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 .
Election/Restrictions
Applicant's election with traverse of Group I in the reply filed on 6/15/2026 is acknowledged. The traversal is on the ground that “Groups I-III share a same or corresponding special technical feature that defines a contribution which each of the claimed inventions, considered as a whole, makes over the prior art, for reasons that will be made of record during further prosecution of this application” (response, bridging paragraph between page 1-2). This is not found persuasive because the restriction did cite prior art showing that the shared technical feature is not special.
The requirement is still deemed proper and is therefore made FINAL.
Claim Status
Claims 1-11, 13, 15-16, 26-28, 30 and 32-32 are pending.
Claims 15-16, 26-28, 30 and 32-32 are withdrawn from examination as being art of non-elected groups.
Claims 1-11 and 13 are being examined.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
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.
Claim 10 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 10 recites, “… is derived from Pseudomonas syringae spp…. “. It is not clear to the Examiner if the claim includes only one species of Pseudomonas, i.e., Pseudomonas syringae; or any two or more species (Pseudomonas spp.) of the genus Pseudomonas is within the meets and bounds of the claim.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-6, 10-11 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Ratu et al. (Rhizobia use a pathogenic‑like effector to hijack leguminous nodulation signalling, 2021, Scientific Reports, 11:2034) in view of Bai et al. (Bacterial type III secretion system as a protein delivery tool for a broad range of biomedical applications, 2018, Biotechnol. Adv., 36:482–493) and Schmidt et al. (The evolving landscape around genome editing in agriculture, 2020, EMBO Reports, 21:e50680).
Ratu et al. describes certain rhizobia (i.e., rhizobium cells, as recited in claim 1) comprising Bradyrhizobium elkanii USDA61 (p.2, para 4, line 2; Supplementary Tables 1-4) hijack leguminous nodulation signaling via its type III secretion system (T3SS) and delivers effector proteins (T3Es) into host (plant) cells (abstract, line 3-5) in plants like soybean (abstract, line 7-8). Ratu et al. describes generating recombinant B. elkanii and A. tumefaciens (p.10, para 2, line 3) by transforming the cells with different plasmids (p.10, line 2; Supplementary Table 4) including pSUPSCAKm (p.10, last para, line 4), pK18mobsacB (p.11, first para, line 1), pBjGroEL4::DsRed , and pBjGroEL4::bel2-5 (p.11, para 2, line 1-2; Supplementary Table 4).
Ratu et al. describes the promoter of the Bel2-5 gene encoding the Bel2-5 effector protein (p.2, para 1, line 4-5; supplementary data 1 a-b), which suppresses plant immune responses for root-nodule symbiosis (p.2, para 1, line 10-12) (as recited in claim 6), is conserved among all T3SS-harboring rhizobia bacteria and the promoter region of the Bel2-5 gene contains tts box (p.2, last para, line 1-2; Supplementary Table 1). Bel2-5 not only dampens several plant defense related responses (which reads on to “suppresses innate immunity in plants” as recited in claim 5), as a phytopathogenic effector, but also plays a distinct role in the induction of a complete set of cytokinin biosynthesis-related genes essential for nodule organogenesis (p.2, para 3, last 3 lines). Bel2-5 resembles the T3 effector protein (T3E), XopD, of plant pathogenic Xanthomonas spp. (abstract, line 6-7; p.2, para 3, line 2-3) (as recited in claim 10).
In rhizobia, the expression of genes encoding T3 effectors (T3Es) and T3SS components is regulated by the transcriptional activator (polypeptide) TtsI and host-derived flavonoids (p .4, last para, line 1-2). The TtsI directly binds to a cis-element tts box in the promoter region (reads on to “T3SS-specific promoter”, as recited in claim 1) of tts clusters upon induction by host flavonoids (p .4, last para, line 2-3). The plasmid pBjGroEL4::DsRed and pBjGroEL4::bel2-5 (part of pBjGroEL4 plasmid) comprises the promoter of the T3SS-specific Bel2-5 gene (p.11, para 2, line 1-2; Supplementary Table 4). Ratu et al. describes constructing expression plant vector(s) encoding Bel2-5 fused with enhanced GFP (Bel2-5-eGFP) and transiently expressed it in Nicotiana benthamiana leaves using Agrobacterium tumefaciens (cell) (as recited in claim 13) mediated transformation (p.6, para 3, line 2-4). It is known in the art that any vector including the Gateway compatible destination vector pB7FWG2, as described by Ratu et al. (p.10, para 2, line 2-3; Supplementary Table 4, p.53), comprising a polynucleotide sequence or transgene used in Agrobacterium tumefaciens-mediated transformation is flanked by at least one (generally two) T-DNA (as recited in claim 11).
Ratu et al. teaches that the N-terminal region is conserved among rhizobia (p.9, para 3, line 7) contains a nuclear localization signal (NLS) motif at 788–796 aa in Bel2-5 (Fig. 2) which would enable nuclear translocation of the (Bel2-5) protein (and any protein fused in-frame with it) to the nucleus of the host plant cells including soybean (G. max cv. Enrei) cells along with any protein like GFP (or eGFP) fused to it (p.6, para 3; Fig. 3).
However, Ritu et al. does not describe any site-specific DNA modifying enzyme including any CRISPR (Cas) endonuclease.
Bai et al. describes several protein delivery tools based on bacterial type III secretion system (T3SS) (abstract). Bai et al. teaches that all type III effector proteins harbor a N-terminal secretion signal sequence (p.3, para 1, line 13-14) which supports the observation of Ratu et al. showing a nuclear localization signal (NLS) motif at 788–796 aa in Bel2-5 protein. Bai et al. describes delivering a site-specific DNA modifying recombinase enzyme (as recited in claim 2) Cre protein by using a T3SS-Cre delivery tool (p.8, para 4 in its entirety; Fig. 6; p.29). Bai et al. also describes other site-specific DNA modifying enzymes including a CRISPR endonuclease, Cas9 (p.10, para 3) (as recited in claims 2-4) and transcription activator-like effector nuclease (TALEN) to be delivered into different host cells including plant cells (p.9, line 5-6) and non-plant host cells like HeLa cells (p.9, line 7-11). Bai et al. describes that the structure of the type III secretion nanomachine (or injectisome) is highly conserved among Gram-negative bacteria including in Pseudomonas spp (p.2, para 3, line 4-6) (as recited in claim 10). Bai et al. describes that TALEN is a protein fusion of a DNA-binding domain from transcription activator-like effector (TALE) and a DNA cleavage domain from FokI endonuclease (p.9, para 1, line 1-3). In nature, TALEs are secreted by Xanthomonas spp. (as recited in claim 10) via their T3SS when they infect plants ((p.9, line 5-6).
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to modify the recombinant B. elkanii USDA61 rhizobium cells, as described by Ratu et al., by replacing the polynucleotide encoding eGFP with a polynucleotide sequence (which would read on to “first nucleic acid sequence”, as recited in claim 1) encoding Cas9 CRISPR endonuclease to deliver the Cas9 protein (as described by Bai et al.) into the soybean cells, as described by Ratu et al. The whole construct would have been driven by the promoter of the T3SS-specific Bel2-5 gene, as described by Ratu et al. The polynucleotide encoding the Bel2-5 protein having the T3SS secretion signal (or just the N-terminal half comprising the NLS sequence and the secretion signal) would read on to the “second nucleic acid sequence operably linked to a T3SS-specific (Bel2-5 promoter) promoter”, as recited in claim 5. It is known in the art that all effector proteins, which can initiate root-nodule symbiosis, do suppress innate immunity in a plant host. Thus, the heterologous Bel2-5 protein in B. elkanii which is able to initiate root-nodule symbiosis requires evasion or suppression of the host (soybean) immune responses (p.2, para 1, line 8-10), as recited in claim 5.
Before the effective filing date, an ordinarily skilled artisan would have been motivated to undertake targeted genome editing using CRISPR-Cas9 technique in a commercially important soybean variety using the T3 secretion system of a rhizobium like B. elkanii and its effector protein Bel2-5 fused to a CRISPR endonuclease like Cas9. This would have produced genome edited or transgenic soybean lines without integrating any transgene (expressing either the Cas9 and/or the effector protein) in the host (soybean) genome which would have been advantageous in many countries e.g., countries in the European Union, New Zealand etc. (Schmidt et al.; Fig1) that legally ban GMO but do not consider genome edited plants without any transgene as GMO.
Claims 7 is rejected under 35 U.S.C. 103 as being unpatentable over Ratu et al. in view of Bai et al. and Schmidt et al., as applied to claims 1-6, 10-11 and 13 above, and further in view of Seikh et al. (Mitogen-activated protein kinase activation after effector recognition in tomato, 2018, Moriones, E., Fernandez Munoz, R., Beuzon, CR., (Eds.). Acta Hortic., pp. 99-104; 5th International Symposium on Tomato Diseases - Perspectives and Future Directions in Tomato Protection. Malaga, SPAIN. June 13 -16, 2016. Int Soc Horticultural Sci.), Das et al. (A protocol for functional study of genes in Brassica juncea by Agrobacterium-mediated transient expression: applicability in other Brassicaceae, 2020, Journal of Plant Biochemistry and Biotechnology, 29:368–379) and Fouts et al. (Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor, 2002, PNAS, 99:2275–2280).
Ratu et al. in view of Bai et al. and Schmidt et al. describe a recombinant Rhizobium cell (B. elkanii) having a bacterial type Ill secretion system (T3SS) and comprising at least two nucleic acid sequences fused together while the first sequence encodes a site-specific DNA modifying enzyme (e.g., TALEN or Cas9) comprising a T3SS (secretion) signal sequence and the second sequence encodes a heterologous (T3) effector protein like Bel2-5 that suppresses innate immunity in plants. The polynucleotide sequence is under the control of a T3SS specific promoter recognized by the Rhizobium cell, as discussed above.
However, Ratu et al. in view of Bai et al. does not describe any of the AvrPto, AvrPtoB, or HopAO1 effector proteins.
Seikh et al. teaches phytopathogenic bacterium Pseudomonas syringae pv. tomato secretes effector proteins AvrPto and AvrPtoB into tomato cells using type III secretion system (T3SS) to target the immune machinery (reads on to “suppress innate immunity”) in the plant (abstract, line 1-4; p.1, para 1, line 6-10).
Das et al. describes a method to facilitate overexpression of foreign proteins in B. juncea (abstract) and in A. thaliana leaves by expressing a bacterial immune suppressor AvrPto (or AvrPto1) (p.370, left column, para 1, line 23-26), which is also a Brassicaceae plant like B. juncea, and known for its recalcitrance towards Agrobacterium-mediated transient transformation (abstract).
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to modify the method, as described by Ratu et al., by replacing the polynucleotide sequence (comprising the coding region of the gene with its promoter region) encoding the Bel2-5 effector protein with the polynucleotide sequence (gene with its promoter region) encoding one of the effector proteins (AvrPto or AvrPtoB) from P. syringae pv. Tomato (as described by Seikh et al. and Das et al.) in P. syringae pv. Tomato having the type II secretion system (T3SS), as described by Seikh et al. The genome and the promoters of P. syringae pv. Tomato is known in the art (Fouts et al.; title and abstract).
Before the effective filing date, the ordinarily skilled artisan would have been motivated to express one of the effector proteins (AvrPto or AvrPtoB) fused with Cas9 endonuclease driven by the native promoter of the gene encoding the effector gene in P. syringae pv. Tomato with a realistic objective to facilitate overexpression of foreign proteins including fusion proteins where a Cas9 or TALEN proteins are fused to other protein(s) including T3Es like AvrPto or AvrPtoB. This would be beneficial for the plants including Solanaceous plants like tomato that are affected by P. syringae including P. syringae pv. Tomato.
Claims 8-9 are rejected under 35 U.S.C. 103 as being unpatentable over Ratu et al. in view of Bai et al. and Schmidt et al., as applied to claims 1-6, 10-11 and 13 above, and further in view of Tenea et al. (Overexpression of Several Arabidopsis Histone Genes Increases Agrobacterium-Mediated Transformation and Transgene Expression in Plants, 2009, The Plant Cell, 21:3350–3367) and Schmidt et al. (The evolving landscape around genome editing in agriculture, 2020, EMBO Reports, 21:e50680).
Ratu et al. in view of Bai et al. and Schmidt et al. describe a recombinant Rhizobium cell (B. elkanii) having a bacterial type Ill secretion system (T3SS) and comprising at least two nucleic acid sequences fused together. The first two nucleic acid sequence encodes a site-specific DNA modifying enzyme (e.g., TALEN or Cas9) comprising a T3SS (secretion) signal sequence while the second sequence encodes a heterologous (T3) effector protein like Bel2-5 that suppresses innate immunity in plants. The polynucleotide sequence is under the control of a T3SS specific promoter recognized by the Rhizobium cell, as discussed above.
However, Ratu et al. in view of Bai et al. and Schmidt et al. do not describe any HTA1 plant protein.
Tenea et al. describes that overexpression of a plant (Arabidopsis) AtHTA1 increases transformation frequency (abstract, line 3; p. 832, right column, para 1, line 2-4). It also teaches certain histones including the H2A-1 variant of H2A histone encoded by the HAT1 gene (p. 3351, left column, para 2, line 1-2) enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation (abstract, last 3 lines). Protecting an incoming transgene DNA during the initial stages of transformation and increasing the efficiency of Agrobacterium-mediated transformation read on to “suppress innate immunity”, as described by the Applicant (spec, p.1, para 0004, last 4 lines).
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to modify the method, as described by Ratu et al. in view of Bai et al. and Schmidt et al., by expressing the heterologous HTA1 protein (as described by Tenea et al.) fused to the N-terminal region of the Bel2-5 protein comprising the NLS and the secretion signal sequence using a rhizobium (e.g., B. elkanii) based plant transformation technique.
Before the effective filing date, the ordinarily skilled artisan would have been motivated to express the heterologous (Arabidopsis) HTA1 protein fused to the N-terminal region of the Bel2-5 protein comprising the NLS and the secretion signal sequence using a rhizobium-based plant transformation technique with a realistic expectation to further increase genome editing or transformation efficiency in a commercially important soybean variety. This would have produced genome edited or transgenic soybean lines without integrating any transgene encoding either the heterologous Cas9 and/or the HAT1 gene(s) in the host (soybean) genome which would have been advantageous in many countries e.g., countries in the European Union, New Zealand etc. (Schmidt et al.; Fig1), that legally ban GMO and do not consider genome edited plants without any transgene as GMO.
Conclusion
No claim is allowed.
Communication
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAY CHATTERJEE whose telephone number is (703)756-1329. The examiner can normally be reached (Mon - Fri) 8.30 am to 5.30 pm..
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J.C.
Jay Chatterjee
2
/Jay Chatterjee/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Supervisory Patent Examiner, Art Units 1661 & 1662
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