Unequal contribution of two paralogous centromeric histones to function the cowpea centromere

Ishii, Takayoshi; Juranić, Martina; Maheshwari, Shamoni; Bustamante, Fernanda de Oliveira; Vogt, Maximilian; Salinas-Gamboa, Rigel; Dreißig, Steven; Gursanscky, Nial R.; How, Tracy; Fuchs, Joerg; Schubert, Veit; Spriggs, Andrew; Calzada, Jean Philippe Vielle; Comai, Luca; Koltunow, Anna M.; Houben, Andreas

doi:10.1101/2020.01.07.897074

Cited by 6 publications

(6 citation statements)

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“…In the GE cross, both the variants colocalized on five chromosomes inherited from the wild-type parent in interphase (Figure 5B), G2 (Figure S4B), and prometaphase cells (Figure 5C). At the higher resolution provided by 3D-SIM, the two variants only overlapped partially (Figures 5C and 5D), suggesting the formation of centromeric subdomains enriched with one or the other protein, consistent with previous observations with natural or artificial combinations of CENH3 types (Ishii et al, 2015, 2020; Maheshwari et al, 2017). Often, faint GFP-ts signals are colocalized with faint CENH3 signals (Figure S3C).…”

Section: Resultssupporting

confidence: 90%

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Biased removal and loading of centromeric histone H3 during reproduction underlies uniparental genome elimination

Ravi

Bondada

et al. 2021

Preprint

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Uniparental genome elimination is a dramatic case of centromeric failure, resulting in the postzygotic loss of a parental chromosome set. Genome partitioning during cell division relies on centromere attachment to spindle fibers through kinetochores. Centromeres are epigenetically specified by CENP-A (CENH3), a conserved centromeric specific histone H3 variant. In Arabidopsis, CENH3 modification results in haploid inducers, whose genome is eliminated frequently when crossed to the wild type. To investigate the underlying mechanism, we dissected the timing and molecular features of genome elimination. In zygotes and early embryos from genome elimination crosses, CENH3 occupied only the centromeres contributed by the wild-type parent. Haploid inducer chromosomes had defective kinetochores and missegregated, often forming micronuclei. This uniparental loss of centromere identity is initiated by the removal of altered CENH3 at fertilization, while wild-type CENH3 persists and maintains strong centromeric identity. Weak centromeres were capable of rebuilding functional kinetochores, but often failed when in competition with normal ones. We induced a similar weak state by mitotic dilution of wild-type CENH3. Furthermore, weakness was suppressed by crosses of haploid inducers to other variants of haploid inducers, and enhanced by mutations in VIM1, a ubiquitin ligase known to modify CENH3 and centromeric DNA methylation.. The differential stability of altered CENH3 during reproduction has important genetic and evolutionary implications.

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Section: Resultssupporting

confidence: 90%

“…The copyright holder for this this version posted February 24, 2021. ; https://doi.org/10.1101/2021.02.24.432754 doi: bioRxiv preprint combinations of CENH3 types (Ishii et al, 2015(Ishii et al, , 2020Maheshwari et al, 2017) . Often, faint GFP-ts signals are colocalized with faint CENH3 signals ( Figure S3C).…”

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confidence: 99%

Biased removal and loading of centromeric histone H3 during reproduction underlies uniparental genome elimination

Ravi

Bondada

et al. 2021

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“…The newly developed genome and transcriptome resources, in combination with CRISPR/Cas9 gene editing can support progression of varietal improvement programs [17,26,40]. Gene editing together with the low efficiency transformation method has successfully uncovered roles of two cowpea centromeric histone H3 genes [19]. Cowpea has a generation time of up to 14 weeks or more, depending on the variety, there is limited information concerning the efficiency of gene editing vectors for cowpea, and designed target guides need testing for efficiency.…”

Section: Introductionmentioning

confidence: 99%

A detached leaf assay for testing transient gene expression and gene editing in cowpea (Vigna unguiculata [L.] Walp.)

et al. 2020

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Background: The legume cowpea (Vigna unguiculata L.) is extensively grown in sub-Saharan Africa. Cowpea, like many legumes has proved recalcitrant to plant transformation. A rapid transient leaf assay was developed for testing gene expression and editing constructs prior to stable cowpea transformation, to accelerate cowpea and legume crop improvement. Results: Attempts to develop a transient protoplast system for cowpea were unsuccessful. Leaflets from plants 3-4 weeks post-germination were age selected to establish a rapid Agrobacterium (Agro) infiltration-mediated transient system for efficacy testing of gene expression and CRISPR/Cas9 gene editing constructs. In planta, Agro-infiltration of leaflets with fluorescent expression constructs, resulted in necrosis. By contrast, Agro-infiltration of detached leaflets with an Arabidopsis (At) ubiquitin3 promoter:ZsGreen construct, followed by culture on solid nutrient medium resulted in fluorescence in over 48% of leaf cells. Expression efficiency was leaf age-dependent. Three cowpea meiosis genes were identified for CRISPR/Cas9 gene-editing, with the forward aim of meiosis-knock out for asexual seed induction in cowpea. Constructs were designed and tested containing candidate gene-specific guide RNAs, expressed using either the cowpea or Arabidopsis U6 promoters with Cas9 expression directed by either the Arabidopsis 40S ribosomal protein or parsley ubiquitin4-2 promoters. Leaflets were infiltrated with test gene-editing constructs and analytical methods developed to identify gene-specific mutations. A construct that produced mutations predicted to induce functional knockout of in the VuSPO11-1 meiosis gene was tested for efficacy in primary transgenic cowpea plants using a previously established stable transformation protocol. Vuspo11-1 mutants were identified, that cytologically phenocopied spo11-1 mutants previously characterized in Arabidopsis, and rice. Importantly, a biallelic male and female sterile mutant was identified in primary transgenics, exhibiting the expected defects in 100% of examined male and female meiocytes. Conclusion: The transient, detached cowpea leaf assay, and supporting analytical methods developed, provide a rapid and reproducible means for testing gene expression constructs, and constructs for inducing mutagenesis in

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“…A comparison of the amino acid sequences of αCENH3 and βCENH3 aligned using FASTA showed that their similarity ranges from 65 to 70% in most species (Table 2). This is substantially lower than the 75% similarity between the two paralogous proteins, CenH3-1 and CenH3-2, in Fabeae species [19] and 91% similarity between the proteins CENH3.1 and CENH3.2 in Vigna unguiculata [21]. However, S. sibirica and S. breviflora have markedly higher similarity values relative to other species, 77.5% and 75.2%, respectively.…”

Section: Lineage-specific Evolutionmentioning

confidence: 73%

“…Subsequently, CENH3 duplications were shown for diploid plants in different taxa: Brassica [18], Pisum and Lathyrus [19], Mimulus [20], Vigna [21]. Both copies produce functional CENH3 proteins, which fully colocalize with each other.…”

Section: Introductionmentioning

confidence: 99%

Editorial Office BMC Biology The Origin and Evolution of a Two-Component System of Paralogous Genes Encoding the Centromeric Histone CENH3 in Cereals

Elisafenko¹,

Евтушенко²,

Вершинин³

2021

Preprint

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Background: The cereal family Poaceae is one of the largest and most diverse angiosperm families. The central component of centromere specification and function is the centromere-specific histone H3 (CENH3). Some cereal species (maize, rice) have one copy of the CENH3 gene, while some (wheat, barley, rye) have two. We applied a homology-based approach to sequenced cereal genomes, in order to finally trace the mutual evolution of the structure of the CENH3 genes and the nearby regions in various tribes. Results: We have established that the syntenic group or the CENH3 locus with the CENH3 gene and the boundaries defined by the Cdpk2 and bZip genes first appeared around 50 Mya in a common ancestor of the subfamilies Bambusoideae, Oryzoideae and Pooideae. This locus came to Pooideae with one copy of CENH3 in the most ancient tribes Nardeae and Meliceae. The βCENH3 gene as a part of the locus appeared in the tribes Stipeae and Brachypodieae around 35-40 Mya. The duplication was accompanied by changes in the exon-intron structure. Purifying selection acts mostly on αCENH3s, while βCENH3s form more heterogeneous structures, in which clade-specific amino acid motifs are present. In barley species, the βCENH3 gene assumed an inverted orientation relative to αCENH3 and the Cdpk2 gene was substituted with Cbp3c. As the evolutionary and breeding processes went on, the locus was growing in size due to an increasing distance between αCENH3 and βCENH3 because of a massive insertion of the main LTR-containing retrotransposon superfamilies, gypsy and copia, without any evolutionary preference on either of them. A comparison of the molecular structure of the locus in the A, B and D subgenomes of the hexaploid wheat T. aestivum showed that invasion by mobile elements and concomitant rearrangements took place in an independent way even in evolutionarily close species. Conclusions: The CENH3 duplication in cereals was accompanied by changes in the exon-intron structure of the βCENH3 paralog, which it was not in other plant taxa. The observed general tendency towards the expansion of the CENH3 locus reveals an amazing diversity of ways in which different species implement the scenario described in this paper.

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Unequal contribution of two paralogous centromeric histones to function the cowpea centromere

Cited by 6 publications

References 64 publications

Biased removal and loading of centromeric histone H3 during reproduction underlies uniparental genome elimination

Biased removal and loading of centromeric histone H3 during reproduction underlies uniparental genome elimination

A detached leaf assay for testing transient gene expression and gene editing in cowpea (Vigna unguiculata [L.] Walp.)

Editorial Office BMC Biology The Origin and Evolution of a Two-Component System of Paralogous Genes Encoding the Centromeric Histone CENH3 in Cereals

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