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 .
Status of Claims
Claims 1-17 & 25-28 are under examination on the merits.
Claims 18-24 are withdrawn from consideration as being drawn to an invention nonelected without traverse in the response filed 10/18/2024.
The objection to the sequence disclosure is withdrawn in light of Applicant’s amendment.
The objections to the specification are withdrawn in light of Applicant’s amendments.
The objections to claims 15-16 are withdrawn in light of Applicant’s amendments.
The rejection of claims 9 & 25-28 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, for enablement is withdrawn in light of Applicant’s amendments.
The rejection of claim 15 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite is withdrawn in light of Applicant’s amendments.
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 1-14 & 25-28 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.
The term “low stringency hybridization conditions” in claims 1 (lines 9-10) 9 (lines 9-10), 25 (lines 10-11), 26 (lines 9-10), 27 (lines 8-9), and 28 (lines 9-10) is a relative term which renders the claims indefinite. The term “low stringency” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. While the instant specification provides examples of conditions involved in hybridization protocols (page 22, paragraph [088]), the instant specification does not provide an exact definition of what is encompassed by “low stringency hybridization conditions” and instead says that conditions may be adjusted. Because one of ordinary skill in the art would not be reasonably apprised of what hybridization conditions are encompassed by the limitation, claims 1-14 & 25-28 are indefinite.
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.
Claim 1-14 & 25-28 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 12/6/2024, as applied to claims 1-14 & 25-28. Applicant' s arguments filed 3/25/2025 have been fully considered but they are not persuasive.
Claims 1-14 & 25-28 require a lectin-domain containing protein or fragment thereof comprising an amino acid sequence of at least 85% sequence identity to SEQ ID NO: 2 or a polynucleotide segment that hybridizes under low stringency hybridization conditions to a polynucleotide of SEQ ID NO: 1.
The claim is drawn to a genus of nucleic acids that hybridize to SEQ ID NO:1 under low stringency conditions. Applicant has described an example of low stringency conditions as salt concentration in the wash step of about 2.0 x SSC at 50°C and temperature in the wash step at room temperature, about 22°C (page 22, paragraph [088]). Applicant says that either or both salt concentration or temperature may be varied to adjust stringency. Because Applicant has not specified the conditions for low stringency hybridization, the claim is broad and reads on virtually any nucleic acid capable of producing membrane invagination when expressed.
Furthermore, the claim is drawn to protein or fragment thereof comprising an amino acid sequence of at least 85% sequence identity to SEQ ID NOs: 2 and 4-12. Proteins with 85% identity to the 287 amino acid-long SEQ ID NO: 2 would have 43 substitutions relative to SEQ ID NO: 2. The specification envisions fragments comprising as few as 50 amino acids (page 22, paragraph [089]).
Thus, given the breadth described above, the claims merely require a nucleic acid encoding any protein with a lectin domain, wherein the nucleic acid is capable of producing membrane invagination when expressed.
Applicant has described 10 amino acid sequences of lectin-domain containing proteins, ranging from 55-75% identity (SEQ ID NOs: 2 and 4-12). See alignment below of SEQ ID NO: 2 and 8. Thus, the specification does not describe species over the full scope of claimed amino acids. The specification describes LDP1 and LDP2 proteins has having extracellular lectin domains and short transmembrane domains but no intracellular kinase domain (page 15, paragraph [069]). Applicant describes the function of LDP1 to induce membrane invagination by clustering (page 15, paragraph [069]). The specification discloses that the purified lectin domain is sufficient to cause membrane invagination in the presence of S. meliloti (page 93, paragraph [0149]; figure 5). The instant specification does not disclose DNA molecules encoding a protein of SEQ ID NOs: 2 and 4-12 that would produce membrane invagination when expressed, other than when an LDP1 and LDP2 protein is expressed.
US-17-932-775-8
Filing date in PALM: 2022-09-16
Sequence 8, US/17932775
GENERAL INFORMATION
APPLICANT: University of Freiburg (en)
TITLE OF INVENTION: Proteins for Regulation of Symbiotic Infection and Associated Regulatory Elements (en)
FILE REFERENCE: AGOE:004US
CURRENT APPLICATION NUMBER: US/17/932,775
CURRENT FILING DATE: 2022-09-16
NUMBER OF SEQ ID NOS: 25
SEQ ID NO 8
LENGTH: 288
TYPE: PRT
FEATURE:
NAME/KEY: source
LOCATION: 1..288
QUALIFIERS: mol_type = protein
organism = Glycine max
ALIGNMENT:
Query Match 55.3%; Score 830.5; Length 288;
Best Local Similarity 57.3%;
Matches 168; Conservative 42; Mismatches 72; Indels 11; Gaps 7;
Qy 1 MALSNLKSNRTLS-SSLITIFI-ISLFLQYHNIKSQSSWQSRQVPRS--ETVAFSITEFE 56
|| | | | | | || || | ||:|::||| | | | ||| : |:
Db 1 MAFPNSKPNLLQSLSPLIKFFIPFLLLLQHHSVKSQ---QQPPSPMSAYETVGIDFSFFD 57
Qy 57 KENPDIFLRGDTSISDGILRLTKTDQSGKPLPNTVGRATYLTPIHIWDKTSGELADFSTS 116
|::|:: | |: |:| | |||| ||| |||:|::||| ::||||:|:| :| ||||::
Db 58 KDDPNVLLIGNASVSGGALRLTNTDQLGKPVPHSVGRVVHITPIHLWNKNNGHLADFTSD 117
Qy 117 FSFIVNTNDSDLHGDGFAFYL--GPLHFDVPKNSSGGYLGLFDPENAFPPSKTPILAIEF 174
|||:|| | | ||||||:| |:| :|||||||||||| || | ||| |:||||
Db 118 FSFVVNPKGSALRGDGFAFFLTSANLNFLIPKNSSGGYLGLFKPETALDPSKNQIVAIEF 177
Qy 175 DGFTNEWDPPSSFQSPHIGIDVGSIVSLEYAQWPINFVPRNALGEANINYNSESKRLSVF 234
| |||:||| | ||||:|||| || |: || | ||: |::||||| ||||||
Db 178 DSFTNDWDPNSPNQSPHVGIDVDSIKSVATVPWPSELEPDNAVAHASLNYNSEDKRLSVF 237
Qy 235 VAYPGTQWNSTRVSVVVDLRSVLPEWVRIGFSATTGELVETHDIINWSFESAL 287
| || : |:| || :||||:|||||: :||||:||:|||||||:|||||:||
Db 238 VGYPDNR-NAT-VSAIVDLRNVLPEWISVGFSASTGDLVETHDILNWSFEAAL 288
Lectin-domain containing proteins are known in the art. Loris et al (1998) Biochimica et Biophysica Acta. 1383:9–36 teaches that lectins in legumes have a domain consisting of β-pleated sheets attached to a carbohydrate binding region (page 11, left column, lines 14-16) that has both a calcium and transition metal ion (page 11, right column, lines 14-16). Proteins from the same lectin family without the metal binding sites do not have carbohydrate recognition activity (page 14, right column, lines 30-33). Lectins can bind monosaccharides, oligosaccharides, and adenine-derived plant hormones, and carbohydrate specificities are determined by a core of residues and a variable loop and quaternary structure (abstract; page 22, table 4). Lectin proteins are involved in diverse processes including infection (page 10, left column, lines 2-7).
It is also known in the art that lectin-domain containing proteins play a role in host/symbiont specificity. For example, Vershinina et al (2012. Symbiosis. 56:25–33) teaches that lectins mediate specificity between host plants and rhizobial strains (page 26, left column, lines 4-7), and that introducing a lectin gene from pea into tobacco, tomato, and rape can increase the adherence to roots of R. leguminosarum but not R. galegae (page 29, figure 4). The art does not disclose the specific features of SEQ ID NOs: 2 and 4-12 that would produce membrane invagination and the specification fails to make up for this lack of knowledge in the art. The art also does not disclose DNA molecules encoding a protein of SEQ ID NOs: 2 and 4-12 that would produce membrane invagination when expressed.
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 amino acids that encode a lectin-domain containing protein 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 low stringency hybridization conditions are defined in paragraph [0088] of the specification (Response page 12, paragraph 3).
This argument is not found persuasive, because the conditions provided in paragraph [0088] are not a definition but an example of what "a" low stringency condition could be. Because there is no specific definition of low stringency conditions for hybridization to SEQ ID NO: 1, claims 1-14 & 25-28 still encompass a genus of recombinant DNA molecules that have not been described over the full extent of the genus.
Applicant traverses that Claim 1 part b requires any protein comprising a lectin domain and urges that the requirement that the recited DNA molecule is capable of producing membrane invagination when expressed in a plant cell limits the claims sufficiently (Response page 12 paragraph 4-page 13 paragraph 1).
This argument is not found persuasive, because the previous Action asserts that claims 1-14 & 25-28 merely requires any protein comprising a lectin domain or fragment thereof, not that claim 1 part b specifically merely requires any protein comprising a lectin domain. Claim 1 parts a-c are required in alternative and so the limitations of claim 1 parts a and b are not required if the limitations of part c are met.
As presented above, the imprecision with which the conditions for hybridization have been described in the amended claims and instant specification means that a nucleic acid encoding any protein with a lectin domain, or encoding a fragment of a protein with a lectin domain, could be found to hybridize under low stringency hybridization conditions to a polynucleotide segment having the nucleotide sequence of SEQ ID NO: 1.
The limitation of the function that the recombinant DNA molecule is capable of producing membrane invagination when expressed in a plant cell is noted. The instant specification describes that the LDP1 protein with a lectin domain is capable of producing membrane invagination when expressed in a plant cell (figures 6A-B; paragraph 0152, paragraph [0154]). However, an expressed DNA molecule is not an expressed protein. The instant specification does not describe how an expressed recombinant DNA molecule is capable of producing membrane invagination without an expressed LDP1 protein. Therefore, the instant specification has not described any examples of a recombinant DNA molecule with the claimed function without translation to an LDP1 protein, and the prior art has not described such recombinant DNA molecules, and Applicant has not described the invention as claimed in such a way to demonstrate they were in possession of such a recombinant DNA molecule at the time of filing.
Applicant urges that the specification has provided ten LDP1 sequences in molecular detail and described a short intracellular region and no intracellular kinase domain. Applicant urges that the specification has described the ability of the disclosed LDP1 sequences to produce membrane invagination when expressed in a plant, fulfilling the Written Description requirement (Response page 13, paragraph 2).
This argument is not found persuasive, because claims 1-14 & 25-28 are not limited to a polynucleotide segment encoding an LDP1 protein but encompass a broader genus as outlined above. Any lectin-domain containing protein or fragment thereof encoded by the recombinant DNA molecule of claims 1-14 & 25-28 does not necessarily comprise the domains and activity of an LDP1 protein and cannot be readily expected to comprise the activity of producing membrane invagination across the full breadth of the sequences that hybridize under “low stringency” hybridization conditions to instant SEQ ID NO: 1.
Applicant urges that the specification describes how minor modifications can be made to the disclosed sequences to arrive at variant sequences having the same activity. As an example, Applicant urges that aligning a sequence to the 10 sequences provided by the instant specification and identifying a sequence with at least about 85% sequence identity would define the sequence as having the activity of the reference sequence, or that fragments having at least about 50 amino acids of SEQ ID NOs: 2 or 4-12 maintain the activity of the base sequences (Response page 13 paragraph 3-page 14 paragraph 2).
This argument is not found persuasive. One of ordinary skill in the art could align a sequence or generate a fragment of the provided sequences but this in itself would not demonstrate that the polynucleotide encoding the variant or fragment has the activity of producing membrane invagination when expressed. Furthermore, that one of ordinary skill could generate a fragment or variant and validate experimentally that said sequence or fragment has the same activity as instant SEQ ID NOs: 2 or 4-12 is a question of enablement, not written description, and does not relieve Applicant of the responsibility of describing the claimed genus over the breadth of the claim. Applicant has described no specific fragments of SEQ ID NOs: 2 and 4-12 that maintain the activity of SEQ ID NOs: 2 and 4-12, and applicant has described no specific sequences with 85% identity to SEQ ID NOs: 2 and 4-12 that maintain the activity, so Applicant has not described the full genus and cannot be considered to have been in possession of the full genus at the time of filing.
Applicant urges that the written description requirement is met because Written Description Training Materials Revision 1 (2008) Example 11B said that a partial structure would have put one of skill in the art in possession of the entire genus of nucleic acid sequences encoding polypeptides having at least 85% identity to a given sequence (Response page 14, paragraph 3-page 15 paragraph 1).
This argument is not found persuasive. First, Written Description Training Materials Revision 1 (2008) is not a current standard and has not been considered.
Secondly, the instant specification does not require that the sequences with 85% sequence identity to SEQ ID NOs: 2, 4-12 allow only conservative mutations in the described domains such that an LDP1 domain is preserved (paragraph [084]) or that 50 amino acid fragments include the described domains (paragraph [089]). The instant specification does not describe that these domains are required for the activity of producing membrane invagination.
Applicant has described no specific fragments of SEQ ID NOs: 2 and 4-12 that maintain the recited activity of SEQ ID NOs: 2 and 4-12, and applicant has described no sequences encoding a polypeptide with 85% identity to SEQ ID NOs: 2 and 4-12 that maintain the activity of producing membrane invagination when expressed in a plant cell.
Applicant has not described or provided any additional limitations to the sequence variants or fragments that would ensure that the activity is maintained. Applicant has claimed a broad genus DNA molecules encoding lectin-containing polypeptides or fragments thereof at least capable of hybridizing under indefinite low stringency conditions to SEQ ID NO: 1, and has neither described the features required for the specific activity of the DNA molecule (of producing invagination) as necessary nor described a variety of species across the full scope of the claim capable of producing the specific activity. Because the specification has not described the structure responsible for this function as the responsible features and the art is silent as to the required features, a person of skill in the art could not have been able to identify all the variants and fragments encompassed by the claim with the function of producing membrane invagination and distinguish these from variants and fragments without the function.
Applicant urges that description of a sequence with an activity and identification of domains responsible for the activity fulfils the requirement for written for description (Response page 15, paragraph 2).
This argument is unpersuasive because Applicant has not explicitly described any specific domains as responsible and required for the claimed activity of producing invagination of membranes. Paragraph 0153 describes expression of GFP-LDP1 (the full protein) leading to membrane invagination. Paragraph [0149] describes LDP1 proteins as capable of causing membrane invagination (paragraph [0152]). Moreover, as presented above, claims 1-14 & 25-28 require that the recombinant DNA molecule is capable of producing membrane invagination when expressed, and the instant specification has not described any features of the expressed nucleic acid molecule that would provide the claimed function apart from translation of an LDP protein.
Applicant has described the claimed genus as encompassing variants with as little as 85% sequence identity or fragments with as few as 50 amino acids but has described no such variants or fragments capable of performing the claimed function. There is no art recognized structure function relationship between membrane invagination and the claimed sequences. Applicant has not described the full extent of the claimed genus with the claimed function.
Applicant urges that Written Description Training Materials Revision 1 (2008) provide an example that Applicant believes is similar to the instant case in describing a sequence with a claimed activity and domains responsible for the recited activity along with a prediction in the specification that these domains will result in a protein having the claimed activity. Applicant urges that similarities between an example in the 2008 Written Description Training Materials Revision 1 and the instant specification indicate the sufficiency of the written description of the instant application (Response page 15, paragraph 3-page 16 paragraph 2).
This argument is not persuasive. First, Written Description Training Materials Revision 1 (2008) is not a current standard and has not been considered.
Second, the instant disclosure describes a highly conserved lectin domain (paragraph [035], figure 15) and describes that LDP1 and LDP2 comprise both the extracellular lectin domain and transmembrane domains (paragraph [068]), but does not provide a prediction that the presence of these domains alone will result in a protein having the claimed activity of membrane invagination. The specification teaches that the LDP1 lectin domain can bind rhizobia in solution and associate with a rhizobial exopolysaccharide matrix (paragraph [0156]), which is not the same activity as producing membrane invagination. The specification describes that LDP1 and LDP2 lack an intracellular kinase domain and therefore would not be expected to be involved in signaling cascades (paragraph [069]), but while this describes intracellular kinase domains as unnecessary for membrane invaginations this is not the same as describing whether or not an intracellular kinase domain would impact membrane invaginations. Claims 1-14 & 25-28 require only a fragment of lectin domain-containing protein, they do not explicitly require even a lectin domain. Additionally, claims 1-14 & 25-28 require that the recombinant DNA molecule, when expressed, have the activity of producing membrane invagination. A recombinant DNA molecule does not have polypeptide functional domains as described in the specification.
Third, claims 1-14 & 25-28 are not limited to DNA molecules encoding sequences with 85% sequence identity to SEQ ID NO: 2, because claims 1-14 & 25-28 allow, in alternative, polynucleotide sequences that hybridize under low stringency conditions to SEQ ID NO: 1, wherein low stringency conditions do not have a definite definition. As explained above, the genus of claimed DNA molecules is much broader than sequences encoding polypeptides with 85% sequence identity to SEQ ID NO: 2, and within this breadth of claimed sequences, Applicant has failed to adequately describe which sequences will have the function of producing membrane invaginations.
The instant specification does not support the argument that Applicant was in possession of the claimed genus and the rejection is maintained.
Improper Dependency
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 7 & 11 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Due to Applicant' s amendment of the claims, the rejection is modified from the rejection as set forth in the Office action mailed 12/6/2024, as applied to claims 7 & 11. Applicant' s arguments filed 3/25/2025 have been fully considered but they are not persuasive
Claims 7 and 11 recite species of plant that are neither dicotyledonous nor monocotyledonous: pine (line 6). Because claims 7 and 11 do not include the limitations of claims 6 and 10 to be drawn to monocotyledonous or dicotyledonous plants, they are improperly dependent. Applicant may cancel the claims, amend the claims to place the claims in proper dependent form, rewrite the claims in independent form, or present a sufficient showing that the dependent claims comply with the statutory requirements.
Applicant urges that the removal of reference to pine and fir trees makes the rejection moot (Response page 18 paragraph 1).
This argument is unpersuasive because the amendments have not removed a reference to the generic “pine”, which would not be encompassed by monocotyledonous or dicotyledonous plants or plant cells.
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.
Claims 1-5 are rejected under 35 U.S.C. 103 as being unpatentable over Frenzel et al (2005) MPMI 18(8):771–782, hereafter Frenzel, in view of Uniprot record A9YWS7 LEC10_MEDTR (first available 2/5/2008).
Due to Applicant' s amendment of the claims, the rejection is modified from the rejection as set forth in the Office action mailed 12/6/2024, as applied to claims 1-5. Applicant' s arguments filed 3/25/2025 have been fully considered but they are not persuasive.
Claims 1-5 are drawn to a recombinant DNA molecule comprising a heterologous promoter operably linked to a polynucleotide encoding a lectin-domain protein of SEQ ID NO: 2 capable of producing membrane invagination when expressed in a cell, in operable linkage with a bacterial artificial chromosome or plasmid or in a bacterial host cell of Escherichia coli.
Frenzel (published online 2/5/2007) teaches a cDNA library created from mycorrhizal roots of Medicago truncatula and therefore enriched for mycorrhiza-upregulated genes, ligated into the pGEM-T vector in E. coli (page 779, right column, lines 19-31). Frenzel teaches that lectin genes are strongly induced in mycorrhizal roots, including the MtLEC10 gene (page 776, left column, lines 6-17). Furthermore, Frenzel teaches that MtLEC10 (TC 75254) is detected only in mycorrhizal roots and not in non-mycorrhizal roots (page 775, table 4). Frenzel does not teach the sequence of MtLEC10. Frenzel does not teach that MtLEC10 is capable of producing membrane invagination when a DNA molecule encoding MtLEC10 is expressed in a plant cell.
Uniprot record A9YWS7 LEC10_MEDTR teaches a sequence identical to instant SEQ ID NO: 2. See alignment below.
LEC10_MEDTR
ID LEC10_MEDTR Reviewed; 287 AA.
AC A9YWS7; A0A0C3XFZ3; G7KGH3;
DT 17-JUN-2020, integrated into UniProtKB/Swiss-Prot.
DT 05-FEB-2008, sequence version 1.
DT 24-JAN-2024, entry version 75.
DE RecName: Full=Lectin 10 {ECO:0000303|PubMed:16134889};
DE Short=MtLec10 {ECO:0000303|PubMed:16134889};
DE AltName: Full=Agglutinin LEC10 {ECO:0000305};
GN Name=LEC10 {ECO:0000303|PubMed:16134889};
GN OrderedLocusNames=MTR_5g031160 {ECO:0000312|EMBL:AES95928.2};
GN ORFNames=MtrunA17_Chr5g0411771 {ECO:0000312|EMBL:RHN54888.1};
OS Medicago truncatula (Barrel medic) (Medicago tribuloides).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; fabids; Fabales; Fabaceae; Papilionoideae; 50 kb inversion clade;
OC NPAAA clade; Hologalegina; IRL clade; Trifolieae; Medicago.
OX NCBI_TaxID=3880;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. Jemalong A17;
RX PubMed=16273388; DOI=10.1007/s00438-005-0057-9;
RA Kevei Z., Seres A., Kereszt A., Kalo P., Kiss P., Toth G., Endre G.,
RA Kiss G.B.;
RT "Significant microsynteny with new evolutionary highlights is detected
RT between Arabidopsis and legume model plants despite the lack of
RT macrosynteny.";
RL Mol. Genet. Genomics 274:644-657(2005).
RN [2]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. Jemalong A17;
RX PubMed=22089132; DOI=10.1038/nature10625;
RA Young N.D., Debelle F., Oldroyd G.E.D., Geurts R., Cannon S.B.,
RA Udvardi M.K., Benedito V.A., Mayer K.F.X., Gouzy J., Schoof H.,
RA Van de Peer Y., Proost S., Cook D.R., Meyers B.C., Spannagl M., Cheung F.,
RA De Mita S., Krishnakumar V., Gundlach H., Zhou S., Mudge J., Bharti A.K.,
RA Murray J.D., Naoumkina M.A., Rosen B., Silverstein K.A.T., Tang H.,
RA Rombauts S., Zhao P.X., Zhou P., Barbe V., Bardou P., Bechner M.,
RA Bellec A., Berger A., Berges H., Bidwell S., Bisseling T., Choisne N.,
RA Couloux A., Denny R., Deshpande S., Dai X., Doyle J.J., Dudez A.-M.,
RA Farmer A.D., Fouteau S., Franken C., Gibelin C., Gish J., Goldstein S.,
RA Gonzalez A.J., Green P.J., Hallab A., Hartog M., Hua A., Humphray S.J.,
RA Jeong D.-H., Jing Y., Jocker A., Kenton S.M., Kim D.-J., Klee K., Lai H.,
RA Lang C., Lin S., Macmil S.L., Magdelenat G., Matthews L., McCorrison J.,
RA Monaghan E.L., Mun J.-H., Najar F.Z., Nicholson C., Noirot C.,
RA O'Bleness M., Paule C.R., Poulain J., Prion F., Qin B., Qu C., Retzel E.F.,
RA Riddle C., Sallet E., Samain S., Samson N., Sanders I., Saurat O.,
RA Scarpelli C., Schiex T., Segurens B., Severin A.J., Sherrier D.J., Shi R.,
RA Sims S., Singer S.R., Sinharoy S., Sterck L., Viollet A., Wang B.-B.,
RA Wang K., Wang M., Wang X., Warfsmann J., Weissenbach J., White D.D.,
RA White J.D., Wiley G.B., Wincker P., Xing Y., Yang L., Yao Z., Ying F.,
RA Zhai J., Zhou L., Zuber A., Denarie J., Dixon R.A., May G.D.,
RA Schwartz D.C., Rogers J., Quetier F., Town C.D., Roe B.A.;
RT "The Medicago genome provides insight into the evolution of rhizobial
RT symbioses.";
RL Nature 480:520-524(2011).
RN [3]
RP GENOME REANNOTATION.
RC STRAIN=cv. Jemalong A17;
RX PubMed=24767513; DOI=10.1186/1471-2164-15-312;
RA Tang H., Krishnakumar V., Bidwell S., Rosen B., Chan A., Zhou S.,
RA Gentzbittel L., Childs K.L., Yandell M., Gundlach H., Mayer K.F.,
RA Schwartz D.C., Town C.D.;
RT "An improved genome release (version Mt4.0) for the model legume Medicago
RT truncatula.";
RL BMC Genomics 15:312-312(2014).
RN [4]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. Jemalong A17;
RX PubMed=30397259; DOI=10.1038/s41477-018-0286-7;
RA Pecrix Y., Staton S.E., Sallet E., Lelandais-Briere C., Moreau S.,
RA Carrere S., Blein T., Jardinaud M.F., Latrasse D., Zouine M., Zahm M.,
RA Kreplak J., Mayjonade B., Satge C., Perez M., Cauet S., Marande W.,
RA Chantry-Darmon C., Lopez-Roques C., Bouchez O., Berard A., Debelle F.,
RA Munos S., Bendahmane A., Berges H., Niebel A., Buitink J., Frugier F.,
RA Benhamed M., Crespi M., Gouzy J., Gamas P.;
RT "Whole-genome landscape of Medicago truncatula symbiotic genes.";
RL Nat. Plants 4:1017-1025(2018).
RN [5]
RP INDUCTION BY ARBUSCULAR MYCORRHIZAL FUNGI.
RC STRAIN=cv. Jemalong A17;
RX PubMed=16134889; DOI=10.1094/mpmi-18-0771;
RA Frenzel A., Manthey K., Perlick A.M., Meyer F., Puehler A., Kuester H.,
RA Krajinski F.;
RT "Combined transcriptome profiling reveals a novel family of arbuscular
RT mycorrhizal-specific Medicago truncatula lectin genes.";
RL Mol. Plant Microbe Interact. 18:771-782(2005).
CC -!- FUNCTION: May be involved in arbuscular mycorrhizal (AM) symbiosis with
CC AM fungi. {ECO:0000305|PubMed:16134889}.
CC -!- SUBCELLULAR LOCATION: Membrane {ECO:0000255}; Single-pass membrane
CC protein {ECO:0000255}.
CC -!- INDUCTION: Accumulates in roots during colonization by arbuscular
CC mycorrhizal (AM) fungi (e.g. Glomus intraradices).
CC {ECO:0000269|PubMed:16134889}.
CC -!- SIMILARITY: Belongs to the leguminous lectin family. {ECO:0000305}.
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DR EMBL; EU306659; ABY48148.1; -; Genomic_DNA.
DR EMBL; CM001221; AES95928.2; -; Genomic_DNA.
DR EMBL; PSQE01000005; RHN54888.1; -; Genomic_DNA.
DR RefSeq; XP_003612970.2; XM_003612922.2.
DR AlphaFoldDB; A9YWS7; -.
DR SMR; A9YWS7; -.
DR STRING; 3880.A9YWS7; -.
DR GlyCosmos; A9YWS7; 4 sites, No reported glycans.
DR PaxDb; 3880-AES95928; -.
DR EnsemblPlants; AES95928; AES95928; MTR_5g031160.
DR GeneID; 11405785; -.
DR Gramene; AES95928; AES95928; MTR_5g031160.
DR KEGG; mtr:11405785; -.
DR eggNOG; ENOG502QTX3; Eukaryota.
DR HOGENOM; CLU_000288_62_2_1; -.
DR OrthoDB; 513960at2759; -.
DR Proteomes; UP000002051; Chromosome 5.
DR Proteomes; UP000265566; Chromosome 5.
DR GO; GO:0016020; C:membrane; IEA:UniProtKB-SubCell.
DR GO; GO:0030246; F:carbohydrate binding; IEA:UniProtKB-KW.
DR GO; GO:0009610; P:response to symbiotic fungus; IEP:UniProtKB.
DR CDD; cd06899; lectin_legume_LecRK_Arcelin_ConA; 1.
DR Gene3D; 2.60.120.200; -; 1.
DR InterPro; IPR013320; ConA-like_dom_sf.
DR InterPro; IPR016363; L-lectin.
DR InterPro; IPR000985; Lectin_LegA_CS.
DR InterPro; IPR001220; Legume_lectin_dom.
DR PANTHER; PTHR32401; CONCANAVALIN A-LIKE LECTIN FAMILY PROTEIN; 1.
DR PANTHER; PTHR32401:SF31; LECTIN 6; 1.
DR Pfam; PF00139; Lectin_legB; 1.
DR PIRSF; PIRSF002690; L-type_lectin_plant; 1.
DR SUPFAM; SSF49899; Concanavalin A-like lectins/glucanases; 1.
DR PROSITE; PS00308; LECTIN_LEGUME_ALPHA; 1.
PE 2: Evidence at transcript level;
KW Glycoprotein; Lectin; Membrane; Reference proteome; Transmembrane;
KW Transmembrane helix.
FT CHAIN 1..287
FT /note="Lectin 10"
FT /id="PRO_0000450045"
FT TOPO_DOM 1..11
FT /note="Cytoplasmic"
FT /evidence="ECO:0000305"
FT TRANSMEM 12..31
FT /note="Helical"
FT /evidence="ECO:0000255"
FT TOPO_DOM 32..287
FT /note="Extracellular"
FT /evidence="ECO:0000305"
FT CARBOHYD 124
FT /note="N-linked (GlcNAc...) asparagine"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00498"
FT CARBOHYD 147
FT /note="N-linked (GlcNAc...) asparagine"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00498"
FT CARBOHYD 243
FT /note="N-linked (GlcNAc...) asparagine"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00498"
FT CARBOHYD 280
FT /note="N-linked (GlcNAc...) asparagine"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU00498"
SQ SEQUENCE 287 AA; 31923 MW; 8D96C83144D88E97 CRC64;
Query Match 100.0%; Score 1502; Length 287;
Best Local Similarity 100.0%;
Matches 287; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MALSNLKSNRTLSSSLITIFIISLFLQYHNIKSQSSWQSRQVPRSETVAFSITEFEKENP 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MALSNLKSNRTLSSSLITIFIISLFLQYHNIKSQSSWQSRQVPRSETVAFSITEFEKENP 60
Qy 61 DIFLRGDTSISDGILRLTKTDQSGKPLPNTVGRATYLTPIHIWDKTSGELADFSTSFSFI 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 DIFLRGDTSISDGILRLTKTDQSGKPLPNTVGRATYLTPIHIWDKTSGELADFSTSFSFI 120
Qy 121 VNTNDSDLHGDGFAFYLGPLHFDVPKNSSGGYLGLFDPENAFPPSKTPILAIEFDGFTNE 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 VNTNDSDLHGDGFAFYLGPLHFDVPKNSSGGYLGLFDPENAFPPSKTPILAIEFDGFTNE 180
Qy 181 WDPPSSFQSPHIGIDVGSIVSLEYAQWPINFVPRNALGEANINYNSESKRLSVFVAYPGT 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 WDPPSSFQSPHIGIDVGSIVSLEYAQWPINFVPRNALGEANINYNSESKRLSVFVAYPGT 240
Qy 241 QWNSTRVSVVVDLRSVLPEWVRIGFSATTGELVETHDIINWSFESAL 287
|||||||||||||||||||||||||||||||||||||||||||||||
Db 241 QWNSTRVSVVVDLRSVLPEWVRIGFSATTGELVETHDIINWSFESAL 287
Before the time of filing of the instant application, it would have been obvious to one of ordinary skill that the recombinant DNA molecule comprising a promoter linked to a polynucleotide encoding a protein of instant SEQ ID NO: 2 taught by Frenzel is one with a sequence identical to A9YWS7 LEC10_MEDTR, which has 100% homology to instant SEQ ID NO: 2. The recombinant DNA molecule is present within an Escherichia coli bacterial host cell and in operable linkage with a plasmid. Moreover, claims 1-5 are drawn to the recombinant DNA molecule, not to a cell comprising the recombinant DNA molecule. The recombinant DNA molecule taught by Frenzel and A9YWS7 LEC10_MEDTR encoding a lectin-domain protein, because it has 100% sequence homology to instant SEQ ID NO: 2, would have been capable of producing membrane invagination when expressed in a plant cell. The instant specification teaches that a sequence of SEQ ID NO: 2, which is the amino acid sequence of M. truncatula LDP1 (paragraph [042]), is capable of producing membrane invagination in a plant cell (paragraph [0149-0152]). Thus, the recombinant DNA molecule of claims 1-5 are obvious in view of Frenzel and UniProt record A9YWS7 LEC10_MEDTR.
Applicant urges that because Frenzel does not teach the sequence of MtLEC10 there would be no reason to select an amino acid with the sequence of SEQ ID NO: 2 for further study (Response page 19, paragraph 2).
The argument is unpersuasive, because Frenzel teaches that the protein MtLEC10 is induced in mycorrhizal roots in Medicago truncatula. UniProt record A9YWS7 LEC10_MEDTR teaches a sequence of a Medicago truncatula LEC10 protein. One of ordinary skill in the art would have been motivated to use a Medicago truncatula LEC10 protein with a sequence identity to instant SEQ ID NO: 2 because Frenzel teaches a Medicago truncatula LEC10 protein. One of ordinary skill in the art would not have needed to know the exact sequence of Frenzel’s LEC10 protein in order to be motivated to use a known sequence of a LEC10 protein from the same species.
Applicant urges that because instant SEQ ID NO: 2 lacks an intracellular kinase domain and comprises a short intracellular region, a person of skill in the art would not have expected LDP1 to be directly involved in membrane invagination or the initiation of signaling cascades leading to symbiotic infection, so a person of ordinary skill in the art would not have had any teaching or motivation to select the sequence of UniProt record A9YWS7 LEC10_MEDTR over 500 other lectin proteins in plants. Applicant also urges that Frenzel and UniProt record A9YWS7 LEC10_MEDTR do not teach the beneficial effect of inducing membrane invagination and enhancing symbiotic performance in the presence of rhizobia (Response page 19, paragraph3 -page 21 paragraph 1).
This argument is unpersuasive, because Frenzel does not teach 500 lectin proteins in plants; Frenzel teaches 7 lectin sequences in M. truncatula that are induced by arbuscular mycorrhizal fungi, including MtLec10 by name (page 776, left column, paragraph 2). Frenzel teaches the importance of these lectins in symbiosis and proposes that the identification of numerous lectin-like sequences with increased RNA accumulation in mycorrhizal roots indicates an important role for these proteins during arbuscular mycorrhizal symbiosis (page 779, left column, paragraph 1). One of ordinary skill in the art, reading Frenzel, would have been taught these 7 lectins, including MtLEC10, to be important to symbiosis. One of ordinary skill in the art would have been motivated to use the sequence of UniProt record A9YWS7 LEC10_MEDTR because it is a M. truncatula LEC10 protein, which Frenzel teaches is important to arbuscular mycorrhizal symbiosis in M. truncatula.
One of ordinary skill in the art would not have needed the same motivation as the Applicants in order to arrive at the sequence of UniProt record A9YWS7 LEC10_MEDTR in the expression library taught by Frenzel, namely that MtLEC10 induces membrane invagination and enhances symbiotic performance in the presence of rhizobia. One of ordinary skill in the art would not have needed to understand the mechanism by which MtLEC10 is involved in arbuscular mycorrhizal symbiosis in M. truncatula in order to have motivation to use a protein of this sequence. Frenzel teaches that MtLEC10 is an expressed sequence important to arbuscular mycorrhizal symbiosis in M. truncatula, which would have been motivation to one of ordinary skill in the art to use a MtLEC10 protein with the sequence of UniProt record A9YWS7 LEC10_MEDTR, which is identical to instant SEQ ID NO: 2.
Applicant urges that because the mechanism of clustering expressed LDP1 polypeptide at the cellular bilayer to cause membrane tension and invagination of the cell membrane was not known in the art and would have been unexpected to one of ordinary skill in the art, it would not have been obvious at the time of filing to one of ordinary skill in the art to use a protein of instant SEQ ID NO: 2 (Response page 20, paragraphs 3-4).
One of ordinary skill in the art would not have needed to understand the mechanism by which MtLEC10 is involved in arbuscular mycorrhizal symbiosis in M. truncatula at the time of filing of the instant application in order to have motivation to use a protein of this sequence. Frenzel teaches that MtLEC10 is a protein important to arbuscular mycorrhizal symbiosis in M. truncatula, which would have been motivation to one of ordinary skill in the art to use a MtLEC10 protein with the sequence of UniProt record A9YWS7 LEC10_MEDTR, which is identical to instant SEQ ID NO: 2.
Applicant urges that overexpression of SEQ ID NO: 2 leading to enhanced symbiotic performance of transformed M. truncatula lines is an inventive technical effect because membrane invagination in the presence of rhizobia due to expression of LDP1 is unexpected and one of ordinary skill in the art would not have expected a protein without an intracellular kinase domain to be involved in signaling cascades leading to symbiotic infection. Applicant urges that because Frenzel does not suggest the sequence of SEQ ID NO: 2, the technical effect of the claimed recombinant DNA molecules would have been non-obvious.
Frenzel teaches that lectin-like proteins, including a LEC10 protein, are important for arbuscular mycorrhizal symbiosis in M. truncatula, which would have been motivation to one of ordinary skill in the art to use a MtLEC10 protein with the sequence of UniProt record A9YWS7 LEC10_MEDTR, which is identical to instant SEQ ID NO: 2. A function, or non-function, in signalizing cascades is irrelevant to the motivation provided by Frenzel to use a MtLEC10 protein. Frenzel teaches more than one hypothesized function for the lectin proteins, including as storage for organic nitrogen, and suggests that AM-specific lectins might have different functions than nodule-enhanced lectins (page 779, left column, paragraph 1). One of ordinary skill in the art would have been taught by Frenzel that lectins play roles in endosymbiosis besides signaling cascades. Whether or not the activity of producing membrane invagination would have been obvious to one of ordinary skill in the art, the importance of MtLEC10 to arbuscular mycorrhizal symbiosis in M. truncatula would have been obvious before the time of filing of the instant application.
Claims 1-8 & 10-14 are rejected under 35 U.S.C. 103 as being unpatentable over Etzler et al US 6849777 B1 (filed 9/6/2000, granted 2/1/2005), hereafter Etzler, in view of Frenzel et al (2005) MPMI 18(8):771–782, hereafter Frenzel, and in view of Uniprot record A9YWS7 LEC10_MEDTR (first available 2/5/2008).
Due to Applicant' s amendment of the claims, the rejection is modified from the rejection as set forth in the Office action mailed 12/6/2024, as applied to claims 1-8 & 10-14. Applicant' s arguments filed 3/25/2025 have been fully considered but they are not persuasive.
Claims 1-3 & 6-7 are drawn to a recombinant DNA molecule encoding a lectin-domain containing protein of SEQ ID NO: 2, expressed in a plant cell to increase symbiotic infection. Claims 8, 10 & 14 are drawn to a plant or plant cell comprising the DNA molecule susceptible to symbiotic infection. Claims 12-13 are drawn to a transgenic seed and a method of making progeny seed. Claims 4-5 are drawn to the recombinant DNA molecule in a bacterial host.
Etzler teaches a method of increasing mycorrhizal infection in a plant comprising introducing into a plant an expression cassette containing a promoter operably linked to a heterologous polynucleotide for a LNP protein (Etzler claim 1, lines 1-8). Etzler teaches that the mycorrhizal fungus symbiont can be Glomus intraradices (Etzler claim 6, lines 1-2; and column 20 lines 18-38), although Etzler also teaches a method of associating transgenic plants with Mesorhizobium loti bacteria (column 17, lines 1-6 table 1; column 19, line 31-column 20, line 16).
Etzler teaches that LNP is a root lectin protein (column 1, line 66) involved in rhizobium-legume biosynthesis (column 2, lines 5-6) and nodulation (table 1 and column 18, lines 7-17). Etzler teaches that LNP is able to bind Nod factors (column 16, line 43). Etzler teaches suitable heterologous promoters, including the CaMV 35S promoter and teaches a motivation for using a heterologous promoter to achieve constitutive and ubiquitous expression (column 13, lines 48-51).
Etzler teaches stably transformed lines of L. japonicus, a dicot, via Agrobacterium transformation (column 19, lines 6-30) and teaches planting and growing seeds from these lines specifically to the T1 and T2 generation (column 19, lines 20-37 and table 1). Etzler teaches a variant of the method of increasing mycorrhizal infection where the cassette is introduced into the plant through a sexual cross (Etzler claim 4).
Etzler does not teach instant SEQ ID NO: 2. Etzler does not teach the transformation of M. truncatula. Etzler does not specifically teach a method of producing progeny seed comprising plant a first seed, growing a plant from the seed, and harvesting the progeny seed from the plants.
The teachings of Uniprot record A9YWS7 LEC10_MEDTR and Frenzel are discussed above.
Before the time of filing of the instant application, it would have been obvious to one having ordinary skill in the art to substitute the lectin-domain containing protein taught by Uniprot record A9YWS7 LEC10_MEDTR for the lectin protein used by Etzler to increase mycorrhizal infection in a plant. One of ordinary skill in the art would have expected the proteins to be functional equivalents because both are lectins expressed in the roots and involved in mycorrhizal colonization (Frenzel page 776, left column, lines 6-17, Etzler column 17 line 43-45). One of ordinary skill in the art would have been reasonably confident of success substituting one lectin protein with another of similar function.
Regarding claims 1-3 and 6, Etzler teaches the lectin protein polynucleotide operably linked to a plant promoter in an expression cassette in a plant to increase mycorrhizal infection (Etzler claim 1). The expression cassette reads on expressing the recombinant DNA molecule in a plant cell (instant claim 2, line 2) and on the recombinant DNA in a plant host cell (instant claim 3, line 2). Etzler teaches an example in Lotus japonicus (column 19, lines 6-30), which reads on a dicot cell (instant claim 6). It would be obvious to use the CaMV35S promoter as taught by Etzler in the expression cassette (column 18, line 66), which would read on a recombinant DNA molecule comprising a heterologous promoter operably linked to a gene for a lectin-domain protein (instant claim 1, lines 1-2). The recombinant DNA molecule taught by Frenzel and A9YWS7 LEC10_MEDTR encoding a lectin-domain protein, because it has 100% sequence homology to instant SEQ ID NO: 2, would have been capable