Co-expression of nitrogenase proteins in cotton (Gossypium hirsutum L.)

PLOS ONE RESEARCH ARTICLE Co-expression of nitrogenase proteins in cotton (Gossypium hirsutum L.) Yimin Shang1☯, Wenfang Guo2☯, Xiaomeng Liu1, Lei Ma1, Dehu Liu2, Sanfeng Chen ID1* 1 College of Biological Sciences, China Agricultural University, Beijing, China, 2 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China ☯ These authors contributed equally to this work. * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Shang Y, Guo W, Liu X, Ma L, Liu D, Chen S (2023) Co-expression of nitrogenase proteins in cotton (Gossypium hirsutum L.). PLoS ONE 18(8): e0290556. https://doi.org/10.1371/journal. pone.0290556 Editor: Basavantraya N. Devanna, ICAR-National Rice Research Institute, INDIA Received: June 8, 2023 Accepted: August 9, 2023 Published: August 24, 2023 Copyright: © 2023 Shang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting information files. Funding: This work was supported by the National Key Research and Development Program of China Award (Grant No. 2017YFD0200807. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abstract Chemical nitrogen fertilizer can maintain crop productivity, but overuse of chemical nitrogen fertilizers leads to economic costs and environmental pollution. One approach to reduce use of nitrogen fertilizers is to transfer nitrogenase biosynthetic pathway to non-legume plants. Fe protein encoded by nifH and MoFe protein encoded by nifD and nifK are two structural components of nitrogenase. NifB encoded by nifB is a critical maturase that catalyzes the first committed step in the biosynthesis of nitrogenase FeMo-cofactor that binds and reduces N2. Expression of the nifB, nifH, nifD and nifK is essential to generate plants that are able to fix atmospheric N2. In this study, the four genes (nifB, nifH, nifD and nifK) from Paenibacillu polymyxaWLY78 were assembled in plant expression vector pCAMBIA1301 via Cre/LoxP recombination system, yielding the recombinant expression vector pCAMBIA1301-nifBHDK. Then, the four nif genes carried in the expression vector were co-introduced into upland cotton R15 using Agrobacterium tumefaciens-mediated transformation. Homozygous transgenic cotton lines B2, B5 and B17 of T3 generation were selected by PCR and RT-PCR. qRT-PCR showed that nifB, nifH, nifD and nifK were co-expressed in the transgenic cottons at similar levels. Western blotting analysis demonstrated that NifB, NifH, NifD and NifK were co-produced in the transgenic cottons. Co-expression of the four critical Nif proteins (NifB, NifH, NifD and NifK) in cottons represents an important step in engineering nitrogenase biosynthetic pathway to non-legume plants. Introduction Nitrogen (N) fertilizer can maintain crop productivity, but overuse of chemical N fertilizers leads to economic costs and environmental pollution [1]. One approach to reduce use of N fertilizers is to transfer biological nitrogen fixation to non-legume crops that can fix nitrogen [2, 3]. Biological nitrogen fixation, conversion of N2 to NH3, is catalyzed by nitrogenase enzyme that is distributed in a few bacteria and archaea. Nitrogenase is composed of two component proteins, MoFe protein (also termed as NifDK protein) and Fe protein (also termed as NifH protein). Fe protein is a homodimer bridged by an intersubunit (4Fe-4S) cluster that serves as the obligate electron donor to MoFe protein. MoFe protein is a heterotetramer that contains PLOS ONE | https://doi.org/10.1371/journal.pone.0290556 August 24, 2023 1 / 12 PLOS ONE Expression of nitrogenase proteins in cotton two metalloclusters: P cluster (8Fe−7S) and FeMo-cofactor (Mo−7Fe−9S−C−homocitrate) which binds and reduces N2 [4, 5]. Studies on N2-fixing models Azotobacter vinelandii and Klebsiella oxytoca have revealed that nitrogenase synthesis and maturation require at least 16 genes. In addition to nifH, nifD and nifK encoding structural subunits of nitrogenase, nifB, nifE nifN, nifX, nifQ, nifV, nifW, nifZ, nifM, nifU, nifS, nifF and nifJ contribute to synthesis of FeMo-cofactor, P cluster and 4Fe-4S cluster and electron transport [6–8]. Our study has revealed that a minimal nif gene cluster composed of 9 genes (nifB nifH nifD nifK nifE nifN nifX hesA nifV) from Paenibacillus polymyxa WLY78 enabled Escherichia coli to fix nitrogen, and deletion analysis has revealed that the six genes (nifB, nifH, nifiD, nifK, nifE and nifN) are essential for nitrogenase activity [9]. FeMo-cofactor is synthesized independently and then inserted into apo-NifDK. NifB is a radical S-adenosyl methionine (SAM) enzyme that catalyzes the formation of NifB-co, a [8Fe-9S-C] cluster which is a precursor for syntheses of FeMocofactor of Mo-nitrogenase [10–13]. NifB proteins have great diversity in their protein architectures among bacteria and archaea [14–16]. There has been a long-standing interest in engineering nitrogenase biosynthetic pathway in non-legume crops that can fix nitrogen [2, 3, 17, 18]. Active nitrogenase Fe protein was detected in tobacco when nifH and nifM from A. vinelandii were co-expressed in tobacco chloroplast [19]. Sixteen nif gengs (nifB, nifD, nifE, nifF, nifH, nifJ, nifK, nifM, nifN, nifQ, nifS, nifU, nifV, nifX, nifY and nifZ) from K. oxytoca were individually expressed using a transient expression system and these Nif proteins were individually targeted to the mitochondrial matrix [20]. NifB-co, a precursor of FeMo-cofactor, was produced in transgenic rice when NifB from the archaea Methanocaldococcus infernus and FdxN from A. vinelandii were coexpressed [21]. Functional nifB was produced in tobacco chloroplasts and mitochondria when nifB from Methanosarcina acetivorans or M. infernus and NifS, NifU and FdxN from A. vinelandii were co-expressed [22]. Although engineering nitrogen fixation genes in non-legume plants shows an attractive prospect, it is still a great challenge to achieve the simultaneous expression of multiple genes in plants. In this study, we use Cre/LoxP recombination system to assemble four genes (nifB, nifH, nifD and nifK) from P. polymyxaWLY78 in expression vector pCAMBIA1301. Then, the four nif genes carried in the expression vector ware co-introduced into upland cotton R15 mediated by Agrobacterium tumefaciens. Homozygous transgenic lines B2, B5 and B17 of T3 generation were identified by PCR and RT-PCR. qRT-PCR and western blotting analysis showed that nifB, nifH, nifD and nifK were co-expressed in transgenic cottons. The Cre/ LoxP recombination system provides one efficient strategy for co-e (...truncated)