Detailed Action
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 24-29 are newly added.
Claims 1-2, 4 and 6-29 are pending.
Claims 16-23 are withdrawn. Claims 1-2, 4, 6-15 and 24-29 are examined.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 7/11/2025 has been entered.
Claim Rejections - 35 USC § 112
Indefiniteness
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 1-2 and 4-15 remain rejected and claims 27-29 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 recites “B-type cytokinin signaling Response Regulator (RR) binding site”. Applicant has not provided a structure that is defined as a “B-type cytokinin signaling Response Regulator (RR) binding site”. This is a common designation which may change over time. Applicant provides examples of this limitation in the Specification, but does not provide a definition. It is not clear what structure or structures are required to comprise this limitation.
Claim 1 recites “NODULE INCEPTION (NIN) protein or a NIN-like protein (NLP protein)”. Applicant has not provided a structure that is defined as a “NODULE INCEPTION (NIN) protein or a NIN-like protein (NLP protein)”. This is a common designation which may change over time. Applicant provides examples of this limitation in the Specification, but does not provide a definition. It is not clear what structure or structures are required to comprise this limitation.
Claim 13 recites “one or more CYCLOPS response elements”. Applicant has not provided a structure that is defined as a CYCLOPS response element. “CYCLOPS” is a common designation which may change over time. Applicant has provided examples of CYCLOPS elements in the Specification but has not provided a definition.
Dependent claims that fail to recite additional limitations overcoming the indefiniteness are also unclear.
Applicant’s arguments regarding rejection under 35 USC 112(b)
Applicant's arguments filed 7/11/2025 have been fully considered but they are not persuasive.
Regarding Applicant’s argument in the paragraph bridging pages 9-10 of the Remarks filed 7/11/2025, that the designation changing over time is not relevant as long as the use is consistent with the ordinary and customary meaning given to the term by those of ordinary skill in the art at the time of the invention, it is not clear what is encompassed by the limitation instantly claimed without a structural requirement such as a sequence. Given the lack of clarity in the art for this term, the 5-nucleotide conserved element provided by Applicant in the Specification does not obviate the rejection because one of ordinary skill in the art would recognize that not every sequence comprising one of the embodiments of the 5-nucleotide sequence of (A/G)GAT(C/T) will necessarily be B-type cytokinin signaling Response Regulator (RR) binding site.
Regarding Applicant’s argument that Ni, referenced by Examiner in the response to arguments made by Applicant regarding the Final rejection under 35 USC 112(b), is not specifically drawn to B-type cytokinin signaling Response Regulator (RR) binding sites, this reference was presented to demonstrate the lack of structure across the genus of B-type cytokinin signaling Response Regulator (RR) sequences. Given the lack of clarity in the art for this term, the 5-nucleotide conserved element provided by Applicant in the Specification does not obviate the rejection because one of ordinary skill in the art would recognize that not every sequence comprising one of the embodiments of the 5-nucleotide sequence of (A/G)GAT(C/T) will necessarily be B-type cytokinin signaling Response Regulator (RR) binding site.
Regarding Applicant’s argument that B-type cytokinin signaling Response Regulator (RR) binding sites were shown in the art to be conserved in rice and that B-type cytokinin signaling Response Regulator (RR) binding sites have been designed synthetically, this argument has been fully considered but it is not persuasive. The finding of B-type cytokinin signaling Response Regulator (RR) binding sites to be conserved across rice plants does not obviate the rejection. While a structural similarity was found across rice plants, such a study demonstrates that there is a lack of clarity and confirmation of structure associated with B-type cytokinin signaling Response Regulator (RR) binding sites is necessary at least in other plants if not also in rice despite the study cited by Applicant given the potential diversity of B-type cytokinin signaling Response Regulator (RR) sequences.
Regarding Applicant’s argument on page 11 in paragraph two, that newly added claim 24 includes sequence limitations, it is noted that this claim has not been included in the rejection under 35 USC 112(b).
Regarding Applicant’s argument in the paragraph bridging pages 11 and 12, that newly added claim 25 recites a sequence limitation with the terms NIN and NLP, it is noted that this claim has not been included in the rejection under 35 USC 112(b); however, reciting a structure in a dependent claim does not obviate the rejection for the other claims.
Regarding Applicant’s argument in the paragraph bridging pages 12 and 13, that newly added claim 26 recites a sequence limitation with the term CYCLOPS response element, it is noted that this claim has not been included in the rejection under 35 USC 112(b); however, reciting a structure in a dependent claim does not obviate the rejection for the other claims.
Written description
The following is a quotation of the first paragraph of 35 U.S.C. 112(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.
Claims 1-2, 4 and 6-15 remain rejected and claims 24-29 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.
Due to Applicant's amendment of the claims, the rejection is modified from the rejection set forth in the Office action mailed 3/12/2025, as applied to claims 1-2, 4 and 6-15.
Applicant claims a plant comprising one or more genetic alterations that increases activity of a nodule inception (NIN) protein or a NIN-like protein (NLP) in response to cytokinin signaling compared to a wild-type (WT) plant without a genetic alteration. The plant comprises a nucleic acid encoding the NIN protein or NLP wherein the one or more alterations comprises addition of one or more cytokinin response elements (CREs) operably linked to the nucleic acid. At least one of the CREs is a B-type cytokinin signaling response regulator (RR) binding site. Claim two specifies that the genetic alteration comprises addition of at least two CREs. Claim four specifies that the CREs are within 1 to 100 nucleotides of each other. Claim five specifies that the nucleic acid is operably linked to a promoter that is operably linked to the CREs. Claim six specifies that the promoter and CREs are within 110,000 nucleotides to 100 nucleotides of each other. Claim seven specifies that the nucleic acid encodes a NIN/NLP1, NLP2-3, NLP4 or a basal NIN/NLP orthogroup protein. Claim eight specifies that the nucleic acid encoding the NIN protein or NLP is endogenous. Claim nine specifies that the nucleic acid encoding the NIN protein or NLP is heterologous. Claim 10 specifies that the promoter is endogenous. Claim 11 specifies that the promoter is heterologous. Claim 12 specifies that the cytokinin signaling or induction of the cytokinin signaling pathway in a root pericycle, endodermis, cortex or epidermis induces nodule organogenesis. Claim 13 specifies that the plant comprises one or more CYCLOPS response elements (REs) operably linked to the nucleic acid, wherein CYCLOPS expression in a root epidermis induces rhizobium infection. Claim 14 specifies that the plant is selected from a Markush group of monocot plants. Claim 15 specifies that the plant is selected from a Markush group of dicot plants. Claims 24-26 each specify a structure for the B-type cytokinin signaling RR binding site, NIN protein or NLP and CYCLOPS response element, respectively. Claim 27 specifies that the NIN or NLP activates NF-YA1. Claim 28 specifies that the B-type cytokinin signaling RR binding site is bound by a B-type RR1 polypeptide. Claim 29 specifies that the CYCLOPS response element is bound by a CYCLOPS polypeptide.
Applicant describes genetically altering M. truncatula nodule-negative (nod-) mutant, FN8113, which Applicant also refers to as “daphne-like” [0097]. Applicant describes complementing the M. truncatula nin-1 mutant to demonstrate the importance of the region upstream of the nucleic acid encoding the NIN protein [0085].
Applicant describes observation of baseline NIN or NLP activity in response to cytokinin signaling in WT ecotype Jemalong A17 comprised a 37-fold increase of NIN expression and a 116-fold increase of NFYA1 expression after synthetic cytokinin application in the form of benzylaminopurine (BAP). By comparison, NIN and NFYA1 expression levels were unchanged after BAP application in daphne-like mutant.
Applicant describes the importance of multiple regions to nodule formation by comparison of WT to multiple plants which either lack one or more regions or have had function rescued by the addition of one or more regions to a mutant plant. Applicant describes introducing the construct ProNIN5kb:NIN to Mt nin-1 roots and observed excessive thread formation however, did not observe nodule formation except in one plant out of 41; cell divisions were not induced in pericycle, endodermis and inner cortical layers [0098]. Applicant describes introduction of construct proNIN2.2kb:NIN nin-1 null mutant had root hairs with tight curls and colonies but few threads, highlighting the importance of the previously known single putative CYCLOPS/Mt IPD3 binding site located about 3kb upstream of the NIN start codon [0098]. Applicant describes nin-1 roots transformed with NIN driven by the -5kb promoter in which the putative CYCLOPS binding site was deleted (ProNIN5kb(4 cyclops):NIN) were observed as having a number of curled root hairs with a microcolony that formed an infection thread decrease from 70% to 7% (n=434). Applicant describes that this result showed that the putative CYCLOPS binding site within the NIN promoter was essential for thread formation [0099]. Applicant describes introducing three conserved regions (3C region) (~4kb) fused to upstream 5kb region (proNIN3C-5kb:NIN) to nin-1 by hairy root transformation resulted in 21 out of 26 transformed roots forming an average of 8 nodules per root [0101]. Applicant describes the CE region (~1kb) was found to have several putative cytokine response elements. Applicant describes nin-1 transformed with ProNIN¬ce-5kb:NIN resulted in 18 out of 37 transgenic roots having formed an average of 8 nodules per root which is a similar number of nodules as the WT comprises [0101].
Applicant describes transforming daphne-like with ProNIN¬ce-5kb:NIN, resulted in 15 out of 17 plants observed having an average of 7 nodules per plant [0102]. Applicant describes that daphne-like phenotype, comprising a 2.49 Mbp insertion that interferes with function of CE region comprised 37 out of 45 roots having an average of 4 nodules per root [0103]. Applicant describes testing three parts (D1, D2 and D3) of a conserved 472bp region within the CE region and observing that deletion of D1 eliminated nodules and deletion of D3 reduced the number of roots with nodules and average number of nodules per root [0104]. Applicant describes transforming daphne-like to express ProNINCE-5kb:GUS and ProNIN5kb:GUS and observing that daphne-like had NIN induced in the epidermis but not inner cell layer, indicated by a lack of nodules. Applicant describes that plants comprising ProNIN5kb:GUS only showed GUS expression in the epidermis and outer cortex and that plants expressing ProNINCE-5kb:GUS showed expression in the epidermis, outer cortex and in the pericycle, indicating that CE is necessary for NIN expression in the pericycle [0108]. Applicant describes that NIN is also required in the epidermis to induce expression in the pericycle [0109]. Applicant describes that the threshold level of NIN expression can be reached by induced expression of the 5kb promoter region which includes the putative CYCLOPS binding site [0112]. Applicant describes the CRE1 receptor in the pericycle perceives cytokinin and activates the B-type RR1, which further activates NIN expression [0113].
Applicant does not describe a genetic alteration that increases activity of a NIN protein or NLP in response to cytokinin signaling compared to a WT plant. Applicant has not demonstrated a genetic alteration increasing activity of a NIN protein other than rescuing deficiencies from mutation, i.e., wherein activity is increased compared to loss-of-function phenotypes such as daphne-like and nin-1. Applicant does not describe the plant comprising a heterologous NIN or NLP nor a heterologous promoter.
Applicant does not describe a representative number of species across the claimed genus nor a structure that, if retained, would increase NIN protein or NLP activity in response to cytokinin signaling across the broadly claimed scope of the invention. Applicant has only taught specific sequences that restore NIN protein or NLP activity in response to cytokinin signaling in Medicago truncatula daphne-like mutant and nin-1 mutant by transforming to comprise at least D1 and D2 regions of CE in addition to the 5kb promoter upstream of the NIN start codon or alternatively, by transforming to comprise the full CE region in addition to the 5kb promoter upstream of the NIN start codon. Applicant has not described a plant with an increased NIN protein or NLP activity in response to cytokinin signaling compared to a WT in any plant species. Applicant has only described a plant with increased NIN protein or NLP activity in response to cytokinin signaling in Medicago truncatula in comparison to loss-of-function mutants wherein specific sequences are comprised. The described embodiment is not representative of the breadth of the claimed genus such that any plant, nor the specified plants of claims 14 and 15, would comprise increased NIN protein or NLP activity in response to cytokinin signaling by comprising one or more of any CREs operably linked to the nucleic acid encoding the NIN protein wherein at least one CRE is RR. Applicant has not described a representative number of species nor a structure function relationship wherein only one of three structures from the list of: b-type cytokinin signaling RR binding site, NIN protein or NLP or CYCLOPS response element is limited by a sequence wherein each of the other two are not. Prior art does not resolve the deficiency of Applicant’s description. It is known in the art that NIN and NLPs are only partially functionally equivalent (Liu and Bisseling. Genes. 11(777). Page 3, paragraph 1), and in some cases they are not functionally equivalent (Liu and Bisseling. Paragraph bridging pages 9 and 10). It is known in the art that NIN and NLPs comprise differences across plant species (Liu and Bisseling. Figure 1). Given the breadth of the genera encompassed by the claims, the described species are not sufficiently representative to allow one of ordinary skill in the art to envision which members of the claimed genus possess the claimed function. Applicant has not provided a structure-function relationship beyond a Medicago truncatula comprising specific necessary sequences as discussed above comprising multiple CREs resulting only in WT-level of NIN protein or NLP activity in response to cytokinin signaling.
Therefore, Applicant has not satisfied the written description requirement.
Applicant’s arguments regarding rejection under 35 USC 112(a) for failing to comply with the written description requirement
Applicant's arguments filed 7/11/2025 have been fully considered but they are not persuasive.
Regarding Applicant’s argument in paragraph in the paragraph bridging pages 13 and 14 of the Remarks filed 7/11/2025 and the first paragraph of page 15, that the Examiner has not established a prima facie case that Applicant failed to comply with the written description requirement, this argument has been fully considered but it is not persuasive for the reasons provided in the rejection above. While Applicant has described the reduction to practice of a limited number of embodiments as addressed in the rejection above, it is not sufficient to demonstrate any member of the genera claimed would possess the claimed function given the breadth of scope of the claims.
Regarding Applicant’s argument on page 14, that 521 sequences encoding exemplary NIN and NLP proteins have been provided in the Specification, this argument has been fully considered but it is not persuasive. The identification of exemplary sequences for one of the elements in the claimed invention does not limit the scope of the claims lacking structural limitations to a scope which is adequately described by Applicant’s disclosed examples which have been reduced to practice.
Closest prior art
The prior art did not teach a genetically altered plant wherein the one or more alterations increased activity of NIN or NLP in the root pericycle cell layer in response to cytokinin signaling. While it was known in the art that expression of NIN is strongly activated during nodulation and is induced by cytokinin in legume plants (Suzaki et al. Front. Plant Sci.4(42). 2013; IDS filed 11/19/2021; page 3, left column, paragraph 2) and that cytokinin responsive elements (CREs), such as B-type response regulator (RR), were known to be within the promoter of some cytokinin-induced genes (Gamas et al. Trends in Plant Science. 22(9) 792-802. 2017; page 796, paragraph 1) and that CYCLOPS acts as a regulator to directly activate the expression of NIN, which was known to be critical for nodule organogenesis in M. truncatula (Gamas; page 793, Box 1), there is no suggestion or teaching in the prior art that would have led one of ordinary skill in the art to expect that operably linking a B-type response regulator to a promoter expressing a NIN would result in an increase of NIN protein in the root pericycle of a plant as instantly claimed.
Conclusion
Claims 1-2, 4, 6-15 and 24-29 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DAVID R BYRNES whose telephone number is (571)270-3935. The examiner can normally be reached 9:00 - 5:00 M-F.
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/DAVID R BYRNES/Examiner, Art Unit 1662