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 1-6, 8, 10-11, 14, 20-21, 24, 29-30, 32, 37-38, 40 & 44 are under examination on the merits.
Nucleotide and/or Amino Acid Sequence Disclosures
This application contains sequence disclosures that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.831(b). However, this application fails to comply with the requirements of 37 CFR 1.831 through 1.834.
Sequence identifiers are missing from the listed DNA sequences on page 14 (line 22) through page 17 (line 12). All polynucleotide sequences longer than 10 nucleotides must be listed in the sequence listing file, and where the description or claims of a patent application discuss a sequence that is set forth in the "Sequence Listing XML" in accordance with paragraph (a) of this section, reference must be made to the sequence by use of the sequence identifier, preceded by "SEQ ID NO:" or the like in the text of the description or claims, even if the sequence is also embedded in the text of the description or claims of the patent application. See MPEP 2412.04.
Full compliance with the sequence rules is required in response to this Office action. A complete response to this Office action must include both compliance with the sequence rules and a response to the issues set forth herein. Failure to fully comply with both of these requirements in the time period set forth in this Office action will be held to be non-responsive.
Claim Objections
Claims 1-4 & 32 are objected to because of the following informalities:
Claim 1 (line 3) & claim 32 (line 3): --an-- should be inserted before “endoplasmic”.
Claim 2 (line 4): “a endoplasmic” should read --an endoplasmic--.
Claim 2 (line 6): “where” should read --wherein--.
Claim 3 (line 2): --a-- should be inserted before “localization”.
Claim 4 (line 2): --a-- should be inserted before “vacuole”.
Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 40 & 44 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claim(s) does/do not fall within at least one of the four categories of patent eligible subject matter because claims 40 & 44 are drawn to a “use” of the transgenic plant in claim 1 for increasing resistance with no active, positively recited steps delimiting how the use is actually practiced.
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 2, 21, 24, 29-30, 40 & 44 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. All dependent claims are included in the rejections below.
Regarding claim 2, the text within the parentheses (line 2) renders the claim indefinite because it is unclear whether the limitation within the parentheses is a requirement of the claimed invention. In addition, the phrase "such as" also renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Regarding claim 21, the text within the parentheses (line 3) renders the claim indefinite because it is unclear whether the species listed within the parentheses are part the claimed invention or merely an example. More than one species of plant can be considered a potato. The parentheses in claims 30 and 38, in contrast, recite the common name for a species of insect, but it is definite that the claims require the recited species.
Claim 24 recites the limitation "the plant pest" in line 2. There is insufficient antecedent basis for this limitation in the claim, because claim 1 does not recite a plant pest.
Claim 40 is drawn to a use of the transgenic plant of claim 1 without setting forth any steps involved in the process. Merely reciting a use without any active, positive steps delimiting how this use is actually practiced is not definite. See MPEP 2173.05(q). Claim 44 further limits the plant pest recited by claim 40 but does not provide any additional step in the process.
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 1-6, 8, 10-11, 14, 20-21, 24, 29-30, 32, 37-38, 40 & 44 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.
A. Claims 1-6, 8, 10-11, 14, 20-21, 24, 29-30, 32, 37-38, 40 & 44 are drawn to a transgenic plant or a progeny thereof which expresses or is capable of expressing pleurotolysin A2 (PlyA2) and pleurotolysin B (PlyB), a method of killing a plant pest comprising expressing PlyA2 and PlyB in a transgenic plant, and a use of the transgenic plant. Claim 5 further requires that the PlyA2 is a polypeptide comprising an amino acid sequence having at least 70% identity with the sequence encoded by instant SEQ ID NO: 1. Claim 6 further requires that the PlyA2 is a polypeptide comprising an amino acid sequence having at least 70% identity with the sequence encoded by instant SEQ ID NO: 2.
The claims broadly require a PlyA2 with 70% sequence identity with the sequence encoded by instant SEQ ID NO: 1 or 2.
A polypeptide having 70% sequence identity with the sequence encoded by instant SEQ ID NO: 1 or 2 encompasses sequences that have 41 substitutions relative to the 138 amino acid long sequences encoded by SEQ ID NOs: 1 & 2.
The instant specification describes proteins of the aegerolysin family to include ostreolysin A6, PlyA2, and erylysin A. The specification describes that aegerolysins bind to a specific membrane lipid receptor, ceramide phosphoethanolamine, in insect gut epithelial cells, leading to binding of the MACPF-protein partner and formation of multimeric aegerolysin/MACPF transmembrane pores that permeabilize insect gut cell membranes (page 2, lines 15-19). The specification describes that unlike Cry toxins from B. thuringiensis, which bind to membrane protein receptors, aegerolysin-based protein complexes bind to a lipid receptor (page 2, lines 15-22). The specification describes aegerolysin-based protein complex as non-toxic for mammals and other non-target organisms (page 2, lines 21-23).
The instant specification describes two sequences encoding PlyA2: SEQ ID NO: 1 and SEQ ID NO: 2. The amino acid sequences encoded by SEQ ID NO: 1 & 2 differ at two positions, K85R and K52R, in order to eliminate potential ubiquitination sites in PlyA2 (page 23, lines 8-9). No structural features required for the binding activity of the PlyA2 are described in the specification, and no features to differentiate a PlyA2 from another aegerolysin are described.
Aegerolysins are described in the art. UniProt record A0AAW0BBF6_9AGAR describes the sequence of an ostreolysin A6, which is an aegerolysin but not a pleurotolysin, with 78% similarity to the amino acid sequence encoded by instant SEQ ID NO: 1. See alignment below. UniProt record D0FZZ2_PLEER (available 12/15/2006) describes a sequence of a Erylysin A protein, which is further annotated as belonging to the pleurotolysin family, which has 111 matches relative to instant SEQ ID NO: 1. See second alignment below. However, this sequence is still labeled Erylysin A protein rather than a PlyA2 protein.
A0AAW0BBF6_9AGAR
ID A0AAW0BBF6_9AGAR Unreviewed; 148 AA.
AC A0AAW0BBF6;
DT 27-NOV-2024, integrated into UniProtKB/TrEMBL.
DT 27-NOV-2024, sequence version 1.
DT 28-JAN-2026, entry version 6.
DE SubName: Full=Ostreolysin A6 {ECO:0000313|EMBL:KAK7023384.1};
GN Name=OLYA6_6 {ECO:0000313|EMBL:KAK7023384.1};
GN ORFNames=VNI00_016799 {ECO:0000313|EMBL:KAK7023384.1};
OS Paramarasmius palmivorus.
OC Eukaryota; Fungi; Dikarya; Basidiomycota; Agaricomycotina; Agaricomycetes;
OC Agaricomycetidae; Agaricales; Marasmiineae; Marasmiaceae; Paramarasmius.
OX NCBI_TaxID=297713 {ECO:0000313|EMBL:KAK7023384.1, ECO:0000313|Proteomes:UP001383192};
RN [1] {ECO:0000313|EMBL:KAK7023384.1, ECO:0000313|Proteomes:UP001383192}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=GH-12 {ECO:0000313|EMBL:KAK7023384.1,
RC ECO:0000313|Proteomes:UP001383192};
RA Baruah I.K., Bukari Y., Amoako-Attah I., Meinhardt L.W., Bailey B.A.,
RA Cohen S.P.;
RT "A draft genome for a cacao thread blight-causing isolate of Paramarasmius
RT palmivorus.";
RL Submitted (JAN-2024) to the EMBL/GenBank/DDBJ databases.
CC -!- SIMILARITY: Belongs to the aegerolysin family.
CC {ECO:0000256|ARBA:ARBA00010795}.
CC -!- CAUTION: The sequence shown here is derived from an EMBL/GenBank/DDBJ
CC whole genome shotgun (WGS) entry which is preliminary data.
CC {ECO:0000313|EMBL:KAK7023384.1}.
CC ---------------------------------------------------------------------------
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DR EMBL; JAYKXP010000141; KAK7023384.1; -; Genomic_DNA.
DR AlphaFoldDB; A0AAW0BBF6; -.
DR Proteomes; UP001383192; Unassembled WGS sequence.
DR GO; GO:0019836; P:symbiont-mediated hemolysis of host erythrocyte; IEA:InterPro.
DR Gene3D; 2.60.270.50; -; 1.
DR InterPro; IPR009413; Aegerolysin-typ.
DR Pfam; PF06355; Aegerolysin; 1.
DR PIRSF; PIRSF007951; Hemolysin, aegerolysin type; 1.
PE 3: Inferred from homology;
KW Reference proteome {ECO:0000313|Proteomes:UP001383192}.
SQ SEQUENCE 148 AA; 16293 MW; 2584047ACE4D06BB CRC64;
Length: 148
Score: 526.00 Matches: 95
Percent Similarity: 78.7% Conservative: 12
Best Local Similarity: 69.9% Mismatches: 29
Query Match: 70.1% Indels: 0
Gaps: 0
US-19-479-699-1 (1-417) x A0AAW0BBF6_9AGAR (1-148)
Qy 7 TATGCTCAGTGGGTTATCATCATAATCCACAACGTCGGCTCAAAGGACGTTAAAATTGTC 66
||||||||||||||||||||| ||| |||||||||::: :::|||||||||
Db 13 TyrAlaGlnTrpValIleIleAsnIleLysAsnValGlyThrAlaAsnValLysIleLys 32
Qy 67 AATCTCAAACCAAGTTGGGGTAAATTACACGCTGACGGTGATAAAGACACCGAAGTGTCT 126
|||||| |||||||||||||||||||||::: ||||||||| |||||||||
Db 33 AsnLeuAspValSerTrpGlyLysLeuHisAlaAsnAspAspLysAspLysGluValSer 52
Qy 127 GCATCAAAGTATGAGGGGACCGTTATCAAGCCCGATGAAAAACTCCAAATCAATGCTTGC 186
:::|||||| |||||| |||||| |||::::::|||||||||
Db 53 LysAspValPheGluGlyArgValIleThrAlaAspGluThrLeuArgMetAsnAlaCys 72
Qy 187 GGACGTTCAGACGCAGCAGAAGGAACAACTGGAACTTTCGATCTAGTTGACCCAGCCGAC 246
|||||| ::: |||||||||||| ||||||||| ||| |||
Db 73 GlyArgLysSerSerProSerGlyThrThrGlyAspPheAspLeuCysAspValAspAsp 92
Qy 247 GGTGATAAGCAAGTCAGACATTTCTACTGGGATTGTCCTTGGGGAAGCAAAGCTAACACT 306
||||||::: :::||||||||||||||||||||||||||||||||| ||||||
Db 93 GlyAspArgValIleArgHisPheTyrTrpAspCysProTrpGlySerSerLysAsnThr 112
Qy 307 TGGACAGTCTCAGGAAGCAACACTAAATGGATGATTGAGTATTCAGGACAGAATTTGGAC 366
|||||||||||||||||||||:::|||||||||:::|||||||||||| |||||||||
Db 113 TrpThrValSerGlySerAsnSerLysTrpMetValGluTyrSerGlyAlaAsnLeuAsp 132
Qy 367 TCTGGTGCATTAGGGACAATTACTGTTGATACTTTAAAAAAAGGAAAC 414
||||||||||||||||||||||||||| :::||||||||||||
Db 133 GlyGlyAlaLeuGlyThrIleThrValAspValMetLysLysGlyAsn 148
ID D0FZZ2_PLEER Unreviewed; 138 AA.
AC D0FZZ2;
DT 15-DEC-2009, integrated into UniProtKB/TrEMBL.
DT 15-DEC-2009, sequence version 1.
DT 10-JUN-2026, entry version 26.
DE SubName: Full=Erylysin A {ECO:0000313|EMBL:BAI45247.1};
DE SubName: Full=Pe.ostreolysin {ECO:0000313|EMBL:BAN83907.1};
GN Name=eryA {ECO:0000313|EMBL:BAI45247.1};
OS Pleurotus eryngii (Boletus of the steppes).
OC Eukaryota; Fungi; Dikarya; Basidiomycota; Agaricomycotina; Agaricomycetes;
OC Agaricomycetidae; Agaricales; Pleurotineae; Pleurotaceae; Pleurotus.
OX NCBI_TaxID=5323 {ECO:0000313|EMBL:BAI45247.1};
RN [1] {ECO:0000313|EMBL:BAI45247.1}
RP NUCLEOTIDE SEQUENCE.
RA Shibata T., Kudou M., Hoshi Y., Kudou A., Nanashima N., Miyairi K.;
RT "Isolation, characterization of a Two-component Hemolysin, Erylysin A and
RT B, from an edible mushroom, Pleurotus eryngii.";
RL Submitted (OCT-2009) to the EMBL/GenBank/DDBJ databases.
RN [2] {ECO:0000313|EMBL:BAN83907.1}
RP NUCLEOTIDE SEQUENCE.
RX PubMed=23918047; DOI=10.1194/jlr.D041731;
RA Bhat H.B., Kishimoto T., Abe M., Makino A., Inaba T., Murate M., Dohmae N.,
RA Kurahashi A., Nishibori K., Fujimori F., Greimel P., Ishitsuka R.,
RA Kobayashi T.;
RT "Binding of a pleurotolysin ortholog from Pleurotus eryngii to
RT sphingomyelin and cholesterol-rich membrane domains.";
RL J. Lipid Res. 54:2933-2943(2013).
RN [3] {ECO:0000313|EMBL:BAN83907.1}
RP NUCLEOTIDE SEQUENCE.
RA Kurahashi A., Sato M., Kobayashi T., Nishibori K., Fujimori F.;
RT "Homologous genes, Pe.pleurotolysin A and Pe.ostreolysin, are both
RT specifically and highly expressed in primordia and young fruiting bodies of
RT Pleurotus eryngii.";
RL Mycoscience 55:113-117(2014).
CC -!- SIMILARITY: Belongs to the aegerolysin family.
CC {ECO:0000256|ARBA:ARBA00010795}.
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DR EMBL; AB526802; BAI45247.1; -; mRNA.
DR EMBL; AB777518; BAN83907.1; -; mRNA.
DR TCDB; 1.C.97.1.2; the pleurotolysin pore-forming (pleurotolysin) family.
DR OrthoDB; 2727348at2759; -.
DR GO; GO:0019836; P:symbiont-mediated hemolysis of host erythrocyte; IEA:InterPro.
DR Gene3D; 2.60.270.50; -; 1.
DR InterPro; IPR009413; Aegerolysin-typ.
DR Pfam; PF06355; Aegerolysin; 1.
DR PIRSF; PIRSF007951; Hemolysin, aegerolysin type; 1.
PE 2: Evidence at transcript level;
SQ SEQUENCE 138 AA; 15090 MW; 20DF9DAB2778962C CRC64;
Length: 138
Score: 618.00 Matches: 111
Percent Similarity: 89.9% Conservative: 13
Best Local Similarity: 80.4% Mismatches: 14
Query Match: 82.4% Indels: 0
Gaps: 0
US-19-479-699-1 (1-417) x A0A9P6A0N8_PLEER (1-138)
Qy 1 ATGGCTTATGCTCAGTGGGTTATCATCATAATCCACAACGTCGGCTCAAAGGACGTTAAA 60
|||||||||||||||||||||||||||:::||||||||||||||| ::::::||||||
Db 1 MetAlaTyrAlaGlnTrpValIleIleLeuIleHisAsnValGlyGlnGlnAsnValLys 20
Qy 61 ATTGTCAATCTCAAACCAAGTTGGGGTAAATTACACGCTGACGGTGATAAAGACACCGAA 120
||| |||||| |||||||||||||||:::||||||||||||||||||||||||
Db 21 IleLysAsnLeuAsnAlaSerTrpGlyLysLeuTyrAlaAspGlyAspLysAspThrGlu 40
Qy 121 GTGTCTGCATCAAAGTATGAGGGGACCGTTATCAAGCCCGATGAAAAACTCCAAATCAAT 180
||| |||||||||||||||||| |||||| ||||||:::::::::|||||||||
Db 41 ValProAlaSerLysTyrGluGlyMetValIleAlaProAspAspGlnValGlnIleAsn 60
Qy 181 GCTTGCGGACGTTCAGACGCAGCAGAAGGAACAACTGGAACTTTCGATCTAGTTGACCCA 240
|||||||||||| |||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaCysGlyArgGluAspAlaAlaGluGlyThrThrGlyThrPheAspLeuValAspPro 80
Qy 241 GCCGACGGTGATAAGCAAGTCAGACATTTCTACTGGGATTGTCCTTGGGGAAGCAAAGCT 300
||| ||||||||||||||||||||| ||||||||||||||||||:::||||||
Db 81 AsnAspSerAspLysGlnValArgHisPheAlaTrpAspCysProTrpGlyThrLysAla 100
Qy 301 AACACTTGGACAGTCTCAGGAAGCAACACTAAATGGATGATTGAGTATTCAGGACAGAAT 360
|||:::||| ||| |||||||||:::||||||||||||||||||:::|||||||||
Db 101 AsnSerTrpValValGlyGlySerAsnSerLysTrpMetIleGluTyrThrGlyGlnAsn 120
Qy 361 TTGGACTCTGGTGCATTAGGGACAATTACTGTTGATACTTTAAAAAAAGGAAAC 414
|||||||||||||||||||||||||||||||||:::||||||::: ||||||
Db 121 LeuAspSerGlyAlaLeuGlyThrIleThrValAsnThrLeuArgIleGlyAsn 138
Aegerolysins, and pleurotolysin A2, are described in the art. Approximately 400 members are known in the aegerolysin protein family (Jotic et al (2021) Toxins. 13(7). 455. Published 6/29/2021, hereafter Jotic; page 8, paragraph 2). Proteins from the aegerolysin family have low isoelectric points, similar molecular weights, and are heat-labile, but most aegerolysins exert biological properties in monomeric forms while pleurotolysin A acts as a bi-component cytolysin with pleurotolysin B (Berne et al (2009) Protein Science. 18(4): 694-706 (published 2/12/2009) page 697, right column, paragraph 4).
Jotic describes that ostreolysin A6 is from P. ostreatus, while PlyA2 and erylysin A are both from P. eryngii, and all can form complexes with PlyB (Jotic page 2, paragraph 3). Cry34Ab1 is structurally related to the aegeroylsins (Jotic page 2, paragraph 3). However, the insecticidal effect of Pleurotus aegerolysins is suggested to be due to aegerolysin-based complexes binding to and permeabilizing ceramide phosphoethanolamine (CPE) but not other membrane lipids, and the formation of transmembrane pores (Jotic page 2, paragraphs 4-5 and page 8, paragraph 3). Erylysin A exclusively recognizes CPE/cholesterol, while ostreolysin A6 and PlyA2 interact with both sphingomyelin/cholesterol and CPE/cholesterol complexes (Jotic page 2, paragraph 6).
Differences in the primary structures of aegerolysins dictate their avidity and selectivity towards specific membrane lipids (Jotic page 9, paragraph 3). Differences in affinity for MACPF-domain partner proteins may be the result of amino acid differences in the C-terminus of the proteins (Jotic page 9, paragraph 5). However, the specific structural differences that separate a PlyA2 from another aegerolysin with at least 70% sequence identity with SEQ ID NO: 1 or 2 are not described in the art.
The instant specification fails to make up for the lack of description in the prior art. Since the disclosure fails to describe the common structural attributes that identify members of the genus, SEQ ID NOs: 1-2 are insufficient to describe the claimed genus over the scope of the claims.
Because Applicant has not described the structural features that differentiate a pleurotolysin A2 from another aegerolysin with 70% identity to SEQ ID NO: 1 or 2 nor provided examples over the genus of sequences encompassed, one of ordinary skill in the art would not conclude that Applicant was in possession of pleurotolysin A2 over the full scope of the claimed invention.
Hence, Applicant has not, in fact, described pleurotolysin A2 over the full scope of the claims, and a transgenic plant comprising a pleurotolysin A2 and a method or use requiring such a plant are likewise not described. The specification fails to provide an adequate written description of the claimed invention.
B. Claims 1-6, 8, 10-11, 14, 20-21, 24, 29-30, 32, 37-38, 40 & 44 are drawn to a transgenic plant or a progeny thereof which expresses or is capable of expressing a pleurotolysin B (PlyB), a method of killing a plant pest comprising expressing PlyA2 and PlyB in a transgenic plant, and a use of the transgenic plant. Claim 10 further requires that the PlyB is a polypeptide comprising an amino acid sequence having at least 70% identity with the sequence encoded by instant SEQ ID NO: 4.
The claims broadly require a PlyB with 70% sequence identity with the sequence encoded by SEQ ID NO: 4.
A polypeptide having 70% sequence identity with the sequence encoded by instant SEQ ID NO: 4 encompasses sequences that have 156 substitutions relative to the 523 amino acid-long sequence encoded by instant SEQ ID NO: 4.
The instant specification describes proteins containing the membrane-attack complex/performin (MACPF) domain to include PlyB and erylysin B. The specification describes that MACPF-protein partners bind to aegerolysins to form multimeric aegerolysin/MACPF transmembrane pores that permeabilize insect gut cell membranes (page 2, lines 15-19). One sequence encoding a PlyB polypeptide is provided by the instant specification: SEQ ID NO: 4. No structural features required for the activity of the PlyB are described in the specification beyond the MACPF domain, and no features to differentiate a PlyB from another MACPF domain-containing protein are described.
MACPF domain-containing proteins such as PlyB are described in the art, including a protein annotated as an erylysin B with 94% similarity to the sequence encoded by instant SEQ ID NO: 4 (UniProt record A0A9P6A1T0_PLEER, available 9/13/2023, see alignment below). A PlyB protein variant from Kusaghiporia persicina with 71% similarity to the sequence encoded by instant SEQ ID NO: 4, but only 287 matches, is described by Gerber et al WO 2025129045 (published 6/1/2025; see second alignment below). PlyB proteins with 70% identity to the sequence encoded by SEQ ID NO: 4 are not described over the full scope of encompassed sequences.
A0A9P6A1T0_PLEER
ID A0A9P6A1T0_PLEER Unreviewed; 540 AA.
AC A0A9P6A1T0;
DT 13-SEP-2023, integrated into UniProtKB/TrEMBL.
DT 13-SEP-2023, sequence version 1.
DT 28-JAN-2026, entry version 7.
DE SubName: Full=Erylysin B {ECO:0000313|EMBL:KAF9497696.1};
GN ORFNames=BDN71DRAFT_1387140 {ECO:0000313|EMBL:KAF9497696.1};
OS Pleurotus eryngii (Boletus of the steppes).
OC Eukaryota; Fungi; Dikarya; Basidiomycota; Agaricomycotina; Agaricomycetes;
OC Agaricomycetidae; Agaricales; Pleurotineae; Pleurotaceae; Pleurotus.
OX NCBI_TaxID=5323 {ECO:0000313|EMBL:KAF9497696.1, ECO:0000313|Proteomes:UP000807025};
RN [1] {ECO:0000313|EMBL:KAF9497696.1}
RP NUCLEOTIDE SEQUENCE.
RC STRAIN=ATCC 90797 {ECO:0000313|EMBL:KAF9497696.1};
RG DOE Joint Genome Institute;
RA Ahrendt S., Riley R., Andreopoulos W., Labutti K., Pangilinan J.,
RA Ruiz-Duenas F.J., Barrasa J.M., Sanchez-Garcia M., Camarero S.,
RA Miyauchi S., Serrano A., Linde D., Babiker R., Drula E.,
RA Ayuso-Fernandez I., Pacheco R., Padilla G., Ferreira P., Barriuso J.,
RA Kellner H., Castanera R., Alfaro M., Ramirez L., Pisabarro A.G., Kuo A.,
RA Tritt A., Lipzen A., He G., Yan M., Ng V., Cullen D., Martin F.,
RA Rosso M.-N., Henrissat B., Hibbett D., Martinez A.T., Grigoriev I.V.;
RL Submitted (NOV-2020) to the EMBL/GenBank/DDBJ databases.
CC -!- CAUTION: The sequence shown here is derived from an EMBL/GenBank/DDBJ
CC whole genome shotgun (WGS) entry which is preliminary data.
CC {ECO:0000313|EMBL:KAF9497696.1}.
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DR EMBL; MU154542; KAF9497696.1; -; Genomic_DNA.
DR AlphaFoldDB; A0A9P6A1T0; -.
DR OrthoDB; 3269052at2759; -.
DR Proteomes; UP000807025; Unassembled WGS sequence.
DR InterPro; IPR020864; MACPF.
DR InterPro; IPR054586; MACPF_1_fungal.
DR InterPro; IPR040971; PlyB_C.
DR Pfam; PF22693; MACPF_1; 1.
DR Pfam; PF18684; PlyB_C; 1.
DR PROSITE; PS51412; MACPF_2; 1.
PE 4: Predicted;
KW Reference proteome {ECO:0000313|Proteomes:UP000807025}.
FT DOMAIN 50..372
FT /note="MACPF"
FT /evidence="ECO:0000259|PROSITE:PS51412"
SQ SEQUENCE 540 AA; 59150 MW; 723EFD35E5C408C8 CRC64;
Length: 540
Score: 2547.50 Matches: 500
Percent Similarity: 94.5% Conservative: 11
Best Local Similarity: 92.4% Mismatches: 11
Query Match: 90.9% Indels: 19
Gaps: 3
US-19-479-699-4 (1-1572) x A0A9P6A1T0_PLEER (1-540)
Qy 1 ATGGAAGCCGTTCTATCCCGTCAAGCTGCAACTGCAGAAGCTATAGGTCGTTTTCAAGAT 60
|||||||||||||||||| |||||||||||||||||||||::: ||||||||||||
Db 1 MetGluAlaValLeuSerGlyGlnAlaAlaThrAlaGluAlaValGluArgPheGlnAsp 20
Qy 61 TCATCCACAAGCGTCGGACTAGTAGCC--------------------------------- 87
|||||||||||||||||||||||||||
Db 21 SerSerThrSerValGlyLeuValAlaValSerAlaAsnTrpSerLeuLeuSerProPro 40
Qy 88 ------------------GGCAGTCCCAGTACTAGGATCCGTCGTCAAGCCGATAACGTA 129
|||||||||||| |||||||||||||||||||||||||||
Db 41 ProGluPheThrLeuGlnGlySerProSer---ArgIleArgArgGlnAlaAspAsnVal 59
Qy 130 GTACTCAAATCCACTTCTCAGGCTGGAGACACCCTAAACGACGTAATCCAGGACCCAACA 189
|||||||||||| |||||||||||||||||||||||||||||||||||||||||||||
Db 60 ValLeuLysSerIleSerGlnAlaGlyAspThrLeuAsnAspValIleGlnAspProThr 79
Qy 190 AGGAGAAATAAACTAATAAACGACAACAACCTCCTAAAAGGCATAATTATGGGTCGTGAC 249
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 80 ArgArgAsnLysLeuIleAsnAspAsnAsnLeuLeuLysGlyIleIleMetGlyArgAsp 99
Qy 250 GGACCAGTTCCCTCCTCACGTGAGTTAATTGTGCGACCAGATACACTCAGGGCAATAATT 309
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 100 GlyProValProSerSerArgGluLeuIleValArgProAspThrLeuArgAlaIleIle 119
Qy 310 AACAACAGAGCAACTATAGAAACTACCACCATGGAGGCTGAGTTTACCGAGACATTGATG 369
||||||||||||||||||||||||||||||:::|||||||||||||||||||||||||||
Db 120 AsnAsnArgAlaThrIleGluThrThrThrValGluAlaGluPheThrGluThrLeuMet 139
Qy 370 GAAAGCAATTACAATAGCGCATCCGTTAAGGTATCAGCACCATTTATTACAGCCAACTCC 429
|||||||||||||||||||||||||||||||||||||||||||||:::||||||||||||
Db 140 GluSerAsnTyrAsnSerAlaSerValLysValSerAlaProPheValThrAlaAsnSer 159
Qy 430 GAATATAGCGAGAGTTCCTCCTTCAAAAACACCGAGACTGAGAAATCCATGTATACCTCA 489
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 160 GluTyrSerGluSerSerSerPheLysAsnThrGluThrGluLysSerMetTyrThrSer 179
Qy 490 TCCAGGTATTTATTCCCTCAGGGGCGTATTGATTTTACCACACCAGACAGCGGTTTTGAC 549
||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||
Db 180 SerArgTyrLeuPheProGlnGlyArgIleAspPheThrMetProAspSerGlyPheAsp 199
Qy 550 GATGTCATTAAGTTATCTCCTCAGTTTACAAGTGGAGTACAAGCTGCCTTAGCAAAGGCT 609
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 200 AspValIleLysLeuSerProGlnPheThrSerGlyValGlnAlaAlaLeuAlaLysAla 219
Qy 610 ACCGGGACTGAAAAGCGTGAAGCCTTGCAAAACTTGTTCCAAGAATATGGACACGTTTTC 669
||||||||||||||||||||||||||||||:::|||||| ||||||||||||||||||
Db 220 ThrGlyThrGluLysArgGluAlaLeuGlnAspLeuPheLeuGluTyrGlyHisValPhe 239
Qy 670 AGAACTAAAGTTCACATTGGAGGTGTCCTATCAGCACATACTATGGAAACATTCTCTCGA 729
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 240 ArgThrLysValHisIleGlyGlyValLeuSerAlaHisThrMetGluThrPheSerArg 259
Qy 730 TCCGAAAATGAAACCGAAGTGAAACAAGATGTCAAAGCTGGTCTTGAGGGTGCCGTCAAG 789
||||||||||||||||||||||||||||||:::|||||||||||||||||||||||||||
Db 260 SerGluAsnGluThrGluValLysGlnAspIleLysAlaGlyLeuGluGlyAlaValLys 279
Qy 790 GGTTGGGGAGGCGGGGCTACCGCCGGTCACGGCAATACACAAGGGACCATTACAACAAGT 849
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 280 GlyTrpGlyGlyGlyAlaThrAlaGlyHisGlyAsnThrGlnGlyThrIleThrThrSer 299
Qy 850 CAGAACCGAAAATTGAATGTGAAATACATCGTTAATGGTGGAGACTACACTAAAATCCAG 909
|||||||||||||||:::||||||||||||||||||||||||||||||||||||||||||
Db 300 GlnAsnArgLysLeuAspValLysTyrIleValAsnGlyGlyAspTyrThrLysIleGln 319
Qy 910 AATACAGAAGAGTGGGTGGCATCTACAAATCAAAGCGAGCACTGGCGAGTAATCGAAGTG 969
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 320 LysThrGluGluTrpValAlaSerThrAsnGlnSerGluHisTrpArgValIleGluVal 339
Qy 970 ACCGAAGTAACCGCAGTTGCCGATCTCCTTCCACAACCCATAAGGGGTCAAGTGAAGGAC 1029
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 340 ThrGluValThrAlaValAlaAspLeuLeuProGlnProIleArgGlyGlnValLysAsp 359
Qy 1030 CTATTGAAGCCACTACTTGGGAAGTGGGTCGACGTCGAAAAGGTTCCTGGGTTGGAATCA 1089
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 360 LeuLeuLysProLeuLeuGlyLysTrpValAspValGluLysValProGlyLeuGluSer 379
Qy 1090 TTACCTGTCAGTGTCTATAGACCCAAAGGGGCAATCCCAGCAGGTTGGTTCTGGCTTGGT 1149
|||||||||||||||||||||||| ||||||||||||||||||||||||||||||
Db 380 PheProValSerValTyrArgProLysAspAlaIleProAlaGlyTrpPheTrpLeuGly 399
Qy 1150 GATACAGCAGACGCTTCTAAAGCCCTCTTGGTCAAACCTACCTTACCCGCCCGAAGCGGG 1209
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 400 AspThrAlaAspAlaSerLysAlaLeuLeuValLysProThrLeuProAlaArgSerGly 419
Qy 1210 AGAAATCCCGCCCTTACTAGCCTTCACCAGGGATCCGGAATGACTGAGCAGCCATTTGTC 1269
|||||||||||||||||||||||||||::: |||||||||||||||||||||||||||
Db 420 ArgAsnProAlaLeuThrSerLeuHisGluSerSerGlyMetThrGluGlnProPheVal 439
Qy 1270 GATCTTCCTCAATATCAGTACCTCAGCACATACTTTGGTTCATTTGCTCACGACACCCCC 1329
||||||||||||||||||||||||||||||||||||||||||||||||:::|||||||||
Db 440 AspLeuProGlnTyrGlnTyrLeuSerThrTyrPheGlySerPheAlaTyrAspThrPro 459
Qy 1330 CCTGGCTCTACTCTTCGAGGCCTTCGACCAGATCATGTCTTGCCAGGACGATATGAGATG 1389
||||||||||||||||||||||||||||||||||||:::|||||||||||||||||||||
Db 460 ProGlySerThrLeuArgGlyLeuArgProAspHisIleLeuProGlyArgTyrGluMet 479
Qy 1390 ---CACGGTGATACTATATCTACTGCCGTATACGTTACTCGTCCTGTGGACGTTCCCTTT 1446
||||||||||||||| |||||||||||||||||||||||||||||||||||||||
Db 480 ValHisGlyAspThrIleGlyThrAlaValTyrValThrArgProValAspValProPhe 499
Qy 1447 CCAGAGGACGAGTGTTTTGACTTAAAATCCCTTGTTCGTGTAAAATTGCCCGGTTCCGGT 1506
||||||||||||||||||||||||||||||:::|||||||||||||||||||||||||||
Db 500 ProGluAspGluCysPheAspLeuLysSerValValArgValLysLeuProGlySerGly 519
Qy 1507 AATCCACCCAAGCCCAGGTCTGCATTGAAAAAAAGCATGGTTCTTTTTGATAGTGGGGAA 1566
|||||||||||||||||| |||||||||||||||||||||||||||||||||||||||
Db 520 AsnProProLysProArgTrpAlaLeuLysLysSerMetValLeuPheAspSerGlyGlu 539
Qy 1567 AAG 1569
:::
Db 540 Glu 540
BRN68123
ID BRN68123 standard; protein; 503 AA.
XX
AC BRN68123;
XX
DT 14-AUG-2025 (first entry)
XX
DE Kusaghiporia persicina plyB protein variant, SEQ 14.
XX
KW agriculture; fungicide; fungicide resistance; insecticide;
KW insecticide resistance; microorganism; pest control; pesticide;
KW pesticide resistance; plant breeding; plant insect pest; plant pest;
KW pleurotolysin B protein; plyB protein; protein engineering;
KW protein production; recombinant protein; seed; transgenic plant.
XX
OS Kusaghiporia persicina.
OS Synthetic.
XX
CC PN WO2025129045-A1.
XX
CC PD 19-JUN-2025.
XX
CC PF 13-DEC-2024; 2024WO-US060102.
XX
PR 15-DEC-2023; 2023US-0610901P.
XX
CC PA (GINK-) GINKGO BIOWORKS INC.
XX
CC PI Gerber RM, Peele CG, Lenzen S, Palekar NV, Ren P;
XX
DR WPI; 2025-62969E/056.
DR N-PSDB; BRN68122.
XX
CC PT New polypeptide having pesticidal activity, comprising specified amino
CC PT acid sequence, amino acid sequence having specified sequence identity,
CC PT used for e.g. controlling killing pest population e.g. insect, fungi,
CC PT bacteria, and fall armyworm.
XX
CC PS Claim 1; SEQ ID NO 14; 110pp; English.
XX
CC The present invention relates to a novel polypeptide that is useful for
CC controlling or killing pest populations, such as insects, fungi,
CC bacteria, and the fall armyworm. The polypeptide comprising an amino acid
CC sequence of SEQ ID NO: 2 (see BRN68111), SEQ ID NO: 4 (see BRN68113), SEQ
CC ID NO: 6 (see BRN68115), SEQ ID NO: 8 (see BRN68117), SEQ ID NO: 10 (see
CC BRN68119), SEQ ID NO: 12 (see BRN68121), SEQ ID NO: 14 (see BRN68123),
CC SEQ ID NO: 16 (see BRN68125), or SEQ ID NO: 18 (see BRN68127). The
CC invention further claims: (1) a nucleic acid molecule comprising a
CC nucleotide sequence encoding a polypeptide; (2) a host cell comprising
CC the nucleic acid molecule; (3) a DNA construct comprising a heterologous
CC promoter operably linked to a nucleic acid sequence; (4) a vector
CC comprising the DNA construct; (5) a host cell comprising the DNA
CC construct or the vector; (6) a transgenic plant comprising the host cell;
CC (7) a formulation comprising the polypeptide; (8) a method for
CC controlling or killing a pest population comprising contacting the
CC population with the formulation; (9) a method for producing the
CC polypeptide with pesticidal activity; (10) a plant having stably
CC incorporated into its genome a DNA construct comprising the nucleic acid
CC molecule; (11) a transgenic seed of the plant, where the seed has stably
CC incorporated into its genome the DNA construct; (12) a method for
CC controlling insect pest damage to a plant; (13) a method for increasing
CC yield in a plant comprising growing in a field a plant or seed; and (14)
CC a method of obtaining a polynucleotide that encodes an improved
CC polypeptide having pesticidal activity. The polypeptide decreases pest
CC growth by 5-95 percent and increases plant yield by 1-100 percent.
XX
SQ Sequence 503 AA;
Length: 503
Score: 1519.50 Matches: 287
Percent Similarity: 71.0% Conservative: 83
Best Local Similarity: 55.1% Mismatches: 128
Query Match: 54.2% Indels: 23
Gaps: 3
US-19-479-699-4 (1-1572) x BRN68123 (1-503)
Qy 1 ATGGAAGCCGTTCTATCCCGTCAAGCTGCAACTGCAGAAGCTATAGGTCGTTTTCAAGAT 60
||| :::||| ::: ||| |||::: ::: ||| |||
Db 1 MetLeuLysThrIleSerIleGluGluAlaLysAlaGlnPheValProArgLeuHisAsp 20
Qy 61 TCATCCACAAGCGTCGGACTAGTAGCCGGCAGTCCCAGTACTAGGATCCGTCGTCAAGCC 120
|||
Db 21 TyrSer------------------------------------------------------ 22
Qy 121 GATAACGTAGTACTCAAATCCACTTCTCAG------GCTGGAGACACCCTAAACGACGTA 174
::::::||||||:::||| ||||||:::|||||| |||:::
Db 23 ------AlaLeuMetLysSerSerSerAlaArgValAlaGlyGluThrLeuIleAspIle 40
Qy 175 ATCCAGGACCCAACAAGGAGAAATAAACTAATAAACGACAACAACCTCCTAAAAGGCATA 234
||| |||||| ||| |||||||||:::|||||||||:::|||||||||:::
Db 41 IleAsnAspProValArgCysLysLysLeuIleAspAspAsnAsnValLeuLysGlyVal 60
Qy 235 ATTATGGGTCGTGACGGACCAGTTCCCTCCTCACGTGAGTTAATTGTGCGACCAGATACA 294
:::||| ||| |||::: |||:::|||||||||||||||||||||::::::
Db 61 ValMetSerArgGlyGlnProIleAlaSerAlaArgGluLeuIleValArgProGluSer 80
Qy 295 CTCAGGGCAATAATTAACAACAGAGCAACTATAGAAACTACCACCATGGAGGCTGAGTTT 354
:::|||||| |||||||||||||||:::|||:::||| ||||||
Db 81 TyrAsnCysAlaValAsnAsnAlaAlaThrIleGluThrSerThrValGluLeuGluPhe 100
Qy 355 ACCGAGACATTGATGGAAAGCAATTACAATAGCGCATCCGTTAAGGTATCAGCACCATTT 414
||| |||||||||||| |||||| ::: :::|||||| |||||||||:::
Db 101 ThrArgThrLeuMetGluAspAsnTyrThrThrValAlaValLysAlaSerAlaProTyr 120
Qy 415 ATTACAGCCAACTCCGAATATAGCGAGAGTTCCTCCTTCAAAAACACCGAGACTGAGAAA 474
:::||||||::::::|||||||||::: ||||||:::|||||||||||||||||||||
Db 121 ValThrAlaSerAlaGluTyrSerAspLysSerSerTrpLysAsnThrGluThrGluLys 140
Qy 475 TCCATGTATACCTCATCCAGGTATTTATTCCCTCAGGGGCGTATTGATTTTACCACACCA 534
:::::: ||||||||||||||||||||||||||| |||||||||::: |||
Db 141 ThrIleSerValSerSerArgTyrLeuPheProGlnGlyHisIleAspPheSerAlaPro 160
Qy 535 GACAGCGGTTTTGACGATGTCATTAAGTTATCTCCTCAGTTTACAAGTGGAGTACAAGCT 594
|||||| ::::::|||||||||:::|||||| :::
Db 161 GlySerGlyLeuProGlyAlaValGlnLeuSerProGluPheThrLysAlaIleAspAsn 180
Qy 595 GCCTTAGCAAAGGCTACCGGGACTGAAAAGCGTGAAGCCTTGCAAAACTTGTTCCAAGAA 654
|||||||||||| ||| |||||| ||||||||| :::|||::::::
Db 181 AlaLeuAlaLysProThrAsnValGluLysAlaGluAlaLeuTyrThrValPheGluAsp 200
Qy 655 TATGGACACGTTTTCAGAACTAAAGTTCACATTGGAGGTGTCCTATCAGCACATACTATG 714
|||||||||:::|||||||||:::|||||| |||||| |||::: ||||||
Db 201 TyrGlyHisIlePheArgThrGluValHisPheGlyGlySerLeuThrValHisThrGln 220
Qy 715 GAAACATTCTCTCGATCCGAAAATGAAACCGAAGTGAAACAAGATGTCAAAGCTGGTCTT 774
:::||||||||||||:::|||:::||| ||| |||:::|||||| |||
Db 221 AspThrPheSerArgThrGluAspGluLysThrValThrThrAspIleLysAlaSerLeu 240
Qy 775 GAGGGTGCCGTCAAGGGTTGGGGAGGCGGGGCTACCGCCGGTCACGGCAATACACAAGGG 834
||| :::||| ||||||||| :::|||||||||||||||||| |||
Db 241 GluThrGluLeuLysAsnTrpGlyGlySerValSerAlaGlyHisGlyAsnThrThrGly 260
Qy 835 ACCATTACAACAAGTCAGAACCGAAAATTGAATGTGAAATACATCGTTAATGGTGGAGAC 894
|||||||||||| ||||||||| ||| |||||||||||| |||||||||
Db 261 ThrIleThrThrGlnGlnAsnArgValLeuGluAlaLysTyrIleValTyrGlyGlyAsp 280
Qy 895 TACACTAAAATCCAGAATACAGAAGAGTGGGTGGCATCTACAAATCAAAGCGAGCACTGG 954
|||||||||||||||:::||| |||:::|||||||||:::||| ||||||
Db 281 TyrThrLysIleGlnAspThrProAlaTrpIleAlaSerThrAspGlnTrpValHisTrp 300
Qy 955 CGAGTAATCGAAGTGACCGAAGTAACCGCAGTTGCCGATCTCCTTCCACAACCCATAAGG 1014
||||||::: |||||||||||| |||:::||||||:::||| |||
Db 301 ArgValValGlyValThrGluValIleProThrValAspMetLeuProGluProThrArg 320
Qy 1015 GGTCAAGTGAAGGACCTATTGAAGCCACTACTTGGGAAGTGGGTCGACGTCGAAAAGGTT 1074
|||||| |||||||||||||||:::|||||||||||| ||||||||||||
Db 321 SerIleValLysSerLeuLeuLysProLeuIleGlyLysTrpValProValGluLysVal 340
Qy 1075 CCTGGGTTGGAATCATTACCTGTCAGTGTCTATAGACCCAAAGGGGCAATCCCAGCAGGT 1134
|||||| ::::::||| ||| :::||||||||| :::::: |||
Db 341 ProGlyGlnAspAsnLeuAlaValGlyIleTyrArgProThr---SerLeuGluAspGly 359
Qy 1135 TGGTTCTGGCTTGGTGATACAGCAGACGCTTCTAAAGCCCTCTTGGTCAAACCTACCTTA 1194
|||:::|||||||||::::::||||||:::||| ||||||:::|||||| :::|||
Db 360 TrpTyrTrpLeuGlyAsnSerAlaAspSerSerHisAlaLeuValValLysAlaSerLeu 379
Qy 1195 CCCGCCCGAAGCGGGAGAAATCCCGCCCTTACTAGCCTTCACCAGGGATCCGGAATGACT 1254
||| :::||||||||||||||||||||| ::: |||:::
Db 380 ProProLysSerGlyArgAsnProAlaLeuCysAsnAlaSerAspAsnGlyGlyIleGly 399
Qy 1255 GAGCAGCCATTTGTCGATCTTCCTCAATATCAGTACCTCAGCACATACTTTGGTTCATTT 1314
||| ||||||:::||||||||| :::|||||||||||||||||| |||
Db 400 AlaGlnAlaLysValAspIleProGlnTyrSerPheLeuSerThrTyrPheGlyGlyPhe 419
Qy 1315 GCTCACGACACCCCCCCTGGCTCTACTCTTCGAGGCCTTCGACCAGATCATGTCTTGCCA 1374
|||||| :::|||:::||||||||||||||| |||
Db 420 AspSerSerLeuGluProGlyLysSerLeuLysGlyLeuArgProAspLeuPheAlaPro 439
Qy 1375 GGACGATATGAGATGCACGGTGATACTATATCTACTGCCGTATACGTTACTCGTCCTGTG 1434
|||:::::::::||||||||| ||| ||||||||||||:::|||||||||:::
Db 440 AlaArgTrpAspLeuHisGlyAspGlnIleAspThrAlaValTyrIleThrArgProIle 459
Qy 1435 GACGTTCCCTTTCCAGAGGACGAGTGTTTTGACTTAAAATCCCTTGTTCGTGTAAAATTG 1494
||||||||||||||||||||||||:::|||:::||||||||||||:::
Db 460 ThrGlyValProProGluAspGluCysPheAspLeuGlnSerValValArgValLysIle 479
Qy 1495 CCCGGTTCCGGTAATCCACCCAAGCCCAGGTCTGCATTGAAAAAAAGCATGGTTCTTTTT 1554
|||||||||||||||||||||:::||| ||||||:::||| |||||||||
Db 480 ProGlySerGlyAsnProProArgProMetValAlaLeuArgLysGluMetValLeuVal 499
Qy 1555 GAT 1557
:::
Db 500 Glu 500
PlyB is described in the art for its role in forming complexes with the aegerolysin PlyA. Although most aegerolysins exert biological properties in monomeric forms, pleurotolysin A acts as a bi-component cytolysin with pleurotolysin B (PlyB) (Berne et al (2009) Protein Science. 18(4): 694-706 (published 2/12/2009) page 697, right column, paragraph 4).
Jotic et al (2021) Toxins. 13(7). 455. (published 6/29/2021, hereafter Jotic) descibes PlyB as capable of forming complexes with ostreolysin A6, erylysin A, and PlyA2 (page 2, paragraph 3). Ostreolysin A6 has affinity for its naturally occurring partner and forms more stable complexes with PlyB than with the MACPF-domain erylysin B, but erylysin A had comparable membrane-disrupting potential with either erylysin B or PlyB (Jotic page 9, paragraph 5). Differences in affinity for MACPF-domain partner proteins, such as PlyB, may be the result of amino acid differences in the C-terminus of the aegerolysin proteins (Jotic page 9, paragraph 5). However, the specific structural differences that separate a PlyB from another MACPF domain-containing protein with 70% sequence identity to the sequence encoded by instant SEQ ID NO: 4 are not described in the art.
The instant specification fails to make up for the lack of description in the prior art. Since the disclosure fails to describe the common structural attributes that identify members of the genus, SEQ ID NO: 4 is insufficient to describe the claimed genus over the scope of the claims.
Because Applicant has not described the structural features that differentiate a pleurotolysin B from another protein with 70% identity to the sequence encoded by SEQ ID NO: 4 nor provided examples over the genus of sequences encompassed, one of ordinary skill in the art would not conclude that Applicant was in possession of pleurotolysin B over the full scope of the claimed invention.
Hence, Applicant has not, in fact, described pleurotolysin B over the full scope of the claims, and a transgenic plant comprising a pleurotolysin B and a method or use requiring such a plant are likewise not described. The specification fails to provide an adequate written description of the claimed invention.
Scope of Enablement
Claims 1-6, 8, 10-11, 14, 20-21, 24, 29-30, 32, 37-38, 40 & 44 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a transgenic plant comprising a PlyA2 encoded by SEQ ID NO: 1 or 2 and a PlyB encoded by SEQ ID NO: 4, does not reasonably provide enablement for any PlyA2 with 70% similarity to the sequence encoded by instant SEQ ID NO: 1 or 2 and any PlyB with 70% similarity to the sequence encoded by SEQ ID NO: 4. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Claims 1-6, 8, 10-11, 14, 20-21, 24 & 29-30 are drawn to a transgenic plant which expresses or is capable of expressing pleurotolysin A2 (PlyA2) and pleurotolysin B (PlyB). Claims 32, 37 & 38 are drawn to a method of killing a plant pest comprising expressing in a transgenic plant PlyA2 and PlyB. Claims 40 & 44 are drawn to a use of the transgenic plant. Claim 10 further requires that the PlyB is a polypeptide comprising an amino acid sequence having at least 70% identity with the sequence encoded by instant SEQ ID NO: 4. Claim 5 further requires that the PlyA2 is a polypeptide comprising an amino acid sequence having at least 70% identity with the sequence encoded by instant SEQ ID NO: 1. Claim 6 further requires that the PlyA2 is a polypeptide comprising an amino acid sequence having at least 70% identity with the sequence encoded by instant SEQ ID NO: 2. Claims 32, 37, and 38 recite that a transgenic plant expressing PlyA2 and PlyB inhibit growth or kill a plant pest, specifically Leptinotarsa decemlineata or Diabrotica virgifera, and claims 40 & 44 recite that resistance against an insect pest is increased by using the transgenic plant.
Thus, the claims all broadly require a transgenic plant capable of expressing PlyA2 and PlyB.
The instant specification teaches two sequences encoding a Ply2A (SEQ ID NO: 1 & 2) and one sequence encoding a PlyB (SEQ ID NO: 4). The specification teaches that transgenic potato transformed with genes encoding PlyB, where PlyB is targeted to the endoplasmic reticulum with a KDEL sequence, facilitates improved resistance against CPB (page 3, lines 1-5; page 22, line 24-page 23, line 3). The specification teaches two transgenic potato plants that are unharmed by CPB compared with non-transgenic plants having the same genetic background (figure 1). These lines are taught to be the two best performing transgenic lines (page 23, lines 15-18).
The instant specification teaches that proteins of the aegerolysin family, which include ostreolysin A6, PlyA2, and erylysin A, bind to a specific membrane lipid receptor, ceramide phosphoethanolamine, in insect gut epithelial cells, leading to binding of the MACPF-protein partner, such as PlyB, and formation of multimeric aegerolysin/MACPF transmembrane pores that permeabilize insect gut cell membranes (page 2, lines 15-19). The specification teaches that unlike Cry toxins from B. thuringiensis, which bind to membrane protein receptors, aegerolysin-based protein complexes bind to a lipid receptor (page 2, lines 15-22). The specification teaches the aegerolysin-based protein complex to be non-toxic for mammals and other non-target organisms (page 2, lines 21-23).
The instant specification teaches two sequences encoding PlyA2, SEQ ID NO: 1 and SEQ ID NO: 2, and one sequence encoding PlyB, SEQ ID NO: 4. The amino acid sequences encoded by SEQ ID NOs: 1 & 2 differ at two positions, K85R and K52R, in order to eliminate potential ubiquitination sites in PlyA2 (page 23, lines 8-9). No structural features required for the binding activity of the PlyA2 are taught in the specification. No structural features required for the activity of the PlyB are taught in the specification beyond the MACPF domain. No features to differentiate a PlyA2 from another aegerolysin or PlyB from another MACPF domain-containing protein are taught by the instant specification.
The specification does not teach any Ply2A or PlyB other than the amino acid sequences encoded by SEQ ID NOs: 1, 2 and 4 and no transgenic plants expressing Ply2A or PlyB other than the amino acid sequences encoded by SEQ ID NOs: 1, 2 and 4.
Ply2A and PlyB polypeptides are known in the art. UniProt record A0A9P6CEK4_9AGAR (available 9/13/2023) teaches a pleurotolysin A sequence with 91% similarity to the sequence encoded by instant SEQ ID NO: 1, although it is not taught to be a PlyA2 sequence, specifically. See first alignment below. The art also teaches more divergent aegerolysin sequences such as the sequence of UniProt record A0AAW0BBF6_9AGAR, which is an ostreolysin A6 rather than a pleurotolysin, but has 78% similarity to the amino acid sequence encoded by instant SEQ ID NO: 1. See alignment above. Similarly, the art teaches a PlyB protein variant from Kusaghiporia persicina with 71% similarity to the sequence encoded by instant SEQ ID NO: 4, but only 287 matches, (Gerber et al WO 2025129045, published 6/1/2025; see alignment above). However, the art also teaches an MACPF domain-containing protein annotated as an erylysin B with 94% similarity to the sequence encoded by instant SEQ ID NO: 4 (UniProt record A0A9P6A1T0_PLEER, available 9/13/2023, see alignment below). PlyB proteins with 70% identity to the sequence encoded by SEQ ID NO: 4 and their structural features are not taught over the full scope of encompassed sequences.
A0A9P6CEK4_9AGAR
ID A0A9P6CEK4_9AGAR Unreviewed; 138 AA.
AC A0A9P6CEK4;
DT 13-SEP-2023, integrated into UniProtKB/TrEMBL.
DT 13-SEP-2023, sequence version 1.
DT 28-JAN-2026, entry version 8.
DE SubName: Full=Pleurotolysin A {ECO:0000313|EMBL:KAF9458124.1};
GN ORFNames=BDZ94DRAFT_1174238 {ECO:0000313|EMBL:KAF9458124.1};
OS Collybia nuda.
OC Eukaryota; Fungi; Dikarya; Basidiomycota; Agaricomycotina; Agaricomycetes;
OC Agaricomycetidae; Agaricales; Tricholomatineae; Clitocybaceae; Collybia.
OX NCBI_TaxID=64659 {ECO:0000313|EMBL:KAF9458124.1, ECO:0000313|Proteomes:UP000807353};
RN [1] {ECO:0000313|EMBL:KAF9458124.1}
RP NUCLEOTIDE SEQUENCE.
RC STRAIN=CBS 247.69 {ECO:0000313|EMBL:KAF9458124.1};
RG DOE Joint Genome Institute;
RA Ahrendt S., Riley R., Andreopoulos W., Labutti K., Pangilinan J.,
RA Ruiz-Duenas F.J., Barrasa J.M., Sanchez-Garcia M., Camarero S.,
RA Miyauchi S., Serrano A., Linde D., Babiker R., Drula E.,
RA Ayuso-Fernandez I., Pacheco R., Padilla G., Ferreira P., Barriuso J.,
RA Kellner H., Castanera R., Alfaro M., Ramirez L., Pisabarro A.G., Kuo A.,
RA Tritt A., Lipzen A., He G., Yan M., Ng V., Cullen D., Martin F.,
RA Rosso M.-N., Henrissat B., Hibbett D., Martinez A.T., Grigoriev I.V.;
RL Submitted (NOV-2020) to the EMBL/GenBank/DDBJ databases.
CC -!- SIMILARITY: Belongs to the aegerolysin family.
CC {ECO:0000256|ARBA:ARBA00010795}.
CC -!- CAUTION: The sequence shown here is derived from an EMBL/GenBank/DDBJ
CC whole genome shotgun (WGS) entry which is preliminary data.
CC {ECO:0000313|EMBL:KAF9458124.1}.
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DR EMBL; MU150349; KAF9458124.1; -; Genomic_DNA.
DR AlphaFoldDB; A0A9P6CEK4; -.
DR OrthoDB; 2727348at2759; -.
DR Proteomes; UP000807353; Unassembled WGS sequence.
DR GO; GO:0019836; P:symbiont-mediated hemolysis of host erythrocyte; IEA:InterPro.
DR Gene3D; 2.60.270.50; -; 1.
DR InterPro; IPR009413; Aegerolysin-typ.
DR Pfam; PF06355; Aegerolysin; 1.
DR PIRSF; PIRSF007951; Hemolysin, aegerolysin type; 1.
PE 3: Inferred from homology;
KW Reference proteome {ECO:0000313|Proteomes:UP000807353}.
SQ SEQUENCE 138 AA; 15170 MW; C516CBA0D1620A54 CRC64;
Length: 138
Score: 648.00 Matches: 115
Percent Similarity: 91.3% Conservative: 11
Best Local Similarity: 83.3% Mismatches: 12
Query Match: 86.4% Indels: 0
Gaps: 0
US-19-479-699-1 (1-417) x A0A9P6CEK4_9AGAR (1-138)
Qy 1 ATGGCTTATGCTCAGTGGGTTATCATCATAATCCACAACGTCGGCTCAAAGGACGTTAAA 60
||||||||||||||||||||||||||||||:::|||||| |||:::||| :::|||
Db 1 MetAlaTyrAlaGlnTrpValIleIleIleLeuHisAsnThrGlyAlaLysProMetLys 20
Qy 61 ATTGTCAATCTCAAACCAAGTTGGGGTAAATTACACGCTGACGGTGATAAAGACACCGAA 120
::: |||||||||||||||||||||||||||||||||||||||||||||||| |||
Db 21 LeuGlnAsnLeuLysProSerTrpGlyLysLeuHisAlaAspGlyAspLysAspAsnGlu 40
Qy 121 GTGTCTGCATCAAAGTATGAGGGGACCGTTATCAAGCCCGATGAAAAACTCCAAATCAAT 180
||| |||||| ||||||||| ||||||::: |||||||||:::|||||||||
Db 41 ValLysAlaSerAspTyrGluGlyLysValIleGluAlaAspGluLysIleGlnIleAsn 60
Qy 181 GCTTGCGGACGTTCAGACGCAGCAGAAGGAACAACTGGAACTTTCGATCTAGTTGACCCA 240
||||||||| |||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaCysGlyTrpSerAspAlaAlaGluGlyThrThrGlyThrPheAspLeuValAspSer 80
Qy 241 GCCGACGGTGATAAGCAAGTCAGACATTTCTACTGGGATTGTCCTTGGGGAAGCAAAGCT 300
:::|||||||||||||||:::||||||||||||||||||||||||||||||||||||
Db 81 SerAspGlyAspLysGlnIleArgHisPheTyrTrpAspCysProTrpGlySerLysThr 100
Qy 301 AACACTTGGACAGTCTCAGGAAGCAACACTAAATGGATGATTGAGTATTCAGGACAGAAT 360
|||||||||||||||||||||||||||:::|||||||||:::||||||||||||||||||
Db 101 AsnThrTrpThrValSerGlySerAsnSerLysTrpMetValGluTyrSerGlyGlnAsn 120
Qy 361 TTGGACTCTGGTGCATTAGGGACAATTACTGTTGATACTTTAAAAAAAGGAAAC 414
|||:::|||||||||||||||||||||||||||||| |||||||||||||||
Db 121 LeuAsnSerGlyAlaLeuGlyThrIleThrValAspAlaLeuLysLysGlyAsn 138
The art teaches approximately 400 members in the aegerolysin protein family (Jotic et al (2021) Toxins. 13(7). 455. Published 6/29/2021, hereafter Jotic; page 8, paragraph 2), and pleurotolysin A2 is just one example. Proteins from the aegerolysin family have low isoelectric points, similar molecular weights, and are heat-labile, but most aegerolysins exert biological properties in monomeric forms while pleurotolysin A acts as a bi-component cytolysin with pleurotolysin B (Berne et al (2009) Protein Science. 18(4): 694-706 (published 2/12/2009, hereafter Berne) page 697, right column, paragraph 4). However, Jotic teaches that ostreolysin A6, PlyA2 and erylysin A can all form complexes with PlyB (Jotic page 2, paragraph 3).
The insecticidal effect of Pleurotus aegerolysins is suggested to be due to aegerolysin-based complexes binding to and permeabilizing ceramide phosphoethanolamine (CPE) but not other membrane lipids, and the formation of transmembrane pores (Jotic page 2, paragraphs 4-5 and page 8, paragraph 3). Erylysin A exclusively recognizes CPE/cholesterol, while ostreolysin A6 and PlyA2 interact with both sphingomyelin/cholesterol and CPE/cholesterol complexes (Jotic page 2, paragraph 6). The art teaches that differences in the primary structures of aegerolysins dictate their avidity and selectivity towards specific membrane lipids (Jotic page 9, paragraph 3). Differences in affinity for MACPF-domain partner proteins may be the result of amino acid differences in the C-terminus of the aegerolysin proteins (Jotic page 9, paragraph 5). For example, although ostreolysin A6 has affinity for its naturally occurring partner and forms more stable complexes with PlyB than with the MACPF-domain erylysin B, erylysin A had comparable membrane-disrupting potential with either erylysin B or PlyB (Jotic page 9, paragraph 5). However, the specific structural differences that separate a PlyA2 from another aegerolysin with at least 70% identity with the sequence encoded by SEQ ID NO: 1 or 2 or that separate a PlyB from another MACPF-domain partner protein with 70% identity to the sequence encoded by SEQ ID NO: 4 are not taught in the art.
In addition, the activity of pleurotolysins and their MACPF-domain partner proteins with 70% identity to the sequences encoded by SEQ ID NOs: 1-2 or 4 respectively may not be identical to the activity taught in the specification of PlyA2/PlyB. The instant specification teaches PlyA2/PlyB to be non-toxic for mammals and other non-target organisms (page 2, lines 22-24). However, Frangez et al (2017) Toxins. 9(4). 128 (published 4/5/2017) teaches that ostreolysin A/PlyB complexes are toxins that cause coronary vasoconstriction and myocardial hypoxia and lead to respiratory arrest in rodents (abstract, table 1). The membrane activity of pleurotolysin correlates with sphingomyelin content of erythrocyte membranes, exhibiting preferences across a gradient of mammals (Berne page 699, left column, paragraph 4-right column, paragraph 1). Thus, a PlyA2 aegerolysin with as little as 70% identity to the sequence encoded by SEQ ID NO: 1 or 2 might not have the same specificity or biological activity as the PlyA2 and PlyB complex in the example of the instant specification given the lack of teachings regarding required structure.
Given the state of the art and the teachings of the instant specification, a person of ordinary skill in the art would not understand how to make a PlyA2 or PlyB, rather than any pleurotolysin and a MACPF-domain partner, over the full scope of the claims as required. In addition, given the host specificity of PlyA2/PlyB activity, one of ordinary skill in the art would not immediate understand which PlyA2 and PlyB over the full scope of encompassed sequences would function to inhibit growth or kill a plant pest, specifically Leptinotarsa decemlineata or Diabrotica virgifera, as required by claims 32, 37, and 38, or increase resistance against an insect pest, as required by claims 40 & 44. One of ordinary skill would be required to conduct undue experimentation to generate and assay the myriad potential transgenic plants comprising PlyA2 and PlyB proteins over the full scope of the proteins and in combination with each other to determine which, if any, provide resistance to or kill any plant pests. Therefore, the instant invention would not have been enabled for one of ordinary skill in the art over the full scope of the claims at the time of filing of the instant application.
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) 1-3, 5-6, 8, 10-11, 14, 20-21, 24, 29-30, 32, 37-38, 40 & 44 are rejected under 35 U.S.C. 103 as being unpatentable over Panevska et al US 2020/0245628 A1 (published 8/6/2020, more than one year prior to the filing of the instant application; hereafter, Panevska) in view of Zhu et al (CN1229087A, published 9/22/1999, hereafter Zhu. Appended with a machine translation of the Description).
Claims 1-3, 5-6, 8, 10-11, 14, 20-21, 24, 29-30, 32, 37-38, 40 & 44 are drawn to a transgenic plant or progeny which expresses PlyA2 and PlyB with endoplasmic reticulum localization signal, a method comprising expressing in a transgenic plant PlyA2 and PlyB with endoplasmic reticulum localization signal, and a use of a transgenic plant for increasing resistance against a plant pest.
Panevska teaches a motivation to use aegerolysins from Pleurotus such as PlyA2 in combination with PlyB as a biopesticide for controlling Colorado potato beetle (CPB) and Western corn rootworm (WCR) because they can be used as pest-control agents and the chances of resistance evolving to aegerolysins are minute because they interact with the membrane lipid receptor rather than proteins prone to variation (paragraphs [0006-0007]). Resistance has developed to other biological pesticides, such as proteins derived from Bacillus thuringiensis (paragraphs [0002-0003]).
Panevska teaches a method of killing CPB by feeding larvae with potato leaf disks soaked in a protein mixture of PlyA2 and PlyB (paragraphs [0205-0213] and a method of killing WCR by feeding beetles collected from corn fields with an artificial diet mixed with protein complexes of PlyA2 and PlyB (paragraphs [0214-0222]). CPB larvae fed with PlyA2 + PlyB had less weight gain than control CPB larvae fed with buffer (figure 6).
Panevska teaches a sequence for PlyA2 (Panevska SEQ ID NO: 2) with no mismatches and 100% similarity to the sequence encoded by instant SEQ ID NO: 1. Panevska SEQ ID NO: 2 also comprises 98% sequence similarity to the sequence encoded by instant SEQ ID NO: 2. See alignments below.
Sequence 2, US/16649385
Publication No. US20200245628A1
GENERAL INFORMATION
APPLICANT: Univerza v Ljubljani
APPLICANT: Kmetijski institut Slovenije
TITLE OF INVENTION: New bio-pesticides for controlling plant pests
FILE REFERENCE: UL01P004WO
CURRENT APPLICATION NUMBER: US/16/649,385
CURRENT FILING DATE: 2020-03-20
NUMBER OF SEQ ID NOS: 5
SEQ ID NO 2
LENGTH: 138
TYPE: PRT
ORGANISM: Pleurotus eryngii
Length: 138
Score: 749.00 Matches: 138
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 99.9% Indels: 0
Gaps: 0
US-19-479-699-1 (1-417) x US-16-649-385-2 (1-138)
Qy 1 ATGGCTTATGCTCAGTGGGTTATCATCATAATCCACAACGTCGGCTCAAAGGACGTTAAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetAlaTyrAlaGlnTrpValIleIleIleIleHisAsnValGlySerLysAspValLys 20
Qy 61 ATTGTCAATCTCAAACCAAGTTGGGGTAAATTACACGCTGACGGTGATAAAGACACCGAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 IleValAsnLeuLysProSerTrpGlyLysLeuHisAlaAspGlyAspLysAspThrGlu 40
Qy 121 GTGTCTGCATCAAAGTATGAGGGGACCGTTATCAAGCCCGATGAAAAACTCCAAATCAAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 41 ValSerAlaSerLysTyrGluGlyThrValIleLysProAspGluLysLeuGlnIleAsn 60
Qy 181 GCTTGCGGACGTTCAGACGCAGCAGAAGGAACAACTGGAACTTTCGATCTAGTTGACCCA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaCysGlyArgSerAspAlaAlaGluGlyThrThrGlyThrPheAspLeuValAspPro 80
Qy 241 GCCGACGGTGATAAGCAAGTCAGACATTTCTACTGGGATTGTCCTTGGGGAAGCAAAGCT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 81 AlaAspGlyAspLysGlnValArgHisPheTyrTrpAspCysProTrpGlySerLysAla 100
Qy 301 AACACTTGGACAGTCTCAGGAAGCAACACTAAATGGATGATTGAGTATTCAGGACAGAAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 AsnThrTrpThrValSerGlySerAsnThrLysTrpMetIleGluTyrSerGlyGlnAsn 120
Qy 361 TTGGACTCTGGTGCATTAGGGACAATTACTGTTGATACTTTAAAAAAAGGAAAC 414
||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LeuAspSerGlyAlaLeuGlyThrIleThrValAspThrLeuLysLysGlyAsn 138
US-16-649-385-2
Sequence 2, US/16649385
Publication No. US20200245628A1
GENERAL INFORMATION
APPLICANT: Univerza v Ljubljani
APPLICANT: Kmetijski institut Slovenije
TITLE OF INVENTION: New bio-pesticides for controlling plant pests
FILE REFERENCE: UL01P004WO
CURRENT APPLICATION NUMBER: US/16/649,385
CURRENT FILING DATE: 2020-03-20
NUMBER OF SEQ ID NOS: 5
SEQ ID NO 2
LENGTH: 138
TYPE: PRT
ORGANISM: Pleurotus eryngii
Length: 138
Score: 743.00 Matches: 136
Percent Similarity: 100.0% Conservative: 2
Best Local Similarity: 98.6% Mismatches: 0
Query Match: 99.1% Indels: 0
Gaps: 0
US-19-479-699-2 (1-417) x US-16-649-385-2 (1-138)
Qy 1 ATGGCTTATGCTCAGTGGGTTATCATCATAATCCACAACGTCGGCTCAAAGGACGTTAAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetAlaTyrAlaGlnTrpValIleIleIleIleHisAsnValGlySerLysAspValLys 20
Qy 61 ATTGTCAATCTCAAACCAAGTTGGGGTAAATTACACGCTGACGGTGATAAAGACACCGAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 IleValAsnLeuLysProSerTrpGlyLysLeuHisAlaAspGlyAspLysAspThrGlu 40
Qy 121 GTGTCTGCATCAAAGTATGAGGGGACCGTTATCAGACCCGATGAAAAACTCCAAATCAAT 180
|||||||||||||||||||||||||||||||||:::||||||||||||||||||||||||
Db 41 ValSerAlaSerLysTyrGluGlyThrValIleLysProAspGluLysLeuGlnIleAsn 60
Qy 181 GCTTGCGGACGTTCAGACGCAGCAGAAGGAACAACTGGAACTTTCGATCTAGTTGACCCA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaCysGlyArgSerAspAlaAlaGluGlyThrThrGlyThrPheAspLeuValAspPro 80
Qy 241 GCCGACGGTGATAGACAAGTCAGACATTTCTACTGGGATTGTCCTTGGGGAAGCAAAGCT 300
||||||||||||:::|||||||||||||||||||||||||||||||||||||||||||||
Db 81 AlaAspGlyAspLysGlnValArgHisPheTyrTrpAspCysProTrpGlySerLysAla 100
Qy 301 AACACTTGGACAGTCTCAGGAAGCAACACTAAATGGATGATTGAGTATTCAGGACAGAAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 AsnThrTrpThrValSerGlySerAsnThrLysTrpMetIleGluTyrSerGlyGlnAsn 120
Qy 361 TTGGACTCTGGTGCATTAGGGACAATTACTGTTGATACTTTAAAAAAAGGAAAC 414
||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LeuAspSerGlyAlaLeuGlyThrIleThrValAspThrLeuLysLysGlyAsn 138
Panevska teaches a PlyB (Panevska SEQ ID NO: 4) with 100% sequence identity to 523 amino acids encoded by instant SEQ ID NO: 4, or 96% match to the full query. See alignment below.
US-16-649-385-4
Sequence 4, US/16649385
Publication No. US20200245628A1
GENERAL INFORMATION
APPLICANT: Univerza v Ljubljani
APPLICANT: Kmetijski institut Slovenije
TITLE OF INVENTION: New bio-pesticides for controlling plant pests
FILE REFERENCE: UL01P004WO
CURRENT APPLICATION NUMBER: US/16/649,385
CURRENT FILING DATE: 2020-03-20
NUMBER OF SEQ ID NOS: 5
SEQ ID NO 4
LENGTH: 523
TYPE: PRT
ORGANISM: Pleurotus ostreatus
Length: 523
Score: 2694.00 Matches: 523
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 96.2% Indels: 0
Gaps: 0
US-19-479-699-4 (1-1572) x US-16-649-385-4 (1-523)
Qy 1 ATGGAAGCCGTTCTATCCCGTCAAGCTGCAACTGCAGAAGCTATAGGTCGTTTTCAAGAT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetGluAlaValLeuSerArgGlnAlaAlaThrAlaGluAlaIleGlyArgPheGlnAsp 20
Qy 61 TCATCCACAAGCGTCGGACTAGTAGCCGGCAGTCCCAGTACTAGGATCCGTCGTCAAGCC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 SerSerThrSerValGlyLeuValAlaGlySerProSerThrArgIleArgArgGlnAla 40
Qy 121 GATAACGTAGTACTCAAATCCACTTCTCAGGCTGGAGACACCCTAAACGACGTAATCCAG 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 41 AspAsnValValLeuLysSerThrSerGlnAlaGlyAspThrLeuAsnAspValIleGln 60
Qy 181 GACCCAACAAGGAGAAATAAACTAATAAACGACAACAACCTCCTAAAAGGCATAATTATG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AspProThrArgArgAsnLysLeuIleAsnAspAsnAsnLeuLeuLysGlyIleIleMet 80
Qy 241 GGTCGTGACGGACCAGTTCCCTCCTCACGTGAGTTAATTGTGCGACCAGATACACTCAGG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 81 GlyArgAspGlyProValProSerSerArgGluLeuIleValArgProAspThrLeuArg 100
Qy 301 GCAATAATTAACAACAGAGCAACTATAGAAACTACCACCATGGAGGCTGAGTTTACCGAG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 AlaIleIleAsnAsnArgAlaThrIleGluThrThrThrMetGluAlaGluPheThrGlu 120
Qy 361 ACATTGATGGAAAGCAATTACAATAGCGCATCCGTTAAGGTATCAGCACCATTTATTACA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ThrLeuMetGluSerAsnTyrAsnSerAlaSerValLysValSerAlaProPheIleThr 140
Qy 421 GCCAACTCCGAATATAGCGAGAGTTCCTCCTTCAAAAACACCGAGACTGAGAAATCCATG 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 141 AlaAsnSerGluTyrSerGluSerSerSerPheLysAsnThrGluThrGluLysSerMet 160
Qy 481 TATACCTCATCCAGGTATTTATTCCCTCAGGGGCGTATTGATTTTACCACACCAGACAGC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 161 TyrThrSerSerArgTyrLeuPheProGlnGlyArgIleAspPheThrThrProAspSer 180
Qy 541 GGTTTTGACGATGTCATTAAGTTATCTCCTCAGTTTACAAGTGGAGTACAAGCTGCCTTA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 GlyPheAspAspValIleLysLeuSerProGlnPheThrSerGlyValGlnAlaAlaLeu 200
Qy 601 GCAAAGGCTACCGGGACTGAAAAGCGTGAAGCCTTGCAAAACTTGTTCCAAGAATATGGA 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 201 AlaLysAlaThrGlyThrGluLysArgGluAlaLeuGlnAsnLeuPheGlnGluTyrGly 220
Qy 661 CACGTTTTCAGAACTAAAGTTCACATTGGAGGTGTCCTATCAGCACATACTATGGAAACA 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 221 HisValPheArgThrLysValHisIleGlyGlyValLeuSerAlaHisThrMetGluThr 240
Qy 721 TTCTCTCGATCCGAAAATGAAACCGAAGTGAAACAAGATGTCAAAGCTGGTCTTGAGGGT 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 PheSerArgSerGluAsnGluThrGluValLysGlnAspValLysAlaGlyLeuGluGly 260
Qy 781 GCCGTCAAGGGTTGGGGAGGCGGGGCTACCGCCGGTCACGGCAATACACAAGGGACCATT 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 261 AlaValLysGlyTrpGlyGlyGlyAlaThrAlaGlyHisGlyAsnThrGlnGlyThrIle 280
Qy 841 ACAACAAGTCAGAACCGAAAATTGAATGTGAAATACATCGTTAATGGTGGAGACTACACT 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 281 ThrThrSerGlnAsnArgLysLeuAsnValLysTyrIleValAsnGlyGlyAspTyrThr 300
Qy 901 AAAATCCAGAATACAGAAGAGTGGGTGGCATCTACAAATCAAAGCGAGCACTGGCGAGTA 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LysIleGlnAsnThrGluGluTrpValAlaSerThrAsnGlnSerGluHisTrpArgVal 320
Qy 961 ATCGAAGTGACCGAAGTAACCGCAGTTGCCGATCTCCTTCCACAACCCATAAGGGGTCAA 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 321 IleGluValThrGluValThrAlaValAlaAspLeuLeuProGlnProIleArgGlyGln 340
Qy 1021 GTGAAGGACCTATTGAAGCCACTACTTGGGAAGTGGGTCGACGTCGAAAAGGTTCCTGGG 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 341 ValLysAspLeuLeuLysProLeuLeuGlyLysTrpValAspValGluLysValProGly 360
Qy 1081 TTGGAATCATTACCTGTCAGTGTCTATAGACCCAAAGGGGCAATCCCAGCAGGTTGGTTC 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LeuGluSerLeuProValSerValTyrArgProLysGlyAlaIleProAlaGlyTrpPhe 380
Qy 1141 TGGCTTGGTGATACAGCAGACGCTTCTAAAGCCCTCTTGGTCAAACCTACCTTACCCGCC 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 381 TrpLeuGlyAspThrAlaAspAlaSerLysAlaLeuLeuValLysProThrLeuProAla 400
Qy 1201 CGAAGCGGGAGAAATCCCGCCCTTACTAGCCTTCACCAGGGATCCGGAATGACTGAGCAG 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 401 ArgSerGlyArgAsnProAlaLeuThrSerLeuHisGlnGlySerGlyMetThrGluGln 420
Qy 1261 CCATTTGTCGATCTTCCTCAATATCAGTACCTCAGCACATACTTTGGTTCATTTGCTCAC 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 ProPheValAspLeuProGlnTyrGlnTyrLeuSerThrTyrPheGlySerPheAlaHis 440
Qy 1321 GACACCCCCCCTGGCTCTACTCTTCGAGGCCTTCGACCAGATCATGTCTTGCCAGGACGA 1380
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 441 AspThrProProGlySerThrLeuArgGlyLeuArgProAspHisValLeuProGlyArg 460
Qy 1381 TATGAGATGCACGGTGATACTATATCTACTGCCGTATACGTTACTCGTCCTGTGGACGTT 1440
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 461 TyrGluMetHisGlyAspThrIleSerThrAlaValTyrValThrArgProValAspVal 480
Qy 1441 CCCTTTCCAGAGGACGAGTGTTTTGACTTAAAATCCCTTGTTCGTGTAAAATTGCCCGGT 1500
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 ProPheProGluAspGluCysPheAspLeuLysSerLeuValArgValLysLeuProGly 500
Qy 1501 TCCGGTAATCCACCCAAGCCCAGGTCTGCATTGAAAAAAAGCATGGTTCTTTTTGATAGT 1560
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 501 SerGlyAsnProProLysProArgSerAlaLeuLysLysSerMetValLeuPheAspSer 520
Qy 1561 GGGGAAAAG 1569
|||||||||
Db 521 GlyGluLys 523
Panevska teaches an embodiment wherein a transgenic plant or progeny expresses a bi-component protein complex, including a complex comprising PlyA2 and PlyB (paragraph [0073]). Panevska also teaches an embodiment wherein the plant comprises recombinant nucleic acid molecules comprising sequences that encode a bi-component protein complex operably linked to a promoter functional to cause the production of mRNA molecules (paragraph [0074]), including wherein the plant is a potato or maize plant (paragraph [0076]). Panevska teaches that the plant pest controlled by the bi-component protein complex may be an insect of the family Chrysomelidae (paragraph [0133]).
Panevska teaches that promoters employed that are functional in a plant cell to cause the production of an mRNA molecule may be constitutive (paragraph [0189]).
Panevska does not teach expression of PlyB with an endoplasmic reticulum localization signal. Panevska does not teach a transgenic plant expressing the nucleic acid sequences of SEQ ID NO: 1, 2 or 4.
Zhu teaches that the higher-order structure formation of peptides is largely completed within the endoplasmic reticulum (ER), and that those that are retained in the ER contain an ER retention signal at the C-terminus consisting of a four-amino acid residue KDEL or HDEL sequence (paragraph [0007]). Zhu teaches that combining an insect-resistant gene with a signal peptide sequence and a KDEL sequence produces a novel insect-resistant protein with enhanced stability and increases the accumulation of the insect-resistant protein in transgenic plants (paragraph [0008]).
Zhu teaches a nucleotide sequence encoding an insect-resistant fusion protein comprising a signal peptide at the 5’ end and an ER retention signal at the 3’ end (paragraph [0017], figure 3-1). Expression in transgenic tobacco of a modified gene comprising the KDEL sequence was significantly higher than that of the unmodified gene (paragraph [0055]). The localization was effective for multiple insect-resistant proteins (paragraph [0115]).
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the transgenic plants taught by Panevska to add a C-terminal endoplasmic reticulum localization signal to PlyB. One of ordinary skill in the art would have been motivated to add KDEL sequence to locate the anti-insect protein into the endoplasmic reticulum inside the plant’s cells to increase stability and accumulated amount of anti-insect protein in the transgenic plants. One of ordinary skill in the art would have had reasonable expectation of success because fusion anti-insect proteins comprising a KDEL sequence had higher expression in transgenic plants, and localization was effective for both Bt and CpTI proteins.
The transgenic plants of Panevska, in light of Zhu, make obvious the transgenic plants of instant claims 1-3, 5-6, 10 & 20-21. The PlyB and PlyA2 amino acid sequences of Panevska read on polypeptides comprising an amino acid sequence having at least 70% identity with the polypeptide encoded by SEQ ID NOs: 1, 2 and 4. It would have been obvious to one of ordinary skill in the art to use an endoplasmic reticulum localization signal of instant SEQ ID NO: 5, which encodes an amino acid sequence of KDEL, because one of ordinary skill in the art would have known how to design and optimize nucleic acid sequences encoding the localization signal amino acid sequence (instant claim 11).
It is indefinite what the plant pest recited in claims 24, 29 & 30 is, but Panevska makes obvious a transgenic plant expressing PlyA2 and PlyB wherein the plant inhibits the growth of CPB or WCR or controls an insect of the family Chrysomelidae, which reads on a plant of claims 24, 29 and 30. It would furthermore have been obvious to place expression of PlyA2 and/or PlyB under control of a constitutive promoter (claims 8 & 14), because Panevska teaches that constitutive promoters may be used to cause the production of the mRNA of the polypeptides of the bio-component protein complex.
A transgenic plant which expresses PlyA2 and PlyB, wherein PlyB is expressed with an endoplasmic reticulum localization signal, makes obvious the single step method of claim 32 requiring expressing PlyA2 and PlyB. Panevska’s method of feeding CPB and WCR with materials comprising PlyA2 and PlyB also makes obvious the method of instant claims 37 & 38, wherein an insect plant pest of CPB or WCR is inhibited in growth by expressing PlyA2 and PlyB in a transgenic plant, and the use of the transgenic plant of instant claims 40 & 44.
Claims 1-3, 5-6, 8, 10-11, 14, 20-21, 24, 29-30, 32, 37-38, 40 & 44 are obvious over Panevska and Zhu.
Claim(s) 4 is rejected under 35 U.S.C. 103 as being unpatentable over Panevska and Zhu as applied to claims 1-3, 5-6, 8, 10-11, 14, 20-21, 24, 29-30, 32, 37-38, 40 & 44 above, and further in view of Singh et al (2014) Current Trends in Biotechnology and Pharmacy. 8(3): 276-287. (published July 2014, hereafter Singh).
Claim 4 is drawn to a transgenic plant wherein PlyA2 is expressed with vacuole localization signal.
The teachings of Panevska and Zhu are presented above. They do not teach PlyA2 expressed with a vacuole localization signal.
Singh teaches that increasing heterologous expression of delta endotoxins in transgenic plants is pursued as a means to increase efficacy and to delay insect resistance (abstract). Singh teaches that innovations such as deleting mRNA destabilizing sequences have increased transgene expression levels in plants to provide viable insect resistant plants, but increasing expression of insecticidal proteins like Bt to the maximum level possible without affecting normal physiological state of the plant is necessary to delay insect resistance (page 277, left column, paragraph 1). Singh teaches that targeting protein to subcellular organelles can enhance stability of foreign proteins in the cells, increasing quantity of heterologous proteins. The vacuole acts as an alternate site for the storage of proteins in large quantities and is a promising candidate for the storage of heterologous proteins (page 277, left column, paragraph 2-right column, paragraph 1).
Singh teaches targeting Cry1Ac protein to the vacuole with a 69 bp N-terminus peptide targeting sequence from soybean vegetative storage protein (VSPαS) and a 27 bp C-terminal vacuolar targeting sequence of tobacco chitinase A (page 277, right column, paragraph 2-page 278, left column, paragraph 1, figure 1).
Transgenic plants comprising the vacuole localization had no significant difference in expression (page 284, left column, paragraph 2; figure 6). Singh teaches that vacuole localization can be used for expressing the insecticidal Bt toxin on par with cytosol, which could be useful when adopting gene pyramiding (page 284, left column, paragraph 1).
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the PlyA2 expressed by a transgenic plant to express the protein with a vacuole localization signal. One of ordinary skill in the art would have been motivated to use a vacuole localization signal to enhance stability of or increase storage of the foreign proteins in the plant cells. One of ordinary skill in the art would have had reasonable expectation of success, because Bt proteins expressed with a vacuole localization signal was on par with Bt proteins expressed in the cytosol of transgenic plants. Instant claim 4 would have therefore been obvious.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-2, 5, 6, 10, 11, 20, 21, 24, 29, 30, 32, 37, 38, 40 & 44 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 5, 11, 13, & 16-19 of U.S. Patent No. 12,049,637 (hereafter ‘637) in view of Zhu et al (CN1229087A, published 9/22/1999, hereafter Zhu. Appended with a machine translation of the Description).
‘637 claim 1 is drawn to a method for protecting a plant against a plant pest or controlling a plant pest comprising applying a composition comprising a plurality of molecules selected from ostreolysin and pleurotolysin , a plurality of molecules of pleurotolysin B and a suitable carrier to said plant wherein the pleurotolysin A is a polypeptide comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 2 and PlyB is a polypeptide comprising an amino acid sequence havin gat least 95% sequence identity with ‘637 SEQ ID NO: 4, wherein the plant pest is an insect of the family Chrysomelidae. ‘637 claim 5 further requires pleurotolysin A of ‘637 SEQ ID NO: 2.
‘637 claim 11 requires the insect is Leptinotarsa decemlineata and ‘637 claim 13 requires that the insect is Diabrotica virgifera virgifera. ‘637 claim 16 requires that the plant is a crop plant and claims 17-19 require that the plant is a potato plant or maize plant.
‘637 SEQ ID NO: 2 comprises a sequence with 100% similarity to the amino acid sequence encoded by instant SEQ ID NO: 1 (see first alignment below) and >99% match to the amino acid sequence encoded by instant SEQ ID NO: 2 (see second alignment below).
US-16-649-385-2
Sequence 2, US/16649385
Patent No. 12049637
GENERAL INFORMATION
APPLICANT: Univerza v Ljubljani
APPLICANT: Kmetijski institut Slovenije
TITLE OF INVENTION: New bio-pesticides for controlling plant pests
FILE REFERENCE: UL01P004WO
CURRENT APPLICATION NUMBER: US/16/649,385
CURRENT FILING DATE: 2020-03-20
NUMBER OF SEQ ID NOS: 5
SEQ ID NO 2
LENGTH: 138
TYPE: PRT
ORGANISM: Pleurotus eryngii
Length: 138
Score: 749.00 Matches: 138
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 99.9% Indels: 0
Gaps: 0
US-19-479-699-1 (1-417) x US-16-649-385-2 (1-138)
Qy 1 ATGGCTTATGCTCAGTGGGTTATCATCATAATCCACAACGTCGGCTCAAAGGACGTTAAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetAlaTyrAlaGlnTrpValIleIleIleIleHisAsnValGlySerLysAspValLys 20
Qy 61 ATTGTCAATCTCAAACCAAGTTGGGGTAAATTACACGCTGACGGTGATAAAGACACCGAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 IleValAsnLeuLysProSerTrpGlyLysLeuHisAlaAspGlyAspLysAspThrGlu 40
Qy 121 GTGTCTGCATCAAAGTATGAGGGGACCGTTATCAAGCCCGATGAAAAACTCCAAATCAAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 41 ValSerAlaSerLysTyrGluGlyThrValIleLysProAspGluLysLeuGlnIleAsn 60
Qy 181 GCTTGCGGACGTTCAGACGCAGCAGAAGGAACAACTGGAACTTTCGATCTAGTTGACCCA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaCysGlyArgSerAspAlaAlaGluGlyThrThrGlyThrPheAspLeuValAspPro 80
Qy 241 GCCGACGGTGATAAGCAAGTCAGACATTTCTACTGGGATTGTCCTTGGGGAAGCAAAGCT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 81 AlaAspGlyAspLysGlnValArgHisPheTyrTrpAspCysProTrpGlySerLysAla 100
Qy 301 AACACTTGGACAGTCTCAGGAAGCAACACTAAATGGATGATTGAGTATTCAGGACAGAAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 AsnThrTrpThrValSerGlySerAsnThrLysTrpMetIleGluTyrSerGlyGlnAsn 120
Qy 361 TTGGACTCTGGTGCATTAGGGACAATTACTGTTGATACTTTAAAAAAAGGAAAC 414
||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LeuAspSerGlyAlaLeuGlyThrIleThrValAspThrLeuLysLysGlyAsn 138
US-16-649-385-2
Sequence 2, US/16649385
Patent No. 12049637
GENERAL INFORMATION
APPLICANT: Univerza v Ljubljani
APPLICANT: Kmetijski institut Slovenije
TITLE OF INVENTION: New bio-pesticides for controlling plant pests
FILE REFERENCE: UL01P004WO
CURRENT APPLICATION NUMBER: US/16/649,385
CURRENT FILING DATE: 2020-03-20
NUMBER OF SEQ ID NOS: 5
SEQ ID NO 2
LENGTH: 138
TYPE: PRT
ORGANISM: Pleurotus eryngii
Length: 138
Score: 743.00 Matches: 136
Percent Similarity: 100.0% Conservative: 2
Best Local Similarity: 98.6% Mismatches: 0
Query Match: 99.1% Indels: 0
Gaps: 0
US-19-479-699-2 (1-417) x US-16-649-385-2 (1-138)
Qy 1 ATGGCTTATGCTCAGTGGGTTATCATCATAATCCACAACGTCGGCTCAAAGGACGTTAAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetAlaTyrAlaGlnTrpValIleIleIleIleHisAsnValGlySerLysAspValLys 20
Qy 61 ATTGTCAATCTCAAACCAAGTTGGGGTAAATTACACGCTGACGGTGATAAAGACACCGAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 IleValAsnLeuLysProSerTrpGlyLysLeuHisAlaAspGlyAspLysAspThrGlu 40
Qy 121 GTGTCTGCATCAAAGTATGAGGGGACCGTTATCAGACCCGATGAAAAACTCCAAATCAAT 180
|||||||||||||||||||||||||||||||||:::||||||||||||||||||||||||
Db 41 ValSerAlaSerLysTyrGluGlyThrValIleLysProAspGluLysLeuGlnIleAsn 60
Qy 181 GCTTGCGGACGTTCAGACGCAGCAGAAGGAACAACTGGAACTTTCGATCTAGTTGACCCA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaCysGlyArgSerAspAlaAlaGluGlyThrThrGlyThrPheAspLeuValAspPro 80
Qy 241 GCCGACGGTGATAGACAAGTCAGACATTTCTACTGGGATTGTCCTTGGGGAAGCAAAGCT 300
||||||||||||:::|||||||||||||||||||||||||||||||||||||||||||||
Db 81 AlaAspGlyAspLysGlnValArgHisPheTyrTrpAspCysProTrpGlySerLysAla 100
Qy 301 AACACTTGGACAGTCTCAGGAAGCAACACTAAATGGATGATTGAGTATTCAGGACAGAAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 AsnThrTrpThrValSerGlySerAsnThrLysTrpMetIleGluTyrSerGlyGlnAsn 120
Qy 361 TTGGACTCTGGTGCATTAGGGACAATTACTGTTGATACTTTAAAAAAAGGAAAC 414
||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LeuAspSerGlyAlaLeuGlyThrIleThrValAspThrLeuLysLysGlyAsn 138
‘637 SEQ ID NO: 4 comprises a sequence with 96% match to the sequence of instant SEQ ID NO: 4. See alignment below.
US-16-649-385-4
Sequence 4, US/16649385
Patent No. 12049637
GENERAL INFORMATION
APPLICANT: Univerza v Ljubljani
APPLICANT: Kmetijski institut Slovenije
TITLE OF INVENTION: New bio-pesticides for controlling plant pests
FILE REFERENCE: UL01P004WO
CURRENT APPLICATION NUMBER: US/16/649,385
CURRENT FILING DATE: 2020-03-20
NUMBER OF SEQ ID NOS: 5
SEQ ID NO 4
LENGTH: 523
TYPE: PRT
ORGANISM: Pleurotus ostreatus
Length: 523
Score: 2694.00 Matches: 523
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 96.2% Indels: 0
Gaps: 0
US-19-479-699-4 (1-1572) x US-16-649-385-4 (1-523)
Qy 1 ATGGAAGCCGTTCTATCCCGTCAAGCTGCAACTGCAGAAGCTATAGGTCGTTTTCAAGAT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetGluAlaValLeuSerArgGlnAlaAlaThrAlaGluAlaIleGlyArgPheGlnAsp 20
Qy 61 TCATCCACAAGCGTCGGACTAGTAGCCGGCAGTCCCAGTACTAGGATCCGTCGTCAAGCC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 SerSerThrSerValGlyLeuValAlaGlySerProSerThrArgIleArgArgGlnAla 40
Qy 121 GATAACGTAGTACTCAAATCCACTTCTCAGGCTGGAGACACCCTAAACGACGTAATCCAG 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 41 AspAsnValValLeuLysSerThrSerGlnAlaGlyAspThrLeuAsnAspValIleGln 60
Qy 181 GACCCAACAAGGAGAAATAAACTAATAAACGACAACAACCTCCTAAAAGGCATAATTATG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AspProThrArgArgAsnLysLeuIleAsnAspAsnAsnLeuLeuLysGlyIleIleMet 80
Qy 241 GGTCGTGACGGACCAGTTCCCTCCTCACGTGAGTTAATTGTGCGACCAGATACACTCAGG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 81 GlyArgAspGlyProValProSerSerArgGluLeuIleValArgProAspThrLeuArg 100
Qy 301 GCAATAATTAACAACAGAGCAACTATAGAAACTACCACCATGGAGGCTGAGTTTACCGAG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 AlaIleIleAsnAsnArgAlaThrIleGluThrThrThrMetGluAlaGluPheThrGlu 120
Qy 361 ACATTGATGGAAAGCAATTACAATAGCGCATCCGTTAAGGTATCAGCACCATTTATTACA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ThrLeuMetGluSerAsnTyrAsnSerAlaSerValLysValSerAlaProPheIleThr 140
Qy 421 GCCAACTCCGAATATAGCGAGAGTTCCTCCTTCAAAAACACCGAGACTGAGAAATCCATG 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 141 AlaAsnSerGluTyrSerGluSerSerSerPheLysAsnThrGluThrGluLysSerMet 160
Qy 481 TATACCTCATCCAGGTATTTATTCCCTCAGGGGCGTATTGATTTTACCACACCAGACAGC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 161 TyrThrSerSerArgTyrLeuPheProGlnGlyArgIleAspPheThrThrProAspSer 180
Qy 541 GGTTTTGACGATGTCATTAAGTTATCTCCTCAGTTTACAAGTGGAGTACAAGCTGCCTTA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 GlyPheAspAspValIleLysLeuSerProGlnPheThrSerGlyValGlnAlaAlaLeu 200
Qy 601 GCAAAGGCTACCGGGACTGAAAAGCGTGAAGCCTTGCAAAACTTGTTCCAAGAATATGGA 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 201 AlaLysAlaThrGlyThrGluLysArgGluAlaLeuGlnAsnLeuPheGlnGluTyrGly 220
Qy 661 CACGTTTTCAGAACTAAAGTTCACATTGGAGGTGTCCTATCAGCACATACTATGGAAACA 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 221 HisValPheArgThrLysValHisIleGlyGlyValLeuSerAlaHisThrMetGluThr 240
Qy 721 TTCTCTCGATCCGAAAATGAAACCGAAGTGAAACAAGATGTCAAAGCTGGTCTTGAGGGT 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 PheSerArgSerGluAsnGluThrGluValLysGlnAspValLysAlaGlyLeuGluGly 260
Qy 781 GCCGTCAAGGGTTGGGGAGGCGGGGCTACCGCCGGTCACGGCAATACACAAGGGACCATT 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 261 AlaValLysGlyTrpGlyGlyGlyAlaThrAlaGlyHisGlyAsnThrGlnGlyThrIle 280
Qy 841 ACAACAAGTCAGAACCGAAAATTGAATGTGAAATACATCGTTAATGGTGGAGACTACACT 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 281 ThrThrSerGlnAsnArgLysLeuAsnValLysTyrIleValAsnGlyGlyAspTyrThr 300
Qy 901 AAAATCCAGAATACAGAAGAGTGGGTGGCATCTACAAATCAAAGCGAGCACTGGCGAGTA 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LysIleGlnAsnThrGluGluTrpValAlaSerThrAsnGlnSerGluHisTrpArgVal 320
Qy 961 ATCGAAGTGACCGAAGTAACCGCAGTTGCCGATCTCCTTCCACAACCCATAAGGGGTCAA 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 321 IleGluValThrGluValThrAlaValAlaAspLeuLeuProGlnProIleArgGlyGln 340
Qy 1021 GTGAAGGACCTATTGAAGCCACTACTTGGGAAGTGGGTCGACGTCGAAAAGGTTCCTGGG 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 341 ValLysAspLeuLeuLysProLeuLeuGlyLysTrpValAspValGluLysValProGly 360
Qy 1081 TTGGAATCATTACCTGTCAGTGTCTATAGACCCAAAGGGGCAATCCCAGCAGGTTGGTTC 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LeuGluSerLeuProValSerValTyrArgProLysGlyAlaIleProAlaGlyTrpPhe 380
Qy 1141 TGGCTTGGTGATACAGCAGACGCTTCTAAAGCCCTCTTGGTCAAACCTACCTTACCCGCC 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 381 TrpLeuGlyAspThrAlaAspAlaSerLysAlaLeuLeuValLysProThrLeuProAla 400
Qy 1201 CGAAGCGGGAGAAATCCCGCCCTTACTAGCCTTCACCAGGGATCCGGAATGACTGAGCAG 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 401 ArgSerGlyArgAsnProAlaLeuThrSerLeuHisGlnGlySerGlyMetThrGluGln 420
Qy 1261 CCATTTGTCGATCTTCCTCAATATCAGTACCTCAGCACATACTTTGGTTCATTTGCTCAC 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 ProPheValAspLeuProGlnTyrGlnTyrLeuSerThrTyrPheGlySerPheAlaHis 440
Qy 1321 GACACCCCCCCTGGCTCTACTCTTCGAGGCCTTCGACCAGATCATGTCTTGCCAGGACGA 1380
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 441 AspThrProProGlySerThrLeuArgGlyLeuArgProAspHisValLeuProGlyArg 460
Qy 1381 TATGAGATGCACGGTGATACTATATCTACTGCCGTATACGTTACTCGTCCTGTGGACGTT 1440
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 461 TyrGluMetHisGlyAspThrIleSerThrAlaValTyrValThrArgProValAspVal 480
Qy 1441 CCCTTTCCAGAGGACGAGTGTTTTGACTTAAAATCCCTTGTTCGTGTAAAATTGCCCGGT 1500
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 ProPheProGluAspGluCysPheAspLeuLysSerLeuValArgValLysLeuProGly 500
Qy 1501 TCCGGTAATCCACCCAAGCCCAGGTCTGCATTGAAAAAAAGCATGGTTCTTTTTGATAGT 1560
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 501 SerGlyAsnProProLysProArgSerAlaLeuLysLysSerMetValLeuPheAspSer 520
Qy 1561 GGGGAAAAG 1569
|||||||||
Db 521 GlyGluLys 523
‘637 does not require a plant expressing the PlyA2 and PlyB molecules or a PlyB molecule with endoplasmic reticulum localization signal.
Zhu teaches a method of creating a transgenic plant using a gene encoding an insect-resistant protein and an expression vector containing the encoding gene (paragraph [0001]). Zhu teaches an expression vector containing the encoding gene comprising a P-35S promoter (figure 14).
Zhu teaches that plant genetic engineering to introduce an exogenous gene into a recipient plant, including genetic engineering insect-resistant plants, has great application prospects in reducing environmental pollution caused by pesticides and reducing the losses to crop production caused by pests (paragraph [0002]).
Zhu teaches that the higher-order structure formation of peptides is largely completed within the endoplasmic reticulum (ER), and that those that are retained in the ER contain an ER retention signal at the C-terminus consisting of a four-amino acid residue KDEL or HDEL sequence (paragraph [0007]). Zhu teaches that combining an insect-resistant gene with a signal peptide sequence and a KDEL sequence produces a novel insect-resistant protein with enhanced stability and increases the accumulation of the insect-resistant protein in transgenic plants (paragraph [0008]).
Zhu teaches a nucleotide sequence encoding an insect-resistant fusion protein comprising a signal peptide at the 5’ end and an ER retention signal at the 3’ end (paragraph [0017], figure 3-1). Expression in transgenic tobacco of a modified gene comprising the KDEL sequence was significantly higher than that of the unmodified gene (paragraph [0055]). The localization was effective for multiple insect-resistant proteins (paragraph [0115]).
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method taught by Panevska to generate a transgenic plant expressing the PlyA and PlyB molecules, including with an endoplasmic reticulum localization signal on PlyB. One of ordinary skill in the art would have been motivated to express the insecticide PlyA and PlyB molecules in a plant to reduce crop losses to pests and further motivated to add a KDEL sequence to locate the anti-insect protein into the endoplasmic reticulum inside the plant’s cells to increase stability and accumulated amount of anti-insect protein in the transgenic plants. One of ordinary skill in the art would have had reasonable expectation of success because fusion anti-insect proteins comprising a KDEL sequence had higher expression in transgenic plants.
‘637 SEQ ID NO: 2 and 4 read on a PlyA2 encoded by instant SEQ ID NOs 1 or 2 and instant SEQ ID NO: 4 respectively. It would have been obvious to transform the plant with a polynucleotide operably linked to at least one promoter that is functional in a transgenic plant to cause the production of mRNA molecules, because the protein would need to be expressed in order to provide insect resistance, as taught by Zhu. It would have been obvious to one of ordinary skill in the art to use an endoplasmic reticulum localization signal of instant SEQ ID NO: 5, which encodes an amino acid sequence of KDEL, because one of ordinary skill in the art would have known how to design and optimize nucleic acid sequences encoding the localization signal amino acid sequence (instant claim 11). A transgenic plant which expresses PlyA2 and PlyB, wherein PlyB is expressed with an endoplasmic reticulum localization signal, makes obvious the single step method of claim 32 requiring expressing PlyA2 and PlyB.
Thus, in light of Zhu, ‘637 makes obvious the transgenic plant of instant claims 1-2, 5-6, 10, 11, 20-21, 24, and 29-30, the method of inhibiting growth of a plant pest, which reads on controlling, of claims 32, 37 & 38, and the use of the transgenic plant in claims 40 & 44.
Claims 1-2, 5, 6, 10, 11, 20, 21, 24, 29, 32, 37 & 38 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 6-9, 12 & 14-20 of copending Application No. 18/644,512 in view of Zhu et al (CN1229087A, published 9/22/1999, hereafter Zhu. Appended with a machine translation of the Description).
This is a provisional nonstatutory double patenting rejection.
‘512 claim 1 is drawn to a method comprising the step of transforming a plant by introducing into a genome one or more nucleic acid constructs encoding a bi-component protein complex consisting of a plurality of molecules selected from the group consisting of ostreolysin and pleurotolysin A and molecules of pleurotolysin B.
‘512 claim 6 is drawn to the method wherein the pleurotolysin A is a polypeptide comprising an amino acid sequence having at least 95% sequence identity with ‘512 SEQ ID NO: 2. ‘512 SEQ ID NO: 2 is a polypeptide with 100% identity to the amino acid encoded by instant SEQ ID NO: 1 and 98.6% similarity to the amino acid encoded by instant SEQ ID NO: 2 (see alignments below).
Sequence 2, US/18644512
Publication No. US20240287540A1
GENERAL INFORMATION
APPLICANT: University of Ljubljana (en)
TITLE OF INVENTION: New bio-pesticides for controlling plant pests (en)
FILE REFERENCE: ALBG1PUS02CIP / P6758US01
CURRENT APPLICATION NUMBER: US/18/644,512
CURRENT FILING DATE: 2024-04-24
NUMBER OF SEQ ID NOS: 5
SEQ ID NO 2
LENGTH: 138
TYPE: PRT
FEATURE:
NAME/KEY: source
LOCATION: 1..138
QUALIFIERS: mol_type = protein
organism = Pleurotus eryngii
Length: 138
Score: 749.00 Matches: 138
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 99.9% Indels: 0
Gaps: 0
US-19-479-699-1 (1-417) x US-16-649-385-2 (1-138)
Qy 1 ATGGCTTATGCTCAGTGGGTTATCATCATAATCCACAACGTCGGCTCAAAGGACGTTAAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetAlaTyrAlaGlnTrpValIleIleIleIleHisAsnValGlySerLysAspValLys 20
Qy 61 ATTGTCAATCTCAAACCAAGTTGGGGTAAATTACACGCTGACGGTGATAAAGACACCGAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 IleValAsnLeuLysProSerTrpGlyLysLeuHisAlaAspGlyAspLysAspThrGlu 40
Qy 121 GTGTCTGCATCAAAGTATGAGGGGACCGTTATCAAGCCCGATGAAAAACTCCAAATCAAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 41 ValSerAlaSerLysTyrGluGlyThrValIleLysProAspGluLysLeuGlnIleAsn 60
Qy 181 GCTTGCGGACGTTCAGACGCAGCAGAAGGAACAACTGGAACTTTCGATCTAGTTGACCCA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaCysGlyArgSerAspAlaAlaGluGlyThrThrGlyThrPheAspLeuValAspPro 80
Qy 241 GCCGACGGTGATAAGCAAGTCAGACATTTCTACTGGGATTGTCCTTGGGGAAGCAAAGCT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 81 AlaAspGlyAspLysGlnValArgHisPheTyrTrpAspCysProTrpGlySerLysAla 100
Qy 301 AACACTTGGACAGTCTCAGGAAGCAACACTAAATGGATGATTGAGTATTCAGGACAGAAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 AsnThrTrpThrValSerGlySerAsnThrLysTrpMetIleGluTyrSerGlyGlnAsn 120
Qy 361 TTGGACTCTGGTGCATTAGGGACAATTACTGTTGATACTTTAAAAAAAGGAAAC 414
||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LeuAspSerGlyAlaLeuGlyThrIleThrValAspThrLeuLysLysGlyAsn 138
Sequence 2, US/18644512
Publication No. US20240287540A1
GENERAL INFORMATION
APPLICANT: University of Ljubljana (en)
TITLE OF INVENTION: New bio-pesticides for controlling plant pests (en)
FILE REFERENCE: ALBG1PUS02CIP / P6758US01
CURRENT APPLICATION NUMBER: US/18/644,512
CURRENT FILING DATE: 2024-04-24
NUMBER OF SEQ ID NOS: 5
SEQ ID NO 2
LENGTH: 138
TYPE: PRT
FEATURE:
NAME/KEY: source
LOCATION: 1..138
QUALIFIERS: mol_type = protein
organism = Pleurotus eryngii
Length: 138
Score: 743.00 Matches: 136
Percent Similarity: 100.0% Conservative: 2
Best Local Similarity: 98.6% Mismatches: 0
Query Match: 99.1% Indels: 0
Gaps: 0
US-19-479-699-2 (1-417) x US-16-649-385-2 (1-138)
Qy 1 ATGGCTTATGCTCAGTGGGTTATCATCATAATCCACAACGTCGGCTCAAAGGACGTTAAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetAlaTyrAlaGlnTrpValIleIleIleIleHisAsnValGlySerLysAspValLys 20
Qy 61 ATTGTCAATCTCAAACCAAGTTGGGGTAAATTACACGCTGACGGTGATAAAGACACCGAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 IleValAsnLeuLysProSerTrpGlyLysLeuHisAlaAspGlyAspLysAspThrGlu 40
Qy 121 GTGTCTGCATCAAAGTATGAGGGGACCGTTATCAGACCCGATGAAAAACTCCAAATCAAT 180
|||||||||||||||||||||||||||||||||:::||||||||||||||||||||||||
Db 41 ValSerAlaSerLysTyrGluGlyThrValIleLysProAspGluLysLeuGlnIleAsn 60
Qy 181 GCTTGCGGACGTTCAGACGCAGCAGAAGGAACAACTGGAACTTTCGATCTAGTTGACCCA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaCysGlyArgSerAspAlaAlaGluGlyThrThrGlyThrPheAspLeuValAspPro 80
Qy 241 GCCGACGGTGATAGACAAGTCAGACATTTCTACTGGGATTGTCCTTGGGGAAGCAAAGCT 300
||||||||||||:::|||||||||||||||||||||||||||||||||||||||||||||
Db 81 AlaAspGlyAspLysGlnValArgHisPheTyrTrpAspCysProTrpGlySerLysAla 100
Qy 301 AACACTTGGACAGTCTCAGGAAGCAACACTAAATGGATGATTGAGTATTCAGGACAGAAT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 AsnThrTrpThrValSerGlySerAsnThrLysTrpMetIleGluTyrSerGlyGlnAsn 120
Qy 361 TTGGACTCTGGTGCATTAGGGACAATTACTGTTGATACTTTAAAAAAAGGAAAC 414
||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LeuAspSerGlyAlaLeuGlyThrIleThrValAspThrLeuLysLysGlyAsn 138
‘512 claim 7 is drawn to the method wherein PlyB is a polypeptide comprising an amino acid sequence having at least 95% sequence identity with ‘512 SEQ ID NO: 4. ‘512 SEQ ID NO: 4 has 96% match to the amino acid sequence encoded by instant SEQ ID NO: 4. See alignment below.
Sequence 4, US/18644512
Publication No. US20240287540A1
GENERAL INFORMATION
APPLICANT: University of Ljubljana (en)
TITLE OF INVENTION: New bio-pesticides for controlling plant pests (en)
FILE REFERENCE: ALBG1PUS02CIP / P6758US01
CURRENT APPLICATION NUMBER: US/18/644,512
CURRENT FILING DATE: 2024-04-24
NUMBER OF SEQ ID NOS: 5
SEQ ID NO 4
LENGTH: 523
TYPE: PRT
FEATURE:
NAME/KEY: source
LOCATION: 1..523
QUALIFIERS: mol_type = protein
organism = Pleurotus ostreatus
Length: 523
Score: 2694.00 Matches: 523
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 96.2% Indels: 0
Gaps: 0
US-19-479-699-4 (1-1572) x US-16-649-385-4 (1-523)
Qy 1 ATGGAAGCCGTTCTATCCCGTCAAGCTGCAACTGCAGAAGCTATAGGTCGTTTTCAAGAT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetGluAlaValLeuSerArgGlnAlaAlaThrAlaGluAlaIleGlyArgPheGlnAsp 20
Qy 61 TCATCCACAAGCGTCGGACTAGTAGCCGGCAGTCCCAGTACTAGGATCCGTCGTCAAGCC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 SerSerThrSerValGlyLeuValAlaGlySerProSerThrArgIleArgArgGlnAla 40
Qy 121 GATAACGTAGTACTCAAATCCACTTCTCAGGCTGGAGACACCCTAAACGACGTAATCCAG 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 41 AspAsnValValLeuLysSerThrSerGlnAlaGlyAspThrLeuAsnAspValIleGln 60
Qy 181 GACCCAACAAGGAGAAATAAACTAATAAACGACAACAACCTCCTAAAAGGCATAATTATG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AspProThrArgArgAsnLysLeuIleAsnAspAsnAsnLeuLeuLysGlyIleIleMet 80
Qy 241 GGTCGTGACGGACCAGTTCCCTCCTCACGTGAGTTAATTGTGCGACCAGATACACTCAGG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 81 GlyArgAspGlyProValProSerSerArgGluLeuIleValArgProAspThrLeuArg 100
Qy 301 GCAATAATTAACAACAGAGCAACTATAGAAACTACCACCATGGAGGCTGAGTTTACCGAG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 AlaIleIleAsnAsnArgAlaThrIleGluThrThrThrMetGluAlaGluPheThrGlu 120
Qy 361 ACATTGATGGAAAGCAATTACAATAGCGCATCCGTTAAGGTATCAGCACCATTTATTACA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ThrLeuMetGluSerAsnTyrAsnSerAlaSerValLysValSerAlaProPheIleThr 140
Qy 421 GCCAACTCCGAATATAGCGAGAGTTCCTCCTTCAAAAACACCGAGACTGAGAAATCCATG 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 141 AlaAsnSerGluTyrSerGluSerSerSerPheLysAsnThrGluThrGluLysSerMet 160
Qy 481 TATACCTCATCCAGGTATTTATTCCCTCAGGGGCGTATTGATTTTACCACACCAGACAGC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 161 TyrThrSerSerArgTyrLeuPheProGlnGlyArgIleAspPheThrThrProAspSer 180
Qy 541 GGTTTTGACGATGTCATTAAGTTATCTCCTCAGTTTACAAGTGGAGTACAAGCTGCCTTA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 GlyPheAspAspValIleLysLeuSerProGlnPheThrSerGlyValGlnAlaAlaLeu 200
Qy 601 GCAAAGGCTACCGGGACTGAAAAGCGTGAAGCCTTGCAAAACTTGTTCCAAGAATATGGA 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 201 AlaLysAlaThrGlyThrGluLysArgGluAlaLeuGlnAsnLeuPheGlnGluTyrGly 220
Qy 661 CACGTTTTCAGAACTAAAGTTCACATTGGAGGTGTCCTATCAGCACATACTATGGAAACA 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 221 HisValPheArgThrLysValHisIleGlyGlyValLeuSerAlaHisThrMetGluThr 240
Qy 721 TTCTCTCGATCCGAAAATGAAACCGAAGTGAAACAAGATGTCAAAGCTGGTCTTGAGGGT 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 PheSerArgSerGluAsnGluThrGluValLysGlnAspValLysAlaGlyLeuGluGly 260
Qy 781 GCCGTCAAGGGTTGGGGAGGCGGGGCTACCGCCGGTCACGGCAATACACAAGGGACCATT 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 261 AlaValLysGlyTrpGlyGlyGlyAlaThrAlaGlyHisGlyAsnThrGlnGlyThrIle 280
Qy 841 ACAACAAGTCAGAACCGAAAATTGAATGTGAAATACATCGTTAATGGTGGAGACTACACT 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 281 ThrThrSerGlnAsnArgLysLeuAsnValLysTyrIleValAsnGlyGlyAspTyrThr 300
Qy 901 AAAATCCAGAATACAGAAGAGTGGGTGGCATCTACAAATCAAAGCGAGCACTGGCGAGTA 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LysIleGlnAsnThrGluGluTrpValAlaSerThrAsnGlnSerGluHisTrpArgVal 320
Qy 961 ATCGAAGTGACCGAAGTAACCGCAGTTGCCGATCTCCTTCCACAACCCATAAGGGGTCAA 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 321 IleGluValThrGluValThrAlaValAlaAspLeuLeuProGlnProIleArgGlyGln 340
Qy 1021 GTGAAGGACCTATTGAAGCCACTACTTGGGAAGTGGGTCGACGTCGAAAAGGTTCCTGGG 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 341 ValLysAspLeuLeuLysProLeuLeuGlyLysTrpValAspValGluLysValProGly 360
Qy 1081 TTGGAATCATTACCTGTCAGTGTCTATAGACCCAAAGGGGCAATCCCAGCAGGTTGGTTC 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LeuGluSerLeuProValSerValTyrArgProLysGlyAlaIleProAlaGlyTrpPhe 380
Qy 1141 TGGCTTGGTGATACAGCAGACGCTTCTAAAGCCCTCTTGGTCAAACCTACCTTACCCGCC 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 381 TrpLeuGlyAspThrAlaAspAlaSerLysAlaLeuLeuValLysProThrLeuProAla 400
Qy 1201 CGAAGCGGGAGAAATCCCGCCCTTACTAGCCTTCACCAGGGATCCGGAATGACTGAGCAG 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 401 ArgSerGlyArgAsnProAlaLeuThrSerLeuHisGlnGlySerGlyMetThrGluGln 420
Qy 1261 CCATTTGTCGATCTTCCTCAATATCAGTACCTCAGCACATACTTTGGTTCATTTGCTCAC 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 ProPheValAspLeuProGlnTyrGlnTyrLeuSerThrTyrPheGlySerPheAlaHis 440
Qy 1321 GACACCCCCCCTGGCTCTACTCTTCGAGGCCTTCGACCAGATCATGTCTTGCCAGGACGA 1380
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 441 AspThrProProGlySerThrLeuArgGlyLeuArgProAspHisValLeuProGlyArg 460
Qy 1381 TATGAGATGCACGGTGATACTATATCTACTGCCGTATACGTTACTCGTCCTGTGGACGTT 1440
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 461 TyrGluMetHisGlyAspThrIleSerThrAlaValTyrValThrArgProValAspVal 480
Qy 1441 CCCTTTCCAGAGGACGAGTGTTTTGACTTAAAATCCCTTGTTCGTGTAAAATTGCCCGGT 1500
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 ProPheProGluAspGluCysPheAspLeuLysSerLeuValArgValLysLeuProGly 500
Qy 1501 TCCGGTAATCCACCCAAGCCCAGGTCTGCATTGAAAAAAAGCATGGTTCTTTTTGATAGT 1560
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 501 SerGlyAsnProProLysProArgSerAlaLeuLysLysSerMetValLeuPheAspSer 520
Qy 1561 GGGGAAAAG 1569
|||||||||
Db 521 GlyGluLys 523
‘512 claims 8, 9, 12, 14, and 15 recite that the plant pest is an insect of the family Chrysomelidae, an insect, Leptinotarsa decemlineata, Diabrotica virgiera virgifera, or selected from the group consisting of Leptinotarsa decemlineata and Diabrotica virgiera virgifera respectively. ‘512 claims 16-18 require that the plant is a crop plant, a potato plant, and a maize plant, respectively. ‘512 claims 19 and 20 are drawn to the transgenic plant which expresses the bi-component protein complex.
‘512 does not teach the transgenic plant expressing a PlyB with an endoplasmic reticulum localization signal.
However, Zhu teaches that the higher-order structure formation of peptides is largely completed within the endoplasmic reticulum (ER), and that those that are retained in the ER contain an ER retention signal at the C-terminus consisting of a four-amino acid residue KDEL or HDEL sequence (paragraph [0007]). Zhu teaches that combining an insect-resistant gene with a signal peptide sequence and a KDEL sequence produces a novel insect-resistant protein with enhanced stability and increases the accumulation of the insect-resistant protein in transgenic plants (paragraph [0008]).
Zhu teaches a nucleotide sequence encoding an insect-resistant fusion protein comprising a signal peptide at the 5’ end and an ER retention signal at the 3’ end (paragraph [0017], figure 3-1). Expression in transgenic tobacco of a modified gene comprising the KDEL sequence was significantly higher than that of the unmodified gene (paragraph [0055]). The localization was effective for multiple insect-resistant proteins (paragraph [0115]). Instant SEQ ID NO: 5 encodes an amino acid sequence of KDEL.
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of ‘512 to add a C-terminal endoplasmic reticulum localization signal, such as instant SEQ ID NO: 5 to PlyB. One of ordinary skill in the art would have been motivated to add a KDEL sequence to locate the anti-insect bi-component protein complex into the endoplasmic reticulum inside the plant’s cells to increase stability and accumulated amount of anti-insect protein in the transgenic plants. One of ordinary skill in the art would have had reasonable expectation of success because fusion anti-insect proteins comprising a KDEL sequence had higher expression in transgenic plants.
Although ‘512 does not specifically require that the plant comprise/express a sequence encoding a PlyA2, it would have been obvious because ‘512 claim 6 requires a PlyA with a polypeptide sequence with 100% identity to a sequence encoded by instant SEQ ID NO: 1. This reads on a PlyA2 of the instant invention. It would have likewise been obvious to transform with a polynucleotide operably linked to a promoter that is function in said transgenic plant to cause the production of mRNA molecules, because ‘512 requires that the bi-component protein complex is expressed.
Thus, the transgenic plant of instant claims 1-2, 5, 6, 10, 11, 20, 21, 24, 29 and 30 and the method comprising expressing PlyA2 and PlyB with an ER localization signal in a plant of instant claims 32, 37 and 38 would have been obvious and unpatentable over co-pending application ‘512 claims 1, 6-9, 12 & 14-20.
Conclusion
No Claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Victoria L DeLeo whose telephone number is (703)756-5998. The examiner can normally be reached M-F 8:00am-4pm EDT.
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/VICTORIA L DELEO/Examiner, Art Unit 1662
/Anne Kubelik/Primary Examiner, Art Unit 1663
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