roots (PR) ratio, a larger root diameter, a higher tolerance to abiotic stress, increased levels of lignin and/or suberin, or a combination thereof as compared with a non-transgenic plant counterpart growing under the same conditions” and that “ABA and PP2CABA enhanced the accumulation of lignin and suberin in endodermal and exodermal cell layers in roots [whereas] lignin is mainly present in peripheral sclerenchyma in wild type plants” (c.f., Claim 1 recitation of “wherein the plant has enhanced lignification of root exodermis compared to a counterpart control plant, ” and claim 9 recitation of “ A plant having increased drought and/or salt tolerance, compared to a counterpart control plant, wherein the plant (a) is mutated or engineered to reduce or knockout expression of a gene)(emphasis provided; para 0014 and 0123).
Yu additionally teaches that ABA is known to suppress LR growth and that “PP2CABA …plays a key role in sensing the ABA signaling in suppression of LR growth” but also “unexpectedly promoted lignification and suberization in cell walls of periphery root tissues by enhancing the expression of genes involved in these processes…including but not limited to SWN1, SWN2, MYB96, CCR, KCS, LTP, POX and LEA3)” (para 0035 and 0128).
Yu teaches “gene knockout” and “homozygous pp2caba mutant…plants [where] PP2CABA transcripts were absent in the homozygous plant, indicating that [the plant bears] a loss-of-function mutant” (emphasis provided; para 0024 and 0119).
Yu does not explicitly teach genetically modifying a gene encoding a SlEXO zinc finger transcription factor to reduce or knockout expression of the SlEXO transcription factor and genetically modifying a gene encoding a heat shock transcription factor B4 Scz transcription factor to reduce or knockout expression of the Scz transcription factor.
Hove teaches, in the summary on page 452, “SCHIZORIZA (SCZ), a single nuclear factor with homology to heat-shock transcription factors that controls the separation of cell fate in a set of stem cells generating different root tissues: root cap, epidermis, cortex, and endodermis” (title; page 452, col 1, summary) and a reference titled “SCHIZORIZA controls an asymmetric cell division and restricts epidermal identity in the Arabidopsis root” (References, 15th).
Hove teaches that “SCZ Is Required for Cortex Fate Specification” (page 455, col 1)
Hove teaches that “after SHR/SCR induced periclinal division, SHR promotes endodermal fate. SCZ [, which is expressed in the cortex,]…suppresses endodermal fate, possibly by downregulation of SHR/SCR expression in the mature ground tissue [and] continues its non-cell-autonomous suppression of epidermis and lateral root cap fate in the mature ground tissue” (page 456, col 2, 1st partial para).
Hove teaches mutagenesis of plant lines followed by “a QC marker-based mutagenesis screen” and that “fine mapping located SCZ to…heat shock transcription factor B4 (HsfB4) locus (At1g46264), based on its stem cell-enriched in silico root expression pattern[…,] Comparison with the corresponding wild-type sequence revealed different mutations in the HsfB4 gene for all scz alleles, including two additional TILLING alleles[…,]the identical phenotype indicates that they are likely HsfB4 mutant null alleles” (c.f., claim 1’s recitation of “(b) genetically modifying a gene encoding a heat shock transcription factor B4 Scz transcription factor to reduce or knockout expression of the Scz transcription factor,” and claim 9 recitation of “mutated or engineered to reduce or knockout expression of a gene encoding a heat shock transcription factor B4 Scz transcription factor” )(emphasis added; page 452, col 2).
Hove further teaches “periclinal ground tissue divisions leading to supernumerary mutant layers” in scz null mutants (Figure 2, caption).
Hove also teaches plants with “double-mutant combinations” comprising of scz null mutants (c.f., claim 9)(page 456, col 1, 2nd para).
BLAST protein searches of NP_175142.1, an amino acid sequence of the polypeptide encoded by the At1g46264 locus taught by Hove, reveals XP_004238144.1 as the closest homolog in Tomato or solanum Lycopersicon. XP 004238144.1 is encoded by a gene with the “GeneID:101250985" (i.e., the gene encoding a heat shock B4 Scz transcription factor) (metadata) and is a 100% match for SEQ ID NO: 3 (c.f., claim 5; data not shown).
Also in the field of transcriptional regulators Lin, published in 2012, teaches “how to improve drought and/or salt tolerance in crops” by inhibiting expression of “zinc finger protein transcription factors, including polypeptides having the amino acid sequence of SEQ ID NO: 2, polypeptides having conserved mutations in the preceding polypeptides, or homologs” where “zinc finger transcription factors include a Csy-2/His-2 type zinc finger structural domain” and wherein said “inhibition is carried out by methods of deletion, mutation, RNAi, antisense or dominant negative regulation” (c.f., claim 1’s recitation of “(a) genetically modifying a gene encoding a zinc finger transcription factor to reduce or knockout expression of the transcription factor,” and claim 9’s “wherein the plant (a) is mutated or engineered to reduce or knockout expression of a gene encoding a …zinc finger transcription factor”)(see Lin claim 9; Paras. 0004, 0009, 0010, 0036, and 0037).
Lin teaches “a highly drought- and salt-tolerant mutant (dst)” comprising a “DST gene containing 2 nucleotide mutations, which lead to 2 amino-acid substitutions (amino acid 69 is mutated from asparagine to aspartic acid, and amino acid 162 is mutated from alanine to threonine) and result in drought and salt resistant phenotype” such that “compared with the wild-type, mutants [plants] have higher drought resistance [and] …increased salt tolerance”, wherein the “DST gene encodes a zinc finger protein (transcription factor) … having a conserved C2H2 type zinc finger domain” and wherein the plants were mutated by “treatment with 0.6% of EMS (ethyl methane sulfonate)” (para 0135-0136 and 0153). This reads on “A plant having increased having increased drought and/or salt tolerance, compared to a counterpart control plant, wherein the plant (a) is mutated or engineered to reduce or knockout expression of a gene encoding a zinc finger transcription factor” limitation of claim 9.
Lin teaches that “DST gene is a negative regulatory factor for drought resistance and salt resistance, inhibiting its expression can enhance resistance to salt or drought stress in plants” and notes that “[DST-]homologous genes all have a conserved C2H2-type zinc finger structural domain, with high similarities at the N terminus, suggesting that DST homologous genes in other plants… would have similar functions as that of rice DST gene” (para 0081-0082).
Dinkins teaches AtZFP10 as affecting root morphology when expressed in tobacco plants.
Dinkins, in the field of C2H2 transcription factors, teaches that “Arabidopsis and tobacco…plants transformed with the KYLX71 vector containing the AtZFP10 (AT2G37740.1) gene [which has a conserved C2H2 domain]…displayed an altered phenotype during regeneration on kanamycin-selective medium (T0 generation). Only a few explants resulted in shoots, leaves on these shoots were smaller and curled compared to wild-type tobacco regeneration. During rooting, the roots were more numerous, thicker and more branched compared to wild-type tobacco” (page 745, col 2). Borrowing Lin’s terms, AtZFP10 expressing plants had a higher lateral roots (LR) to primary roots (PR) ratio compared to wildtype. Dinkins teaches in the conclusion that “target genes for SUP and AtZFP10… probably involve cell division” (page 745, col 2).
NCBI Reference Sequence: NP_181310.1 is the amino acid sequence of “zinc-finger protein 10 [Arabidopsis thaliana]” encoded by the AT2G37740 locus taught by Dinkins and is annotated with “EXPRESSED IN: root; CONTAINS InterPro DOMAIN/s: Zinc finger, C2H2-like (InterPro:IPR015880)” (metadata). Combined with Dinkins’s teachings, one of ordinary skill in the art would understand that root-expressed AtZFP10 probably targets genes involved cell division promoting LR branching where nascent lateral roots have less establishes root barriers.
In tomato, NCBI Accession XP 019071045.1 is the closest ortholog to and is substantially identical to NP_181310.1 (See snippet below) and is encoded by a gene with the “GeneID:101261848" " (i.e., the gene encoding a SlEXO zinc finger transcription factor) (metadata).
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NCBI Accession XP 019071045.1 teaches the sequence of zinc finger protein 10 from the Solanum lycopersicum plant, is annotated as including a Csy-2/His-2 type zinc finger structural domain such that, by Lin’s definition, it is a zinc finger transcription factor, and is a 100% match for SEQ ID NO: 1 (c.f., claim 5)(data not shown).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to try combining the modified teachings of Yu, Hove taken with the evidence of NP_175142.1, and XP_004238144.1 with the teachings of Lin, Dinkins and XP_019071045.1. Reading Hove and Yu, one of ordinary skill in the art would learn that SCZ is expressed in the cortex and radially suppresses cell fate where it reaches the exodermis, in plants that have it, then the epidermis (Yu, figure 10a) and recognize the benefit of knocking down or reducing expression of SCZ, reducing its non-cell-autonomous suppression of endodermal, likely exodermal (seeing as the exodermis is situated between the cortex and the epidermis in plants that have it; see Yu, figure 10a), and epidermal cell fates such that the cells can mature to contain lignin and protect against stresses such as drought and salinity and would be enabled to do so by recognizing XP_004238144.1 as the closest tomato ortholog of SCZ. Reading Lin and Dinkins, after Yu, one of ordinary skill in the art would recognize the benefit of knocking down/out a C2H2 zinc finger transcription factor/protein and/or its orthologs known to be expressed the roots, where silencing of the gene leads to drought tolerance and agronomic trait known to be controlled and promoted by ABA. After having read Lin and Dinkins, the person of ordinary skill in the art would be able to identify XP_019071045.1 as the closest tomato ortholog of Dinkins’s C2H2 zinc finger transcription factor. One would have been motivated to simultaneously reduce or knockout the expression of both XP_004238144.1 and XP_019071045.1, arriving upon the claimed method and plant with a reasonable expectation of success, because gene stacking is known in the art, Yu’s expression of PP2CABA affects multiple genes, Hove teaches double mutants comprising scz mutants, and Lin teaches that “drought and salts are major non-biological (abiotic) stresses, causing significant reduction in quantities and qualities of crops every year [where] drought often accompanies salts” (Hove, page 456, col 1, para 2; Lin, para 0002).
Regarding claim 4, which depends from claim 1, Lin teaches “inhibition…by methods of deletion, mutation, RNAi, antisense or dominant negative regulation” and “provides antagonists for zinc finger transcription factor proteins or polynucleotides [where] antagonists are small interference RNAs, antibodies or antisense oligonucleotides” (para 0037, and 0035).
Regarding claim 6, which depends from claim 1, Lin teaches that “plants are selected from:… Solanaceae…” (para 0017).
Regarding claim 7, which depends from claim 6, Yu teaches that “the transgenic plant can be a…tomato” (para 0015).
Regarding claim 8, which depends from claim 1, Lin teaches “methods for generating transgenic plants with enhanced drought and salt tolerance and transgenic plants generated by these methods” (para 0008).
Regarding claim 10, which depends from claim 9, Lin teaches “Using RNAi to down regulate the expression of [a] gene” (para 0078).
Regarding claim 11, which depends from claim 9, Lin teaches that “plants are selected from:… Solanaceae…” (para 0017).
Regarding claim 12, which depends from claim 11, Yu teaches that “the transgenic plant can be a…tomato” (para 0015).
Claims 2-3, which depend from claim 1, are rejected under 35 U.S.C. 103 as being unpatentable over the combined teachings of Yu, Hove, XP_004238144.1, Lin, Dinkins, and XP_019071045.1 as applied above further in view of Zhang (ZHANG et al , Fusing T5 Exonuclease with Cas9 and Cas12a Increases the Frequency and Size of Deletion at Target Sites, Science China Life Sciences, Volume 63, No 12, December 2020, pp 1918-1927, published 2020).
Yu, Hove, NP_175142.1, XP_004238144.1, Yilmaz, Lin, Dinkins, NP_181310.1, Yang, and XP_019071045.1 teach the method of claim 1 but do not explicitly teach gene editing nor that it comprises CRISPR/Cas gene editing.
However, Zhang is in the field of plant engineering and teaches “T5exo-Cas fusion [proteins],” “CRISPR/Cas systems,” and that “the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease” (Abstract)(i.e., gene editing of the gene encoding SlEXO and/or Scz, gene editing comprising CRISPR/Cas).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Yu, Hove, XP_004238144.1, Lin, Dinkins, and XP_019071045.1 with the method teachings of Zhang in order to take advantage of enhanced genome editing efficiencies of T5exo-Cas fusion CRISPR/Cas systems with a reasonable expectation of success and to “further…elucidate the function…of these genes in plant development” (Dinkins page 749, col 1, first full para).
References Cited but not Applied
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Mary (Mary Prathiba Joseph, et al. The Arabidopsis ZINC FINGER PROTEIN3 Interferes with Abscisic Acid and Light Signaling in Seed Germination and Plant Development, Plant Physiology, Volume 165, Issue 3, July 2014, Pages 1203–1220, https://doi.org/10.1104/pp.113.234294, published 2014) teaches in supplemental Figure 7 that ZPF10 is most expressed in the roots and hypocotyl of seedlings and then in the yet mature siliques of Arabidopsis and in Figure 8 that ABA treatments reduced the transcript levels of ZPF10.
Conclusion
No claims allowed.
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/YVETTE B. TAMUKONG/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Supervisory Patent Examiner, Art Units 1661 & 1662
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