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Characterization of a new rice OsMADS1 null mutant generated by homologous recombination-mediated gene targeting

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Abstract

Main conclusion

A new, stable, null mutant of OsMADS1 generated by homologous recombination-based gene targeting in an indica rice confirms its regulatory role for floral meristem identity, its determinate development and floral organ differentiation.

Abstract

OsMADS1, an E-class MADS-box gene, is an important regulator of rice flower development. Studies of several partial loss-of-function and knockdown mutants show varied floret organ defects and degrees of meristem indeterminacy. The developmental consequences of a true null mutant on floret meristem identity, its determinate development and differentiation of grass-specific organs such as the lemma and palea remain unclear. In this study, we generated an OsMADS1 null mutant by homologous recombination-mediated gene targeting by inserting a selectable marker gene (hpt) in OsMADS1 and replacing parts of its cis-regulatory and coding sequences. A binary vector was constructed with diphtheria toxin A chain gene (DT-A) as a negative marker to eliminate random integrations and the hpt marker for positive selection of homologous recombination. Precise disruption of the endogenous OsMADS1 locus in the rice genome was confirmed by Southern hybridization. The homozygous osmads1ko null mutant displayed severe defects in all floral organs including the lemma and palea. We also noticed striking instances of floral reversion to inflorescence and vegetative states which has not been reported for other mutant alleles of OsMADS1 and further reinforces the role of OsMADS1 in controlling floral meristem determinacy. Our data suggest, OsMADS1 commits and maintains determinate floret development by regulating floral meristem termination, carpel and ovule differentiation genes (OsMADS58, OsMADS13) while its modulation of genes such as OsMADS15, OsIG1 and OsMADS32 could be relevant in the differentiation and development of palea. Further, our study provides an important perspective on developmental stage-dependent modulation of some OsMADS1 target genes.

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Acknowledgements

We thank Dr. Patrick D’ Silva, Department of Biochemistry, Indian Institute of Science, Bangalore, India for the DT-A gene and Dr. K. Dharmalingam, School of Biotechnology, Madurai Kamaraj University for his permission to use the radioisotope facility. This work was funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India [Project entitled “Functional Analysis of Gene Regulatory Networks during Flower and Seed development in rice”, Project No. BT/AB/FG-1 (PH-II)/2009] to KV and UV. UV acknowledges the Department of Biotechnology, Ministry of Science and Technology (DBT-IISc partnership programme to Biological Sciences). University Grants Commission, Govt. of India is acknowledged for Faculty Fellowship to KV [No. F. 18-1/2011 (BSR)]. Indian National Science Academy is acknowledged for the Senior Scientist Fellowship to KV. Student fellowship from Council of Scientific and Industrial Research, Govt. of India supported GLC. We acknowledge the National Centre for Biological Sciences (NCBS), TIFR micro-CT imaging facility and Sunil Prabhakar for helping with micro-CT image analysis. We acknowledge help from Sandhan Prakash for Western blot analyses.

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Authors and Affiliations

  1. Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, 625021, India

    Pachamuthu Kannan, Bharat Bhusan Majhi, Debjani Basu & Karuppannan Veluthambi

  2. Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru, 560012, India

    Grace Lhaineikim Chongloi & Usha Vijayraghavan

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  1. Pachamuthu Kannan
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  2. Grace Lhaineikim Chongloi
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  3. Bharat Bhusan Majhi
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Correspondence to Usha Vijayraghavan.

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Pachamuthu Kannan and Grace Lhaineikim Chongloi are the joint first authors.

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Figure S1

Schematic representation of the strategy employed to construct the pGT-OsMADS1 binary vector to knock out the OsMADS1 gene by homologous recombination-mediated gene targeting. pGT-OsMADS1 T-DNA consists of the hpt gene, OsMADS1 left flank, OsMADS1 right flank and two copies of the DT-A gene. The P35S-DT-A-35S 3’ cassette was excised from pRP8 (lab collection) as an EcoRV fragment and cloned in the EcoRV site of pIC19R to yield pPK8 (Step 1). The 4.28-kb 5’ part of the OsMADS1 sequence was excised from pPK15 (lab collection) as an EcoRV/XhoI fragment and cloned in SmaI/XhoI sites of pPK8 (Step 2). The resultant plasmid pPK16 has the P35S-DT-A-35S 3’ cassette and the 5’ part of OsMADS1. A 5.6-kb fragment containing the P35S-DT-A-35S 3’ cassette along with the 5’ part of OsMADS1 was taken as an EcoRI fragment from pPK16 and cloned in the EcoRI site of pDEBJANI2 (Step 3). pDEBJANI2 is a pPZP101-derived binary plasmid with the hpt positive selectable marker gene. The resultant plasmid was named as pPK18 . The 4.05-kb 3’ part of OsMADS1 sequence was excised from pUOsM2 (lab collection) as a SpeI/XhoI fragment and cloned in the XbaI/XhoI sites of pBHARAT15, which has the P35S-DT-A-35S 3’ cassette (Step 4). This step yielded the plasmid pPK17 which has the P35S-DT-A-35S 3’ + the 3’ part of OsMADS1 (right flank). A 5.4-kb fragment comprising P35S-DT-A-35 3’ + 3’ part of OsMADS1 (right flank) was taken from pPK17 as a HindIII fragment and cloned in the HindIII site of pPK18 (Step 5) to yield pGT-OsMADS1 binary plasmid (TIFF 6596 kb)

Figure S2

Nucleotide sequences used for the construction of pGT-OsMADS1 binary plasmid. a DT-A- negative selectable marker gene. b The 4.28 kb 5’ OsMADS1 sequence (left flank) comprising upstream/promoter sequences, 5’UTR, exon 1 and a part of intron 1 of OsMADS1. c hpt positive selectable marker gene. d The 4.05 kb 3’ OsMADS1right flank which comprises a part of exon 6, intron 7, exon 7, intron 8, exon 8 and 3’UTR of the OsMADS1 gene (DOCX 19 kb)

Figure S3

Southern blot analysis of the T0 GT-1 plant with the DT-A probe and GT-2 plant with the hpt probe. a DNA from wild-type control (C) and the T0 GT-1 plant (same DNA loaded in duplicate lanes labeled as 1 and 2) was digested with BamHI + PstI and the blot was probed with [α-32P]dCTP-labeled DT-A gene. Bi, binary plasmid pGT-OsMADS1 digested with BamHI + PstI. The sizes of 1 kb Plus DNA ladder are marked on the left. b The T0 GT-2 plant DNA digested with HindIII and SacI and control plant DNA digested with HindIII. The blot was probed with the [α-32P]dCTP-labeled hpt gene. The sizes of λ/HindIII fragments are marked on the left (TIFF 2201 kb)

Figure S4

Southern blot analysis to confirm precise homologous recombination between the OsMADS1 genomic locus and the cloned locus-specific DNA in the T-DNA of pGT-OsMADS1. a Total DNA from wild-type and T0 GT-1 plant was digested with SacI, SacII, BamHI and HindIII and the blot was probed with [α-32P]dCTP-labeled 3’OsMADS1. b The wild-type and T0 GT-1 plant DNA was digested with HindIII, EcoRI, PstI and EcoRV and the [α-32P]dCTP-labeled 5’OsMADS1 was used as a probe. The sizes of marker DNA fragments from λ/HindIII digestion are indicated to the left. c and d Schematic representations of the fragments expected to hybridize upon using the 3’OsMADS1 or 5’OsMADS1 locus-specific probes, respectively, are shown below the corresponding blots. C, control wild-type; GT, GT-1 plant (TIFF 2201 kb)

Figure S5

PCR analysis to determine the heterozygous or homozygous status of the OsMADS1 GT locus (osmads1ko) in GT-1 T1 plants. a, b Schematic representation of the locations of primers used for amplification of the native OsMADS1 locus and the osmads1 locus, respectively. c F1 and R1 primers were used to amplify a 1.1-kb fragment of the OsMADS1 native locus. d F2 and R2 primers were used to amplify a 0.9-kb fragment from the osmads1 locus. Both 0.9 and 1.1-kb fragments were amplified in the heterozygous plants. In wild-type plants, only the 1.1-kb fragment is amplified. In osmads1ko plants, only the 0.9-kb fragment is amplified. In c and d +/+, wild-type plant; +/-, heterozygous plant; -/-, homozygous osmads1ko mutant (TIFF 2201 kb)

Figure S6

Western blot detection of OsMADS1 protein in 1-2 cm of various panicle tissues. Cross- reacting OsMADS1 protein bands can be seen in the PB1 wild-type tissues (arrow), probed with crude anti-OsMADS1 serum, but not in the osmads1ko nuclear extract. Non-specific cross reacting band is also detected in both the nucleoprotein extracts and marked with an *. Coomassie stained gel (pre-transfer), is presented as a loading control. pM-Color Prestained Protein Standard, Broad Range (10-250 kDa) (NEB, Massachusetts, USA) (TIFF 1653 kb)

Figure S7

Phenotypic characterization of osmads1ko florets. a Wild-type floret with lemma and palea, a pair of lodicules, 6 stamens and a central carpel. b An opened osmads1ko floret with lemma and palea, 4 reiterated lemma/palea-like organs, 3 stamens and an enlarged carpel. c and d osmads1ko floret with repeated lemma/palea-like organs, multiple carpel or mosaic of lemma/palea and carpel. e andf Toluidene stained histological sections of inflorescences at stage In4 showing branch meristems, spikelet and floret meristems in wild-type (e) and osmads1ko plants (f) (box bracket in e and f shows a primary branch). Scale bar 50 μm (e and f) (TIFF 3168 kb)

Figure S8

Range of severe phenotypes of osmads1ko florets. a Wild-type mature floret. b An osmads1ko floret with continually reiterated lemma/palea-like organs. c and d Show new florets developing within an older floret and even continuing as an inflorescence branch indicating floret indeterminacy (indicated by red box bracket and white arrow). e An osmads1ko plant ~50 days after heading where new florets are sustained within older florets (TIFF 371 kb)

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Kannan, P., Chongloi, G.L., Majhi, B.B. et al. Characterization of a new rice OsMADS1 null mutant generated by homologous recombination-mediated gene targeting. Planta 253, 39 (2021). https://doi.org/10.1007/s00425-020-03547-3

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