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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 3/3/2026 has been entered.
Status of Claims
Claims 1-2, 6-10, 12-18 & 43 are under examination on the merits.
Claims 19-42 are withdrawn without traverse.
The objections to claims 15 and 17 are withdrawn in light of Applicant’s amendments.
The rejection of claim 14 under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 2, 6-10 & 12-16 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 2, 6-10 & 12-18 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, for scope of enablement is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 2, 6, 8-9 & 12-16 under 35 U.S.C. 103 as being unpatentable over Lee et al (2020) Genes & Genomics. 42:1151–1162 in view of UniProtKB/TrEMBL record A0A072VL17_MEDTR and Repetti et al US 2012/0131691 A1, taken with the evidence of Ito et al (2005) Biosci. Biotechnol. Biochem. 69 (2), 382–390 and Cocking et al (2006) In Vitro Cell. Dev. Biol. - Plant. 42:74-82 is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 2, 6-10 & 16-18 under 35 U.S.C. 103 as being unpatentable over Lei et al (2019) J of Integrative Plant Biology. 61(4): 463–477 in view of UniProtKB/TrEMBL record A0A072VL17_MEDTR, taken with the evidence of Kiselev et al (2021) Molecular Biology Reports 48:2235-2241 is withdrawn in light of Applicant’s amendments.
Claim Objections
Claims 7, 8 & 17 are objected to because of the following informalities:
Claim 7 (line 7) & claim 8 (line 7): Medicago should be italicized.
Claim 17 (line 7): “in a plant” should be inserted after “ALOG domain” in keeping with line 4.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
Written Description
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.
Claims 1, 2, 6-10, 12-18 & 43 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 9/5/2025, as applied to claims 1, 2, 6-10 & 12-18. Applicant' s arguments filed 12/4/2025 have been fully considered but they are not persuasive.
Claims 1, 2, 6-10, 12-16 & 43 require a recombinant DNA molecule encoding a light sensitive short hypocotyl (LSH) protein wherein said protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 and comprises an ALOG domain, said polynucleotide segment comprises a sequences having at least 95% sequence identity to at least 200 contiguous nucleotides of SEQ ID NO: 1, 3 or 5-8 and encodes a protein comprising an ALOG domain, or said polynucleotide comprises at least 200 contiguous nucleotides of SEQ ID NO: 1, 3 or 5-8 and encodes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 comprising an ALOG domain. Claims 17-18 require at least one LSH protein having 95% sequence identity to SEQ ID NO: 2 and an ALOG domain or an LSH protein encoded by a nucleic acid sequence having at least 95% sequence identity to at least 200 contiguous nucleotides of SEQ ID NO: 1, 3, 5-8 and encoding a protein comprising an ALOG domain.
An amino acid sequence of at least 95% identity to SEQ ID NO: 2 encompasses amino acid sequences with 11 substitutions relative to the 222 amino acid-long SEQ ID NO: 2.
A polynucleotide segment having 95% sequence identity to SEQ ID NOs: 1, 5 or 7 encompasses polynucleotides with 33 substitutions in the 669 nucleotide-long SEQ ID NOs: 1 & 7 or 60 substitutions in the 1218 nucleotide-long SEQ ID NO: 5. This encompasses sequences in which every substitution results in an amino acid change, which in the case of SEQ ID NO: 1, would result in a protein with 85% sequence identity to SEQ ID NO: 2 or in the case of SEQ ID NO: 5, would result in a protein with 73% sequence identity to SEQ ID NO: 2.
A polynucleotide having 95% sequence identity to at least 200 contiguous nucleotides of SEQ ID NO: 1, 5, or 7 encompasses sequences with 10 substitutions in this contiguous sequence and unlimited substitutions outside of this sequence. Even a polynucleotide with 200 identical contiguous nucleotides would encode a fragment of only 66 amino acids, or 29% the length of SEQ ID NO: 2. Sequences in which every substitution outside of this nucleotide region results in an amino acid change and every one of the 10 substitutions results in an amino acid change could have up to 166 amino acid substitutions relative to the 222 amino-acid long SEQ ID NO: 2 (10 substitutions in the 66 amino acids encoded by the contiguous nucleotides, plus substitutions in every other position). This encompasses sequences with only 25% sequence identity to SEQ ID NO: 2, although the claims require that the polynucleotide encode a protein comprising an ALOG domain or an LSH protein.
The only species of light sensitive short hypocotyl protein described in the specification are LSH1 (SEQ ID NO: 2, encoded by SEQ ID NOs: 1, 5 & 7) and LSH2 (SEQ ID NO: 4, encoded by SEQ ID NOs: 3, 6 & 8). SEQ ID NO: 5 is the gDNA sequence while SEQ ID NO: 1 is the cDNA sequence of the M. truncatula LSH1 protein. SEQ ID NO: 7, the H. vulgare codon-optimized coding sequence, has 76.8% identity to SEQ ID NO: 1. See alignment below. Similarly, SEQ ID NO: 6 is the gDNA sequence and SEQ ID NO: 3 is the cDNA sequence of the M. truncatula LSH2 protein. SEQ ID NO: 8, the H. vulgare codon-optimized sequence, has 86% identity to SEQ ID NO: 3. The instant specification also describes polypeptide sequences comprising a conserved ALOG domain (SEQ ID NOs: 9-84), of lengths ranging from 137 amino acids (SEQ ID NO: 43) to 436 amino acids (SEQ ID NO:24). The ALOG domain polypeptide sequence with highest similarity to SEQ ID NO: 2 (SEQ ID NO: 24) has just 86.5% sequence identity. See second alignment below. Thus, the specification does not describe species over the full scope of the proteins and does not describe the full scope of the claimed nucleic acids.
US-18-320-150-7
Sequence 7, US/18320150
Publication No. US20240011045A1
GENERAL INFORMATION
APPLICANT: Cambridge Enterprise Limited (en)
TITLE OF INVENTION: PROTEINS FOR REGULATION OF SYMBIOTIC NODULE ORGAN IDENTITY (en)
FILE REFERENCE: AGOE:007US
CURRENT APPLICATION NUMBER: US/18/320,150
CURRENT FILING DATE: 2023-05-18
NUMBER OF SEQ ID NOS: 94
SEQ ID NO 7
LENGTH: 669
TYPE: DNA
FEATURE:
NAME/KEY: source
LOCATION: 1..669
QUALIFIERS: mol_type = other DNA
note = Codon optimized LSH1 protein
organism = Hordeum vulgare
Query Match 76.8%; Score 513.8; Length 669;
Best Local Similarity 85.5%;
Matches 572; Conservative 0; Mismatches 97; Indels 0; Gaps 0;
Qy 1 ATGGATTCAATTCAAGATTTTATGGACTCATGTAACTCTGACAACAGTTGCAGCCTCACC 60
||||||||||| |||||||| ||||||||||||||||| |||||||| ||||||||||||
Db 1 ATGGATTCAATCCAAGATTTCATGGACTCATGTAACTCGGACAACAGCTGCAGCCTCACC 60
Qy 61 AACAGCACCATAACTACCAACAGCAGTAACAACAACATCAGCAACGCCATAGTTGGTAGC 120
||| ||||| || ||||| ||||| ||||||||||||||||||||||| || ||| |
Db 61 AACTCAACCATTACCACCAATAGCAGCAACAACAACATCAGCAACGCCATCGTGGGTTCC 120
Qy 121 TCTTCACCTTCTGGCTCTACCACCACAAGCAGCCGCTATGAAAACCAGAAACGCCGTGAC 180
|| || || || ||| ||||| ||||||||||||||||| |||||||| ||||| |||
Db 121 TCGTCCCCGTCGGGCAGCACCACGACAAGCAGCCGCTATGAGAACCAGAAGCGCCGCGAC 180
Qy 181 TGGAACACTTTTGGCCAGTACCTCAAGAATCACCGTCCTCCTCTCTCCCTCTCCAGATGT 240
|||||||| |||||||||||||||||||||||||||||||| |||||||||||||||||
Db 181 TGGAACACGTTTGGCCAGTACCTCAAGAATCACCGTCCTCCGCTCTCCCTCTCCAGATGC 240
Qy 241 AGCGGTGCTCATGTGCTTGAATTTCTCCGGTACTTGGACCAATTTGGCAAGACAAAAGTG 300
||||||||||||||||| || || ||||||||| |||||||||||||||||||||||||
Db 241 AGCGGTGCTCATGTGCTGGAGTTCCTCCGGTACCTGGACCAATTTGGCAAGACAAAAGTC 300
Qy 301 CACACACCAATTTGTCCATTCTATGGACATCCAAACCCTCCAGCACCATGTCCATGTCCA 360
||||| ||||| || || ||||| |||||||| ||||||||||| || ||||| || ||
Db 301 CACACTCCAATCTGCCCGTTCTACGGACATCCCAACCCTCCAGCCCCCTGTCCTTGCCCC 360
Qy 361 TTAAGACAAGCTTGGGGTAGTCTTGATGCACTGATAGGTCGTTTAAGGGCAGCTTTTGAG 420
||||||||||| ||||| ||||||||||| |||||||| || || |||||||| || |||
Db 361 TTAAGACAAGCATGGGGCAGTCTTGATGCCCTGATAGGGCGGTTGAGGGCAGCGTTCGAG 420
Qy 421 GAAAATGGAGGGAAGCCAGAAACAAATCCATTTGGTGCTAGAGCTGTTAGACTTTACCTT 480
||||||||||||||||| ||||| |||||||| || ||||| || || || || |||||
Db 421 GAAAATGGAGGGAAGCCCGAAACTAATCCATTCGGCGCTAGGGCGGTCAGGCTCTACCTG 480
Qy 481 CGTGAGGTTCGTGATCTTCAATCCAAAGCAAGAGGTATTAGTTATGAGAAGAAGAAAAGG 540
|| |||||||| || ||||| ||||| || |||| |||||||| ||||||||||| |||
Db 481 CGGGAGGTTCGGGACCTTCAGTCCAAGGCCCGAGGCATTAGTTACGAGAAGAAGAAGAGG 540
Qy 541 AAACGTCCACCACAACAACAACCACAACAGCTTCAGCTTCAGCAGCAACAACCAATGCAA 600
||||| || || ||||| || || |||||||| ||||| |||||||| || || |||||
Db 541 AAACGCCCCCCGCAACAGCAGCCGCAACAGCTGCAGCTACAGCAGCAGCAGCCCATGCAG 600
Qy 601 TTGCAATTGCCTCTTCATCTTCATCATCACCATCAACATCAGCTTCCACCTCCAGGTGCA 660
||||||||||||| || ||||| || ||||| ||||||||||| ||||| ||||| ||
Db 601 CTGCAATTGCCTCTACACCTTCACCACCACCACCAACATCAGCTCCCACCGCCAGGGGCG 660
Qy 661 ACTCAATAA 669
|| |||| |
Db 661 ACGCAATGA 669
US-18-320-150-24
Sequence 24, US/18320150
Publication No. US20240011045A1
GENERAL INFORMATION
APPLICANT: Cambridge Enterprise Limited (en)
TITLE OF INVENTION: PROTEINS FOR REGULATION OF SYMBIOTIC NODULE ORGAN IDENTITY (en)
FILE REFERENCE: AGOE:007US
CURRENT APPLICATION NUMBER: US/18/320,150
CURRENT FILING DATE: 2023-05-18
NUMBER OF SEQ ID NOS: 94
SEQ ID NO 24
LENGTH: 436
TYPE: PRT
FEATURE:
NAME/KEY: source
LOCATION: 1..436
QUALIFIERS: mol_type = protein
organism = Pisum sativum
Query Match 86.5%; Score 1043.5; Length 436;
Best Local Similarity 88.8%;
Matches 198; Conservative 9; Mismatches 9; Indels 7; Gaps 4;
Qy 1 MDSIQDFMDSCNSDNSCSLTNSTITTNSSN--NNISNAIVGSSSPSGSTTTSSRYENQKR 58
|||||||||||||||||||||||||||::| |: :||:: |||||||| ||||||||||
Db 1 MDSIQDFMDSCNSDNSCSLTNSTITTNNTNNINHNNNALICSSSPSGSTATSSRYENQKR 60
Qy 59 RDWNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPAPCP 118
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 RDWNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPAPCP 120
Qy 119 CPLRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEKK 178
||||||||||||||||||||||||||||:|||||||||||||||||||||||||||||||
Db 121 CPLRQAWGSLDALIGRLRAAFEENGGKPDTNPFGARAVRLYLREVRDLQSKARGISYEKK 180
Qy 179 KRKRPPQQQPQQLQLQQQQPMQ---LQLPLHLHHHHQHQLPPP 218
|||||| || | | || ||:| :|||||| |||||||||
Db 181 KRKRPPPQQQHQ-QQQQHQPLQQLPMQLPLHL-CHHQHQLPPP 221
ARABIDOPSIS LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and rice G1/LIGHT-DEPENDENT SHORT HYPOCOTYLS (ALOG/LSH) are known in the art as plant specific transcription factors that regulate diverse processes in growth, development, and stress response (Upadhyaya et al 2025 J of Experimental Botany. 76(3): 836–850 (published 2/7/2025 after the priority date of the instant application, hereafter Upadhyaya) page 836, right column, paragraph 1 figure 4).
Upadhyaya discloses that ALOG proteins have a Domain of Unknown Function 640 or an ALOG domain (page 837, left column, paragraph 1) comprising 4 alpha-helices and a zinc-ribbon (page 837, left column, paragraph 2) a C-terminus Nuclear Localization Signal (page 837, right column, paragraph 2). Upadhyaya discloses that the ALOG domain is likely a DNA-binding domain recognizing DNA sequences in gene promoters (page 837, left column, paragraph 2). DNA interactions are mediated by contacts made by 3rd alpha helix and NLS (Upadhyaya page 845, left column, paragraph 3). Although the DNA motif bound by ALOG proteins has been identified, the molecular mode of action of ALOGs is unknown (Upadhyaya page 845, right column, paragraph 1).
Lee et al (2024) Current Biology. 34: 825–840 (published 2/26/2024 after the filing date of the instant application, hereafter Lee 2024) describes the biological function of LSH1 and LSH2 as repressors and activators of transcription (page 831, left column, paragraph 2). Lee 2024 describes LSH1 and LSH2 as having an identical ALOG domain similar to AtLSH3 and Pisum sativum SYMMETRIC PETALS 1 (page 826, left column, paragraph 2), but no other structure is described for these proteins.
The instant specification describes Light Sensitive Short Hypocotyl (LSH) proteins as transcription factors and regulators of nodule organ identity (paragraph [049]). The instant specification describes one LSH function to promote NF-YA1 and NOOT1/2 in the cortex (paragraph [050]). The instant specification describes LSH1 and LSH2 as having an ALOG domain (paragraph [0128]). LSH1 and LSH2 are expressed in roots after rhizobial inoculation but not during lateral root development (paragraph [0128]). The instant specification describes loss of function mutants (but not their sequence or the mutation that resulted in the loss of function) and that the loss of function led to an increase in lateral roots (paragraph [0130]). The instant specification describes that NF-YA1 expression can partly rescue lsh1/lsh2 mutants (paragraph [0139]). LSH1 and LSH2 play a role in infectability of cortical tissue and habitability of nodule primordium (paragraph [0132]). The instant specification discloses targets of LSH1 including CRE1, IPT1, RR19, CKX3, PIN1, STYLISH, PINOID, and NOOT1 and that LSH1 binds directly to DNA regions of cytokinin and auxin signaling and biosynthesis genes (paragraph [0144]). No structural features of an LSH protein other than an ALOG domain are described in the instant specification to provide the biological function of the LSH protein. The ALOG domain, which in SEQ ID NO: 2 extends from residue 52 to 179 (specification paragraph 15) is 127 amino acids long. This domain is longer than the 66 amino acids encoded by a nucleotide segment comprising only 200 contiguous nucleotides. Moreover, the instant specification does not describe the presence of an ALOG domain as sufficient to confer the claimed activity, and it is not apparent from the instant disclosure that all light sensitive short hypocotyl proteins encompassed by claims 1, 2, 6-10, 12-16 & 43 would have the function of promoting intercellular cortical infection, intracellular colonization, or nitrogen fixation by bacteria as taught in the instant specification for LSH1 and LSH2.
Liu (2020. The roles of the NOOT-BOP-COCH-LIKE genes in plant development and in the symbiotic organ identity (Doctoral dissertation, Université Paris-Saclay) published 2020, relevant pages 170-207 uploaded; hereafter Liu) describes a Medicago truncatula ALOG family member, Medtr1g075990, involved in negatively regulating nodulation (abstract, page 173 & page 170 paragraph 3). Liu describes that insertional mutants in this Mtalog1 gene have increased nodulation (page 184, paragraph 1-page 185, paragraph 1). Medtr1g075990 (GenBank reference AES61108.2, available 6/18/2014) encodes an ALOG protein with an amino acid sequence with 87% identity to instant SEQ ID NO: 2 (see alignment below), which is within the range of amino acid sequences encompassed by the claims, despite having an activity opposite to the activity recited in the instant claims of increasing intracellular colonization.
Score
Expect
Method
Identities
Positives
Gaps
257 bits(657)
6e-93
Compositional matrix adjust.
141/173(82%)
151/173(87%)
5/173(2%)
Query 13 SDNSCSLTNSTITTNSSNNNISNAIVGSSS---PSGSTTTSSRYENQKRRDWNTFGQYLK 69
+ N + TN+T T+ N NISN SSS P+ +T T SRYENQKRRDWNTFGQYL+
Sbjct 13 TKNIINFTNTT--TSEDNKNISNFTSSSSSAVPPATNTNTLSRYENQKRRDWNTFGQYLR 70
Query 70 NHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPAPCPCPLRQAWGSLD 129
NHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVH+ ICPF+GHPNPPAPCPCPLRQAWGSLD
Sbjct 71 NHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHSQICPFFGHPNPPAPCPCPLRQAWGSLD 130
Query 130 ALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEKKKRKR 182
ALIGRLRAAFEENGGKPE NPFGARAVRL+LREVRD QSKARGISYEKKKRKR
Sbjct 131 ALIGRLRAAFEENGGKPEDNPFGARAVRLFLREVRDSQSKARGISYEKKKRKR 183
Thus, the specification fails to make up for the lack of knowledge in the art regarding shared features of the genus of polynucleotides with the recited activity. One of skill in the art would not recognize that Applicant was in possession of the necessary common attributes or features of the genus in view of the disclosed species.
Hence, Applicant has not, in fact, described nucleic acids encoding a light sensitive short hypocotyl protein or fragment thereof over the full scope of the claims, and the specification fails to provide an adequate written description of the claimed invention. Therefore, given the lack of written description in the specification with regard to the structural and functional characteristics of the claimed compositions, Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Applicant urges that amended claims require that the at least one polynucleotide segment encoding a LSH protein comprising an ALOG domain and urges that in all cases expression produces the specific symbiotic outcomes taught by the specification (remarks page 9, paragraph 3-page 10, paragraph 2).
Applicant urges that the specification conveys possession of the claimed genus of variants and fragments because the specification teaches the common ALOG domain architecture and nodulation-regulatory functions (page 10, paragraph 5) and teaches full length sequences of LSH1 and LSH2 and genomic and codon-optimized sequences encoding LSH1 and 2 (page 11, paragraph 1). Applicant urges that the inventor possesses the ALOG centered structural genus because ALOG domain sequences across species have been provided and the functional characteristics of LSH1 and 2 have been described (page 10, paragraph 2-page 11, paragraph 1). Applicant urges that the disclosed species and variants provides a roadmap of the conserved domain and characteristic motifs to identify the structural boundaries of the invention (remarks page 12, paragraph 3-page 13, paragraph 1).
This argument is unpersuasive, because Applicant has not described amino acid sequence variants or fragments with an ALOG domain with the specific disclosed function of LSH1 and LSH2 to produce an increase in intercellular cortical infection, colonization by nitrogen-fixing bacteria, or increase in nitrogen-fixation by bacteria. A nucleotide sequence having at least 95% sequence identity to at least 200 contiguous nucleotides of SEQ ID NO: 1, 3, 5-8 and encoding a protein comprising an ALOG domain encompasses LSH proteins with as little as 29% sequence identity to SEQ ID NO: 2. The ALOG domain polypeptide sequence disclosed in the specification with the highest similarity to SEQ ID NO: 2 (SEQ ID NO 24) has just 86.5% sequence identity to SEQ ID NO: 2. Applicant has not described variants and fragments of LSH proteins encompassed by the claims that provide the claimed function, other than SEQ ID NO: 2 and 4.
The art teaches that ALOG/LSH proteins in Arabidopsis and rice regulate diverse processes in growth, development, and stress response (Upadhyaya et al (2025) J of Experimental Botany. 76(3): 836-850 page 836, right column, paragraph 1, figure 4). Given the teachings of the art, one of skill in the art would not understand that the ALOG/LSH proteins described in the instant specification show possession of proteins that are involved in the specific function of the invention rather than other processes in growth, development, and stress response. Thus, one of skill in the art would not recognize that Applicant was in possession of the full scope of the genus of claimed LSH proteins at the time of filing of the instant application.
Applicant urges that the description of “high stringency” for hybridization rejection is moot (Remarks page 12, paragraph 2).
This argument is unpersuasive to the current rejection, in which hybridization requirements are not addressed.
Applicant urges that tissue functional promoter and temporal expression limitations track the specification (Remarks page 13, paragraph 2-3).
This argument is unpersuasive because Applicant has not described which of the disclosed homologs LSH1/LSH2 with an ALOG domain would be functional in the root cortex, so it is not apparent from the instant specification that Applicant was in possession of the genus of LSH proteins that, when expressed in the root cortex, would provide the recited phenotype. In addition, a heterologous promoter functional in the root cortex linked to a polynucleotide encoding LSH1/LSH2 constitutes New Matter. See rejection below.
New Matter
Claims 1-2, 6-10, 12-16 & 43 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. This is a New Matter rejection.
A. Claims 1-16 & 43 require a promoter functional in the root cortex of a plant (claim 1, line 2) and that expression of the polynucleotide varies over a 24-hour period under symbiotic conditions (line 19).
The instant specification describes that LSH1 proteins promote cortex-specific expression (paragraph [0050]) and describes how LSH proteins provide cortical activation of NF-YA1 (paragraphs [0010, 0138-1039]). The specification describes a polynucleotide encoding an LSH protein may exhibit varying levels of expression over time (paragraph [088]), although the relation between this variation in expression and symbiotic conditions is not described. Thus, the subject matter of the amended claims, particularly the limitation of a promoter functional in the root cortex of a plant operably linked to a polynucleotide encoding an LSH protein, is not supported in the original disclosure and constitutes New Matter. In response to this rejection, Applicant is required to point specifically to support for the concept in the specification or to cancel the New Matter.
Applicant urges that supporting material is found in the specification in paragraphs [0050-51, 0030, 0032, 0037], figure 21 and the original claims (Remarks, page 8, paragraph 1).
This argument is unpersuasive, because, paragraphs [0030, 0032, and 0037], figure 21, and the original claims do not mention the root cortex or a promoter functional in those cells, and paragraphs [0050-0051] disclose that cortex-specific promotion is a function of LSH1/LSH2. This is distinct from a polynucleotide encoding LSH1/LSH2 operably linked to a promoter functional in the root cortex of a plant, required in amended claims. In addition, paragraphs [0050-51, 0030, 0032, 0037], figure 21 and the original claims do not disclose expression of the claimed polynucleotide segment varies over a 24-hour period under symbiotic conditions.
Claim 43 requires that the gene expression pattern of the heterologous promoter functional in the root cortex of a plant is constitutive, temporal, spatial, developmental, tissue-specific, cell cycle-specific, or chemically responsive expression pattern.
The instant specification describes that regulatory elements characterized by their gene expression pattern may be constitutive, temporally or spatially specific, or responsive to different factors (paragraph [0105]). The specification also describes regulatory elements (SEQ ID NOs: 84-93). The originally field specification does not describe DNA molecules comprising a promoter, specifically, over the recited expression patterns operably linked to at least one polynucleotide segment encoding a LSH protein.
Thus, the limitations of claim 43 represent New Matter. In response to this rejection, Applicant is required to point specifically to support for the concept in the specification or to cancel the New Matter.
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.
Claim(s) 17 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Liu (2020). The roles of the NOOT-BOP-COCH-LIKE genes in plant development and in the symbiotic organ identity (Doctoral dissertation, Université Paris-Saclay). (published 2020, relevant pages 170-207 uploaded, hereafter Liu) taken with the evidence of GenBank reference KEH42512.1 (available 6/18/2014).
Liu discloses a Medicago truncatula ALOG family member involved in negatively regulating nodulation (abstract, page 173). This gene named MtALOG1 (Medtr1g075990) is a close relative to AtLSH4 (page 179, paragraph 3). Liu discloses an additional Medicago gene, MtALOG2 which is Medtr1g069825 and similar to LSH3 (figure 1a), and which is expressed in nodules and aerial parts at low levels (page 180, paragraph 1, figure 1d). Liu discloses that an ALOG gene has a positive role in regulating nodulation in Lotus japonicus (page 178, paragraph 3; page 198, paragraph 2).
Liu discloses that overexpression of Arabidopsis LSH4 and LSH3 induces extra flower differentiation within a flower (page 198, paragraph 2). Liu discloses a method of overexpression of MtALOG1 by transforming plants with the MtALOG1 sequence under the control of the 35S-CaMV promoter (page 203, paragraph 2).
Liu discloses inoculations of Medicago plants with Sinorhizobium medicae strain WSM419 (page 203, paragraph 3). Liu discloses growing M. truncatula plants alongside wildtype plants in a growth chamber on either soil, Buffered Nodulation Media, or perlite and sand, and that the sand-perlite mixture was watered with 1g/L N-free nutritive solution (page 201, paragraph 2). Liu discloses crossing M. truncatula lines (page 201, paragraph 3-page 202, paragraph 1).
GenBank reference KEH42512.1 provides evidence that the Medtr1g069825 gene that Liu discloses is expressed in nodules encodes a protein with an ALOG domain and that comprises a sequence with 100% identity to instant SEQ ID NO: 2. See alignment below.
Score
Expect
Method
Identities
Positives
Gaps
461 bits(1186)
7e-173
Compositional matrix adjust.
222/222(100%)
222/222(100%)
0/222(0%)
Query 1 MDSIQDFMDSCNSDNSCSLTNSTITTNSSNNNISNAIVGSSSPSGSTTTSSRYENQKRRD 60
MDSIQDFMDSCNSDNSCSLTNSTITTNSSNNNISNAIVGSSSPSGSTTTSSRYENQKRRD
Sbjct 1 MDSIQDFMDSCNSDNSCSLTNSTITTNSSNNNISNAIVGSSSPSGSTTTSSRYENQKRRD 60
Query 61 WNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPAPCPCP 120
WNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPAPCPCP
Sbjct 61 WNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPAPCPCP 120
Query 121 LRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEKKKR 180
LRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEKKKR
Sbjct 121 LRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEKKKR 180
Query 181 KRPPQQQPQQLQLQQQQPMQLQLPLHLHHHHQHQLPPPGATQ 222
KRPPQQQPQQLQLQQQQPMQLQLPLHLHHHHQHQLPPPGATQ
Sbjct 181 KRPPQQQPQQLQLQQQQPMQLQLPLHLHHHHQHQLPPPGATQ 222
Because MtALOG2 is expressed in nodules, Liu’s method of growing wildtype plants and inoculating with Sinorhizobium medicae anticipates a method of expressing an LSH protein having at least 95% sequence identity to SEQ ID NO: 2 and comprising an ALOG domain in a plant and contacting said plant with an effective amount of rhizobia bacterium (instant claim 17).
Claim Rejections - 35 USC § 103
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 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.
Claim(s) 1, 2, 6-10, 12-17 & 43 are rejected under 35 U.S.C. 103 as being unpatentable over Kwok et al US 8344210 B2 (patented 1/1/2013, hereafter Kwok) in view of Liu (2020). The roles of the NOOT-BOP-COCH-LIKE genes in plant development and in the symbiotic organ identity (Doctoral dissertation, Université Paris-Saclay) (published 2020, relevant pages 170-207 uploaded, hereafter Liu), GenBank reference KEH42512.1 (available 6/18/2014), and Repetti et al US 2012/0131691 A1 (published 5/24/2012, hereafter Repetti), and taken with the evidence of Ito et al (2005) Biosci. Biotechnol. Biochem. 69 (2), 382–390 (available online 5/22/2014, hereafter Ito), Cocking et al (2006) In Vitro Cell. Dev. Biol. - Plant. 42:74-82 (published Jan-Feb 2006, hereafter Cocking), and Walker et al (2017) Plant Cell. 29(10): 2393-2412 (published October 2017, hereafter Walker).
Due to Applicant' s amendment of the claims, this is a new rejection. Applicant' s arguments filed 12/4/2025 have been fully considered as they pertain to the new rejection, but they are not persuasive.
Claims 1, 2, 6-10, 12-17 & 43 are drawn to a plant cell comprising a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide segment encoding a LSH protein, plants comprising said molecule, a method of producing progeny seed comprising the recombinant DNA molecule, and a method of using a plant expressing such a DNA molecule.
Kwok teaches a motivation to introduce exogenous nucleic acids into plants to confer traits that reduce response to low light, because typical plant response to low light conditions negatively affects crop yield (column, 1, lines 24-48).
Kwok teaches Ceres ANNOT ID no. 828846 as a DNA clone from Arabidopsis thaliana predicted to encode an amino acid polypeptide designated LSH3 (column 37, lines 58-61). This amino acid sequence (Kwok SEQ ID NO: 110) has 82% sequence identity to instant SEQ ID NO: 2. See alignment below.
Score
Expect
Method
Identities
Positives
Gaps
256 bits(655)
5e-92
Compositional matrix adjust.
142/188(76%)
156/188(82%)
8/188(4%)
Query 1 MDSIQDFMDSCNSDNSCSLTNSTITTNSSNNNISNAIVGSSS-----PSGSTTTSSRYEN 55
MD I M+ S +TN I +N+S++ ++ A G ++ S + SSRYEN
Sbjct 1 MDMIPQLMEG--SSAYGGVTNLNIISNNSSS-VTGATGGEATQPLSSSSSPSANSSRYEN 57
Query 56 QKRRDWNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPA 115
QKRRDWNTFGQYL+NHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHT IC FYGHPNPPA
Sbjct 58 QKRRDWNTFGQYLRNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTNICHFYGHPNPPA 117
Query 116 PCPCPLRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISY 175
PCPCPLRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRD+QSKARG+SY
Sbjct 118 PCPCPLRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDMQSKARGVSY 177
Query 176 EKKKRKRP 183
EKKKRKRP
Sbjct 178 EKKKRKRP 185
Kwok teaches constructs comprising Ceres ANNOT ID no. 828846 operably linked to a CaMV 35S promoter and transformed into wild-type Arabidopsis thaliana plants (column 39, lines 10-52). Presence of the vector was confirmed in a transgenic Arabidopsis line by herbicide resistance, PCR amplification, or sequencing (column 40, lines 40-47). Transformed plants had short hypocotyl phenotype under low light conditions (column 45, lines 1-17). Kwok teaches T2 and T3 seedlings from a transformation event with this DNA clone (column 45, lines 6-9).
Kwok teaches a method of assaying for light response by growing transformangs with 16:8 hour light:dark cycles (column 27, lines 32-46).
Kwok envisions the plant being a member of the genus Medicago (column 2, lines 40-47) and teaches a polypeptide from Medicago truncatula that is homologous to another sequence involved in low light response (table 34), although no Medicago homolog to Kwok SEQ ID NO: 110 is taught.
Kwok does not teach an LSH protein with 95% sequence identity to SEQ ID NO: 2, that expression of the polynucleotide segment varies over a 24-hour period including wherein expression is increased during the first 6 or 12 hours of a 12 hour/12 hour light/dark cycle, or the method of producing progeny seed comprising the recombinant DNA molecule explicitly.
Liu teaches a Medicago gene, MtALOG2, Medtr1g069825, which is similar to Arabidopsis LSH3 (figure 1a), and which is expressed in nodules and aerial parts at low levels (page 180, paragraph 1, figure 1d). Liu teaches methods for cultivating Medicago, including growing M. truncatula plants alongside wildtype plants in a growth chamber on either soil, Buffered Nodulation Media, or perlite and sand and inoculating Medicago plants with Sinorhizobium medicae strain WSM419 (page 201, paragraph 2, page 203, paragraph 3). Liu teaches transformation of Medicago (page 203, paragraph 2).
GenBank reference KEH42512.1 teaches that the Medtr1g069825 gene encodes a protein with an ALOG domain and that comprises a sequence with 100% identity to instant SEQ ID NO: 2. See alignment below.
Score
Expect
Method
Identities
Positives
Gaps
461 bits(1186)
7e-173
Compositional matrix adjust.
222/222(100%)
222/222(100%)
0/222(0%)
Query 1 MDSIQDFMDSCNSDNSCSLTNSTITTNSSNNNISNAIVGSSSPSGSTTTSSRYENQKRRD 60
MDSIQDFMDSCNSDNSCSLTNSTITTNSSNNNISNAIVGSSSPSGSTTTSSRYENQKRRD
Sbjct 1 MDSIQDFMDSCNSDNSCSLTNSTITTNSSNNNISNAIVGSSSPSGSTTTSSRYENQKRRD 60
Query 61 WNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPAPCPCP 120
WNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPAPCPCP
Sbjct 61 WNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPAPCPCP 120
Query 121 LRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEKKKR 180
LRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEKKKR
Sbjct 121 LRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEKKKR 180
Query 181 KRPPQQQPQQLQLQQQQPMQLQLPLHLHHHHQHQLPPPGATQ 222
KRPPQQQPQQLQLQQQQPMQLQLPLHLHHHHQHQLPPPGATQ
Sbjct 181 KRPPQQQPQQLQLQQQQPMQLQLPLHLHHHHQHQLPPPGATQ 222
Repetti teaches APRR9 as a light-inducible promoter (paragraph [0081]) and teaches the sequence of the Arabidopsis APRR9 promoter (Repetti SEQ ID NO: 6). Repetti teaches transformation of Arabidopsis with constructs driven by light-inducible promoters (paragraphs [0091-0095]). Repetti teaches that crop species such as soybean expressing polypeptides of interest may have improved traits when a sequence encoding the polypeptide is placed under regulatory control of light-responsive promoters (paragraph [0098]). Repetti teaches that a light-inducible promoter can regulate and optimize overexpression of genes in plants to confer improved traits with reduced or no impact on yield, appearance, quality or fitness compared to plant constitutively overexpressing the polypeptides (paragraph [003-005]).
Ito provides evidence that the promoter of APRR9 in Arabidopsis has diurnal oscillation and is rapidly induced by light. When grown under 12 light/12h dark cycles, APRR9 expression increased sharply in the morning (page 384, left column, paragraph 4-right column, paragraph 1), and was highest in the first 6 hours in the light (fig 1A).
Walker provides evidence that expression of the circadian clock gene Pseudo-response regulator9 (PRR9) in Arabidopsis is invariant between cell types in the roots, which includes the cortex (page 2396, left column, paragraph 3).
Cocking provides evidence that roots of Arabidopsis thaliana can undergo intracellular colonization (figure 1) by the nitrogen fixing bacterium Gluconacetobacter diazotrophicus (page 74, left column, paragraph 1).
Before the filing date of the instant application, it would have been obvious to one of ordinary skill in the art to substitute the CaMV 35S promoter used by Kwok for the light inducible APRR9 promoter taught by Repetti. One would have been motivated to use a light-inducible promoter instead of a constitutive promoter in order to optimize expression in a transgenic plant. One would have had reasonable expectation of success, because APRR9 in an endogenous Arabidopsis promoter.
Before the filing date of the instant application, it would also have been obvious to one of ordinary skill in the art to transform a Medicago plant rather than an Arabidopsis plant. One of ordinary skill in the art would have been motivated to use a Medicago plant, because this was suggested by Kwok. One of ordinary skill in the art would have had reasonable expectation of success, because Medicago truncatula had been successfully transformed prior to the instant filing.
It would have been obvious to one of ordinary skill in the art to substitute the Medicago homolog of GenBank reference KEH42512.1 with 100% identity to instant SEQ ID NO: 2 for the Arabidopsis LSH3 gene. One of ordinary skill in the art would have been motivated to substitute the Medicago endogenous homolog for transformation of a Medicago plant, or even an Arabidopsis plant, through design choice. One of ordinary skill would have had reasonable expectation of success, because a Medicago gene homologous to Arabidopsis LSH3 was known in the art and Medicago homologs of other proteins involved in light response were taught by Kwok.
In this way, a Medicago truncatula, or possibly an Arabidopsis, plant carrying the Medicago ALOG gene of encoding GenBank reference KEH42512.1 operably linked to the APRR9 promoter of Repetti and grown with light/dark cycles makes obvious the transgenic plant cell of instant claim 1, wherein expression varies over a 24-hour period and wherein the heterologous polynucleotide is linked to a promoter functional in the root cortex of a plant.
That the expression of said polynucleotide segment increases intercellular cortical infection, an increase in intracellular colonization by nitrogen-fixing bacteria, or an increase in nitrogen fixation by bacteria is an inherent result of expression of the polynucleotide segment in the recited cell types. Because both Arabidopsis and Medicago form symbiotic relationships, expression increasing nitrogen-fixation by bacteria under symbiotic conditions and expression varying under symbiotic conditions would inherently result from the transformed plants.
The transgenic Arabidopsis, a dicot, also makes obvious instant claims 6, 8, & 9, to a dicotyledonous plant cell, a plant comprising the transgenic plant cell, and a dicot plant respectively. The transgenic Medicago plant makes obvious a Medicago ssp. plant or plant cell of claims 7 & 10. Kwok’s T2 and T3 seedlings make obvious the existence of transgenic seed (claim 14).
The method of Kwok makes obvious instant claim 2, wherein during at least the process of transformation the plant cell comprises a plasmid operably linked to the recombinant DNA molecule.
Under an APRR9 promoter, the LSH protein would increase during the first 6 or 12 hours of a 12 hour/12 hour light/dark cycle (instant claims 12-13). These claims recite a result of growing the claimed plant under a 12 hour/12 hour light/dark cycle but because they are drawn to the plant and not a method, it is not necessary for the plants to be grown under such a cycle for the limitation to be met. This variation reads on expression varying over a 24-hour period. Arabidopsis PRR9 is also a circadian clock gene, and so its promoter reads on one with an expression pattern that is temporal (claim 43).
An Arabidopsis or Medicago plant expressing a protein identical to SEQ ID NO: 2 would inherently be susceptible to intercellular cortical infection or intracellular colonization by nitrogen fixing bacteria (instant claim 16), because Arabidopsis and/or Medicago can be colonized by nitrogen-fixing bacteria which reads on susceptible to intracellular colonization by nitrogen-fixing bacteria.
Before the filing date of the instant application, the method of producing progeny seed of claim 15 would have been obvious to one of ordinary skill in the art. The existence of homozygous T3 seed inherently teaches that a T0 transgenic seed was planted, the plant was grown, and progeny seed comprising the recombinant DNA molecule was harvested from the plant, and that this process was repeated twice.
Finally, it would have been obvious to cultivate transgenic Medicago truncatula expressing the low-light responsive protein as taught by Kwok with the method of cultivating Medicago truncatula of Liu, comprising contacting the plant with an amount of Sinorhizobium bacteria to form nodules. This reads on the method of claim 17 comprising expressing at least one LSH protein having SEQ ID NO: 2 in a plant and contacting said plant with an effective amount of one or more rhizobia bacterium.
Applicant urges that Lee teaches an LSH protein with an ALOG domain which is approximately 82% similar to SEQ ID NO: 2 and so is outside the amended scope of the claims (Remarks, page 17, paragraph 3). Applicant urges that the UniProt record of an identical M. truncatula ALOG/LSH1 protein is merely a sequence record and does not teach or motivate to place this protein under a root cortex-functional diurnal promoter in Arabidopsis or another plant, so Applicant urges that substitution depends on hindsight reasoning (Remarks, page 17, paragraph 5-page 18 paragraph 1).
This argument is unpersuasive, because one cannot show non-obviousness by attacking references individually where, as here, the rejections are based on combinations of references. In re Keller, 208 USPQ 871 (CCPA 1981). Additionally, “[a]ny judgement on obviousness is in a sense necessarily a reconstruction based on hindsight reasoning, but so long as it takes into account only knowledge which was within the level of ordinary skill in the art at the time the claimed invention was made and does not include knowledge gleaned only from applicant’s disclosure, such a reconstruction is proper.” In re McLaughlin 443 F.2d 1392, 1395, 170 USPQ 209, 212 (CCPA 1971).
Applicant urges that Repetti generally describes light-inducible promoters but Ito and Repetti do not demonstrate that APRR9 promoter is functional in the root cortex (Remarks, page 17, paragraph 4).
This argument is unpersuasive, because the APRR9 promoter is functional in the root cortex.
Applicant urges that Cocking does not teach overexpression of LSH1/LSH2 in the root cortex. Applicant urges that the results recited by the claims are not inherent or inevitable but the result of particular spatial and temporal expression of LSH proteins. Applicant urges that the rationale for substituting the CaMV 35S promoter with an APRR9 promoter does not address the claim-critical tissue requirement or show reasonable expectation of success (Remarks, page 18, paragraph 2).
This argument is unpersuasive, because the motivation to combine teachings in the art does not have to be Applicant's own in order to be obvious. If it would have been obvious to express a known protein under control of a known promoter which happens to have the spatial and temporal expression required for the claimed results, then the results would be inherent to an obvious combination of features known in the art. One of ordinary skill in the art would have had reasonable expectation of success in combining the known protein encoding gene with the known protein, because the promoter of Repetti was suggested for introducing a transgene into a legume plant. One of ordinary skill in the art would not have needed to know whether or not the expression of the ALOG protein would result in improved infection, intracellular colonization, or nitrogen fixation for the combination to have been obvious, because achieving those recited results would not have been the motivation to combine the provided references.
Furthermore, Applicant’s remark that the recited phenotypes result from particular spatial and temporal expression of LSH proteins is not commensurate with the evidence provided by the filed disclosure. The specification describes that levels of expression of the LSH protein may vary over time (paragraph [088]); however, examples provided by the specification teach introducing the Medicago LSH1 and LSH2 genes into barley driven by the rice ubiquitin promoter (paragraph [0153]), overexpression of an LSH1 protein under a ubiquitin promoter in Medicago, which led to altered rhizobial infection patterns (paragraph [0145]), and that expression of NF-YA1 under a constitutive UBI promoter was sufficient to result in a partial rescue of lsh1/lsh2 mutants (paragraph [0140]). If constitutive expression of the LSH protein or related proteins is sufficient for embodiments of the instant invention, one of ordinary skill in the art would not conclude that a root cortex-functional diurnal promoter, specifically, is required for the phenotypes recited by the instant invention.
Applicant urges that the amended claims are supported by unexpected technical effects, because overexpression of LSH1 alters root architecture, modifies lateral root primordia, and alters rhizobial infection patterns, which are phenotypes not suggested by Lee, Repetti, Ito, Cocking or the UniProt entry. Applicant urges that the claimed configuration provides an inventive technical effect directly tied to the claimed limitations (Remarks, page 18, paragraph 4-page 19, paragraph 1).
This argument is unpersuasive, because the claims do not require the technical effects of altered root architecture and lateral root primordia development. In addition, the art teaches a motivation to express an LSH protein in a plant to generate low-light tolerant plants and also a motivation to use a light-inducible promoter, including one capable of driving diurnally variable expression in roots, for expression of transgenes. The motivation to combine prior art elements does not need to be Applicant’s own for the combination to be obvious.
Even if expression of a protein with the sequence of instant SEQ ID NO: 2 under the specifically recited spatiotemporal limitations is required for the recited results, and there is not evidence in the specification that it is, see response to Remarks above, expression driven by the known PRR9 promoter would result in root cortex diurnally varying expression even if one of ordinary skill in the art was not aware of the importance of this expression pattern to colonization and nitrogen fixation.
Claim(s) 18 is rejected under 35 U.S.C. 103 as being unpatentable over Kwok, Liu, GenBank reference KEH42512.1, and Repetti as applied to claims 1, 2, 6-10, 12-17 & 43 above, and further in view of Jones et al (2008) PNAS. 105(2): 704-709 (published 1/15/2008, hereafter Jones).
This is a new rejection.
Claim 18 is drawn to a method wherein a plant is contacted with an effective amount of a Sinorhizobium meliloti bacteria.
The teachings of Kwok, Liu, GenBank reference KEH42512.1, and Repetti are presented above. They do not teach contacting a plant with Sinorhizobium meliloti.
Jones teaches that Medicago truncatula and Medicago sativa have a rhizobial symbiotic partner Sinorhizobium meliloti (page 704, left column, paragraph 1). Jones teaches a method wherein M. truncatula plants were grown and then roots were inoculated with S. meliloti (page 709, left column, paragraph 3).
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to substitute Sinorhizobium medicae with S. meliloti in inoculation of Medicago truncatula. One of ordinary skill in the art would have been motivated to inoculate with S. meliloti, because it is a known rhizobial symbiotic partner of Medicago. One of ordinary skill in the art would have had reasonable expectation of success, because inoculation of Medicago with S. meliloti, which reads on contacting a plant with an effective amount, was known and practiced in the art prior to the filing of the instant application, and both bacteria were known symbionts of Medicago truncatula.
Thus, claims , 2, 6-10, 12-18 & 43 would have been obvious over Kwok, Liu, GenBank reference KEH42512.1, Repetti, and Jones.
Claims 1, 2, 6-10, 16-18 & 43 are rejected under 35 U.S.C. 103 as being unpatentable over Lei et al (2019) J of Integrative Plant Biology. 61(4): 463–477 (available 8/21/2018, hereafter Lei; appended PDF includes the supplement) in view of UniProtKB/TrEMBL record A0A072VL17_MEDTR (first available 10/1/2014), taken with the evidence of Kiselev et al (2021) Molecular Biology Reports 48:2235-2241 (published 2/25/2021, hereafter Kiselev) and Battraw et al (1990) Plant Molecular Biology. 15: 527-538. (published October 1990, hereafter Battraw).
This is a new rejection in light of Applicant’s amendments. Applicant’s remarks filed 12/4/2025 have been considered below as they pertain to this rejection, but they are not persuasive.
Claims 1, 2, 6-10, 16-18 & 43 are drawn to a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide segment encoding a LSH protein or fragment thereof, plants comprising said molecule, and a method of increasing intercellular cortical infection or intracellular colonization by nitrogen-fixing bacteria in a plant comprising expressing a LSH protein.
Lei teaches an overexpression plasmid with a pCAMBIA 1302 backbone comprising an LjALOG1 cDNA molecule fused to red florescence protein driven by CaMV 35S promoter (page 473, right column, paragraph 4). Lei teaches transformation of L. japonicus seedlings via Agrobacterium rhizogenes inoculation (page 473, right column, paragraph 5-page 474, left column, paragraph 1). Overexpression of LjALOG1 in transgenic L. japonicus increased nodule number (page 468, right column, paragraph 2).
Lei teaches that the sequence of the L. japonicus ALOG1 gene was from the Lj5g3v1083950.1 record found on the miyakogusa.jp 3.0 release (figure 1; page 473, left column, paragraph 4). Lei teaches the sequence of the LjALOG1 protein (Figure S1), which has 84% sequence identity to SEQ ID NO: 2. See alignment below.
Range 1: 1 to 207GraphicsNext MatchPrevious Match
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
313 bits(802)
3e-114
Compositional matrix adjust.
188/224(84%)
198/224(88%)
20/224(8%)
Query 1 MDSIQDFMDSCNS-DNS-CSLTNSTITTNSSNNNISNAIVGSSSPSGS-TTTSSRYENQK 57
MDSIQ+FM+SCNS DN+ CSL+N+T +NS IVG SSPS S TTTSSRYENQK
Sbjct 1 MDSIQEFMESCNSHDNTNCSLSNTT--SNS-------LIVGGSSPSASVTTTSSRYENQK 51
Query 58 RRDWNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPICPFYGHPNPPAPC 117
RRDWNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTP+CPFYGHPNPPAPC
Sbjct 52 RRDWNTFGQYLKNHRPPLSLSRCSGAHVLEFLRYLDQFGKTKVHTPMCPFYGHPNPPAPC 111
Query 118 PCPLRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEK 177
PCPLRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEK
Sbjct 112 PCPLRQAWGSLDALIGRLRAAFEENGGKPETNPFGARAVRLYLREVRDLQSKARGISYEK 171
Query 178 KKRKRPPQQQPQQLQLQQQQPMQLQLPLHLHHHHQHQLPPPGAT 221
KKRKRP PQQ Q QQQQP+Q+ LP HHHHQ QLPPPGA+
Sbjct 172 KKRKRP---HPQQ-QQQQQQPLQV-LP---HHHHQLQLPPPGAS 207
Lei teaches nodulation experiments comprising inoculating plants with Mesorhizobium loti strain MAFF303099 (page 473, left column, paragraph 2). Lei teaches that M. loti are rhizobia capable of infecting cortical cells (page 467, left column). Lei teaches increased expression of LjALOG1 after inoculation with M. loti in wild type plants (page 467, right column, paragraph 1; figure 6b) and also teaches inoculating lotus hair roots with M. loti after transforming with an overexpression construct (page 473, right column, paragraph 5-page 474, left column, paragraph 1). LjALOG1 overexpression increased nodule formation (figure 9).
Lei does not teach an LSH protein with 95% sequence identity to SEQ ID NO: 2, that expression of the polynucleotide segment varies over a 24-hour period including wherein expression varies throughout the day, that the heterologous promoter is functional in the root cortex, or the method of producing progeny seed comprising the recombinant DNA molecule.
UniProtKB/TrEMBL record A0A072VL17_MEDTR teaches an ALOG domain containing protein from Medicago truncatula with 100% sequence identity to SEQ ID NO: 2. See alignment above.
Kiselev provides evidence that expression of 35S-driven genes varied over a 24-hour period, with highest expression at night (page 2238, left column, paragraph 2).
Battraw provides evidence that CaMV 35S is active in root cortex (abstract, page 531, right column, paragraph 2, figure 7b, page 537, left column, paragraph 2) but only marginally active or absent in the root epidermis (figure 7e).
Before the filing date of the instant application, one of ordinary skill in the art would have been motivated to modify the plants and methods of Lei to substitute a polynucleotide encoding the Medicago truncatula ALOG protein of UniProtKB/TrEMBL record A0A072VL17_MEDTR for the gene encoding the lotus ALOG protein. One of ordinary skill in the art would have been motivated to substitute one ALOG protein-encoding sequence from a legume plant for another as a design choice. One of ordinary skill in the art would have had reasonable expectation of success, because both genes come from legumes and the proteins have sequence similarity > 80%.
Under control of a 35S promoter, the expression of the protein would vary over a 24-hour period and be present in cortex cells of the root. That the expression of said polynucleotide segment increases intercellular cortical infection, an increase in intracellular colonization by nitrogen-fixing bacteria, or an increase in nitrogen fixation by bacteria is an inherent result of expression of the polynucleotide segment in the recited tissues during symbiotic conditions. Lotus plants are known to form symbiotic conditions.
Thus, a transgenic lotus plant carrying a Medicago ALOG-encoding gene reads on a transgenic plant cell comprising a heterologous promoter operably linked to a polynucleotide segment encoding a LSH protein wherein expression varies over a 24-hour period (claim 1), including a dicotyledonous cell or lotus cell (instant claims 6-7), and a plant comprising the cell, including a dicot or lotus plant or a plant susceptible to intercellular cortical infection (claims 8-10 & 16). Because expression of the 35S promoter is active in root cortex and vascular cylinder but only marginally in the root epidermis, this expression pattern reads on a spatial or tissue-specific expression pattern (claim 43).
The method of transformation via Agrobacterium rhizogenes inoculation makes obvious the transgenic plant cell comprising, for some period of time, a plasmid operably linked to the recombinant DNA molecule (claim 2).
The transformed hairy roots carrying an overexpression construct for an ALOG gene inoculated with M. loti reads on the method of instant claims 17-18.
Thus, claims 1, 2, 6-10, 16-18 & 43 are obvious over Lei and UniProtKB/TrEMBL record A0A072VL17_MEDTR taken with the evidence of Kiselev and Battraw.
Applicant urges that the combination of references does not meet the limitations of a heterologous promoter functional in the root cortex and diurnal variation of expression or methods of expressing an LSH protein and contacting the plant with rhizobia or AMF (Remarks, page 19, paragraph 4).
This argument is not persuasive, because diurnal fluctuations and spatial expression of CaMV 35S expression meet the limitations of the claim language, and CaMV 35S is used as a promoter for plant transgenic expression, as evidenced by the cited references. Similarly, the substitution of a homologous ALOG protein from another leguminous plant species that also forms rhizobial nodules would have been an obvious homologous substitution to one of ordinary skill in the art.
Applicant urges that Lei does not teach the specific and mechanistic outcomes claimed arising from spatiotemporally controlled LSH expression in the root cortex (Remarks, page 20, paragraph 1).
This argument is unpersuasive, because the promoter taught by Lei does provide expression that can vary over 24 hours and is found in the root cortex. One cannot show non-obviousness by attacking references individually where, as here, the rejections are based on combinations of references. In re Keller, 208 USPQ 871 (CCPA 1981). Diurnally variable expression of a protein identical to SEQ ID NO: 2 (UniProtKB/TrEMBL A0A07VL17_MEDTR) in the root cortex would inherently result in the specific outcome claimed.
Additionally, Applicant’s remark that the recited phenotypes result from particular spatial and temporal expression of LSH proteins is not commensurate with the evidence provided by the filed disclosure. See response to Remarks above.
Applicant urges that there is no teaching to substitute Lotus ALOG with Medicago LSH1 in a manner that would satisfy the current claims and substitution is not supported by an articulated rationale beyond general homology (Remarks, page 20, paragraph 2).
This argument is unpersuasive, because the substitution of homologous proteins for expression of heterologous genes in plants has been routine and well established in the field prior to the filing of the instant application. The substitution of one protein for another homologous protein of another species would be based on the rationale of design choice.
Applicant urges that Kiselev does not address the claim-critical requirement that the promoter be functional in the root cortex or that diurnal expression be harnessed in that tissue to drive LSH1/LSH2 in a way that produces increases in intercellular cortical infection, intracellular colonization or nitrogen fixation. Applicant urges that incidental fluctuation does not meet the purposeful spatiotemporal control required by the claims or establish reasonable expectation that substituting SEQ ID NO: 2 into Lei’s system would yield the phenotypes recited (Remarks, page 20, paragraph 3 -page 21, paragraph 1).
This argument is unpersuasive, because the intentionality of combining elements is not a patentable limitation. The fact that Applicant recites temporally varying expression for a different purpose does not alter the conclusion that the use of a promoter with temporally varying expression would be prima facie obvious from the purpose disclosed in the reference. In re Lintner, 173 USPQ 560.
Applicant urges that the specification provides evidence that targeted LSH expression yields unexpected root and nodulation phenotypes which were not predictable from Lei’s report or ALOG literature and depend on specific sequence and spatiotemporal expression parameters. Because the prior art does not suggest the combination of elements to produce the claimed phenotypes, Applicant urges the rejection should be withdrawn (Remarks, page 21, paragraphs 2-4).
This argument is unpersuasive, because silence in the art regarding a specific phenotype does not constitute unexpected results. The prior art provides teaching and motivation to transform a plant with a gene encoding an ALOG domain-containing protein under control of a promoter provides the recited expression patterns. Substitution of one homologous ALOG-domain encoding gene for another in another species would have been obvious as presented above. Because the recited phenotypes would inherently result from the expression of a polypeptide of instant SEQ ID NO: 2, under control of a promoter with diurnally variable root expression, under symbiotic conditions, the combination of elements is obvious.
Furthermore, Applicant’s argument that the recited phenotypes depend on specific sequence and spatiotemporal expression parameters is not commensurate with the evidence provided in the instant application, and therefore is not persuasive with respect to the nonobviousness of the claimed invention. See response to Remarks above.
Conclusion
No claims are allowed.
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/VICTORIA L DELEO/Examiner, Art Unit 1662
/Anne Kubelik/Primary Examiner, Art Unit 1663
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