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Synthetic maize centromeres transmit chromosomes across generations

Nature Plants volume 9, pages 433–441 (2023)Cite this article

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Abstract

Centromeres are long, often repetitive regions of genomes that bind kinetochore proteins and ensure normal chromosome segregation. Engineering centromeres that function in vivo has proven to be difficult. Here we describe a tethering approach that activates functional maize centromeres at synthetic sequence arrays. A LexA-CENH3 fusion protein was used to recruit native Centromeric Histone H3 (CENH3) to long arrays of LexO repeats on a chromosome arm. Newly recruited CENH3 was sufficient to organize functional kinetochores that caused chromosome breakage, releasing chromosome fragments that were passed through meiosis and into progeny. Several fragments formed independent neochromosomes with centromeres localized over the LexO repeat arrays. The new centromeres were self-sustaining and transmitted neochromosomes to subsequent generations in the absence of the LexA-CENH3 activator. Our results demonstrate the feasibility of using synthetic centromeres for karyotype engineering applications.

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Fig. 1: LexA-CENH3(v1) recruits native CENH3 to ABS4.
Fig. 2: Segregation errors in LexA-CENH3(v2) ABS4 plants.
Fig. 3: Analysis of independent neochromosomes 4L.

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Data availability

All raw sequencing data generated in this study have been submitted to the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under accession number PRJNA874319. For description of files, see Supplementary Table 2.

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Acknowledgements

We thank the Maize Genetics Cooperation Stock Center for providing maize stocks and M. Tindall Smith for genotyping stocks. This study was supported in part by resources and technical expertise from the Georgia Advanced Computing Resource Center, as well as grants from the National Science Foundation (IOS-1444514 and IOS-2040218).

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Author notes

  1. Fang-Fang Fu

    Present address: Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, China

  2. Na Wang

    Present address: Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai, China

Authors and Affiliations

  1. Department of Genetics, University of Georgia, Athens, GA, USA

    R. Kelly Dawe, Yibing Zeng, Han Zhang & Jianing Liu

  2. Department of Plant Biology, University of Georgia, Athens, GA, USA

    R. Kelly Dawe, Jonathan I. Gent, Fang-Fang Fu, Kyle W. Swentowsky, Dong Won Kim & Na Wang

  3. Institute of Bioinformatics, University of Georgia, Athens, GA, USA

    R. Kelly Dawe & Rebecca D. Piri

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Contributions

R.K.D., J.I.G. and H.Z. conceived and designed experiments. R.K.D., J.I.G., Y.Z., H.Z., F.-F.F., K.W.S., D.W.K., N.W., J.L. and R.D.P. performed experiments. R.K.D., J.I.G., Y.Z., F.-F.F., K.W.S., N.W., J.L. and R.D.P. analysed the data. R.K.D. and J.I.G. wrote the manuscript.

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Correspondence to R. Kelly Dawe.

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Extended data

Extended Data Fig. 1 Confirmation that LexA-CENH3(v1) recruits native CENH3 by a ChIP-seq assay.

The data show CENH3 ChIP enrichment relative to input, measured by Illumina sequencing. Each ChIP is from an individual plant that was heterozygous for ABS4 and the LexA-CENH3(v1) transgene. CentC is a native centromere repeat whereas knob180 and TR-1 are non-centromeric repeats. The Y-axis indicates the numbers of reads in the ChIP sample divided by numbers in input and normalized by the total number of reads.

Extended Data Fig. 2 Complementation of the cenh3 null by LexA-CENH3 transgenes.

A minus sign indicates the lack of LexA-CENH3 transgene. A plus sign indicates the wild-type CENH3 allele. Plants heterozygous for either LexA-CENH3(v1) or LexA-CENH3(v2) were crossed to lines carrying the cenh3 null and heterozygotes self-crossed. The genotypes of progeny are shown to the right. Note that of 67 plants carrying LexA-CENH3(v1), none were homozygous for cenh3, indicating that the transgene cannot complement the null. In contrast, of 20 plants carrying LexA-CENH3(v2), four were homozygous for cenh3. One of these four plants, which proved to be homozygous for LexA-CENH3(v2), looked normal (see Extended Data Fig. 3).

Extended Data Fig. 3 Demonstration that LexA-CENH3(v2) is sufficient for cell division and plant growth.

a). A plant homozygous for the LexA-CENH3(v2) transgene and homozygous for a cenh3 null mutant. The plant was self-crossed to reveal a full ear (below), indicating that the transgene was homozygous (one quarter of the seeds would have been dead if the plant was heterozygous for LexA-CENH3(v2)). The second ear of this plant was used for the third lane of the protein blot in B. b) Protein blot analysis of CENH3 in plants of different genotypes. A minus sign indicates the lack of LexA-CENH3(v2) transgene. A plus sign indicates wild-type CENH3 allele. Native maize CENH3 is 17 kDa and recognized by a maize-specific antibody. The oat CENH3 antibody recognizes the LexA-CENH3 fusion protein only. The predicted ~27 kDa LexA-CENH3 band is observed along with a smaller band of unknown cause/origin. Importantly, native CENH3 was not detectable in the LexA-CENH3(v2)/LexA-CENH3(v2), cenh3/cenh3 null plant (third lane). The lower panel (Coomassie) shows that the amounts of protein loaded into each lane were similar. These staining patterns were observed in two different protein blots using the same protein samples.

Extended Data Fig. 4 Segregation errors in LexA-CENH3(v2) ABS4 plants.

a) Mitotic errors. Root tips from five sectored seeds were processed for FISH and the number of Cent4 and ABS spots counted in interphase nuclei. Two Cent4 (red) spots were observed in all nuclei. ABS (green), which is on one copy of chromosome 4, showed high frequencies of mis-segregation consistent with the model in Fig. 2a. The images shown above are examples. The two cells in the right image are daughter cells of a single division, where the left cell has no ABS and the right cell as two ABS loci. b) Meiotic errors. Three plants heterozygous for LexA-CENH3(v2) and ABS4 were analyzed at meiosis (siblings from a single ear). Only cells in mid-anaphase I, identified by the short distance between segregating chromosomes masses, were tallied. Figures with no recombination between the centromere and ABS4 were identified by having ABS dots only on one side (first column). Cases where recombination occurred and ABS4 loci segregated freely to one pole were identified by having one ABS dot on both sides (second column). Figures with bridges invariably had one or two ABS dots in the midzone, suggesting there had been recombination between centromere and ABS4, and that centromere cohesion at ABS4 restrained movement. At late anaphase I and telophase I, no intact bridges were observed.

Extended Data Fig. 5 Leaf defects observed in plants with both LexA-CENH3(v2) and ABS4.

The leaves of nearly all LexA-CENH3(v2) ABS4 plants looked and felt uneven, with ridges or crinkles. Occasionally the leaves had missing pieces, such as shown here.

Extended Data Fig. 6 Crosses involving c2-GFP.

a) The cross used to generate material for the neochromosome screen. C2 confers purple pigmentation to the outer layers of the endosperm (purple appears black under the blue light used here). The binding of LexA-CENH3 to ABS4 causes errors in chromosome segregation and loss of the linked C2 gene in sectors (arrow). Two other recessive color alleles (c1 and r1) are also segregating in these transgenic lines, which caused many kernels to be colorless regardless of the presence of C2 (in this case only c1 was heterozygous on the female side). b) Fluorescent sectored kernels from A were planted and crossed as females to a c2 tester. Kernels that received both C2 (purple aleurone) and c2-GFP (fluorescent endosperm) were candidates for having inherited a neochromosome. This ear shows such a kernel. Filing off the outer layer of cells removes the purple pigment and makes the underlying fluorescence more obvious (arrow).

Extended Data Fig. 7 Pedigrees of Neo4L chromosomes.

Females are shown on the left side of the crosses. Neo4L-2, Neo4L-3, Neo4L-4 are denoted as Neo4L-X in the pedigree.

Extended Data Table. 1 Segregation data for Neo4L chromosomes
Full size table

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Supplementary Tables 1 and 2, and Data 1.

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Dawe, R.K., Gent, J.I., Zeng, Y. et al. Synthetic maize centromeres transmit chromosomes across generations. Nat. Plants 9, 433–441 (2023). https://doi.org/10.1038/s41477-023-01370-8

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