Rapid creation of CENH3-mediated haploid induction lines using a cytosine base editor (CBE)

• Haploid induction (HI) can create true-breeding lines in a single generation, which can significantly accelerates the breeding process. In recent years, scientists have developed a variety of new techniques to induce haploids through manipulation of CENH3, a variant of the centromere-specific histone H3. One alternative approach is based on CENH3 point mutations derived from EMS/TILLING, which is not lethal and yet is responsible for inducing haploids. However, most residues have been obtained by EMS mutagenesis over a long period of time.
• Recently, a new approach called ‘base editing’ was developed for plants. Here, we report a new method that uses a cytosine base editor (CBE) to create a point mutation of CENH3 as a haploid induction line, which substitutes adenine (A) for guanine (G).
• As proof of the extreme simplicity of this approach to create haploid-induced lines, we identified an L130F substitution within the histone fold domain in Arabidopsis thaliana. Subsequently, we tested the haploid-inducing potential of homozygous L130F plants by pollinating them with Col-0, and obtained 2.9% paternal haploid plants.
• In brief, our innovative technology provides a new perspective for the promotion of CENH3-mediated haploid induction in crops, and also provides a variety of options for breeders. Such conserved point mutations as L130F could be developed into a general instrument for haploid induction in a wide range of plant species. Extending these systems would represent a major advance over haploid production.

     

A variety of changes,including CRISPR/Cas9‐mediated deletions,in CENH3 lead to haploid induction on outcrossing

Creating true‐breeding lines is a critical step in plant breeding. Novel, completely homozygous true‐breeding lines can be generated by doubled haploid technology in single generation. Haploid induction through modification of the centromere‐specific histone 3 variant (CENH3), including chimeric proteins, expression of non‐native CENH3 and single amino acid substitutions, has been shown to induce, on outcrossing to wild type, haploid progeny possessing only the genome of the wild‐type parent, in Arabidopsis thaliana. Here, we report the characterization of 31 additional EMS‐inducible amino acid substitutions in CENH3 for their ability to complement a knockout in the endogenous CENH3 gene and induce haploid progeny when pollinated by the wild type. We also tested the effect of double amino acid changes, which might be generated through a second round of EMS mutagenesis. Finally, we report on the effects of CRISPR/Cas9‐mediated in‐frame deletions in the αN helix of the CENH3 histone fold domain. Remarkably, we found that complete deletion of the αN helix, which is conserved throughout angiosperms, results in plants which exhibit normal growth and fertility while acting as excellent haploid inducers when pollinated by wild‐type pollen. Both of these technologies, CRISPR mutagenesis and EMS mutagenesis, represent non‐transgenic approaches to the generation of haploid inducers.     

A CENH3 mutation promotes meiotic exit and restores fertility in SMG7-deficient Arabidopsis

Meiosis in angiosperm plants is followed by mitotic divisions to form multicellular haploid gametophytes. Termination of meiosis and transition to gametophytic development is, in Arabidopsis, governed by a dedicated mechanism that involves SMG7 and TDM1 proteins. Mutants carrying the smg7-6 allele are semi-fertile due to reduced pollen production. We found that instead of forming tetrads, smg7-6 pollen mother cells undergo multiple rounds of chromosome condensation and spindle assembly at the end of meiosis, resembling aberrant attempts to undergo additional meiotic divisions. A suppressor screen uncovered a mutation in centromeric histone H3 (CENH3) that increased fertility and promoted meiotic exit in smg7-6 plants. The mutation led to inefficient splicing of the CENH3 mRNA and a substantial decrease of CENH3, resulting in smaller centromeres. The reduced level of CENH3 delayed formation of the mitotic spindle but did not have an apparent effect on plant growth and development. We suggest that impaired spindle re-assembly at the end of meiosis limits aberrant divisions in smg7-6 plants and promotes formation of tetrads and viable pollen. Furthermore, the mutant with reduced level of CENH3 was very inefficient haploid inducer indicating that differences in centromere size is not the key determinant of centromere-mediated genome elimination.     

Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain

The centromeric histone H3 (CENH3) substitutes histone H3 within the nucleosomes of active centromeres in all eukaryotes. CENH3 deposition at centromeres is needed to assemble the kinetochore, a complex of conserved proteins responsible for correct chromosome segregation during nuclear division. Histones of regular nucleosomes are loaded during replication in S phase, while CENH3 deposition deviates from this pattern in yeast, human, and Drosophila melanogaster cells. Little is known about when and how CENH3 targets centromeric loci. Therefore, we determined the location and quantity of recombinant enhanced yellow fluorescent protein (EYFP)-CENH3 in mitotic root and endopolyploid leaf nuclei of transgenic Arabidopsis thaliana cells. Our data indicate significant loading of A. thaliana CENH3 during G2 (before splitting into sister kinetochores) rather than during the S or M phase of the cell cycle. The histone fold domain of the C-terminal part of CENH3 is sufficient to target A. thaliana centromeres. A. thaliana EYFP-CENH3 can recognize and target three different centromeric repeats of Arabidopsis lyrata but not field bean (Vicia faba) centromeres.     

Deposition,turnover, and release of CENH3 at <Emphasis Type="Italic">Arabidopsis</Emphasis> centromeres

The kinetochore is a complex multiprotein structure located at centromeres and required for the proper segregation of chromosomes during mitosis and meiosis. An important role in kinetochore assembly and function plays the centromeric histone H3 variant (CENH3). Cell cycle stage of CENH3 deposition to centromeres varies between different organisms. We confirmed by in vivo studies that deposition of Arabidopsis CENH3 takes place at centromeres during G2 and demonstrated that additionally a low turnover of CENH3 occurs along the cell cycle, apparently for replacement of damaged protein. Furthermore, enhanced yellow fluorescent protein (EYFP)-CENH3 of photobleached chromocenters is not replaced by EYFP-CENH3 molecules from unbleached centromeres of the same nucleus, indicating a stable incorporation of CENH3 into centromeric nucleosomes. In differentiated endopolyploid nuclei however, the amount of CENH3 at centromeres declines with age.     

Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.     

Recognition of <Emphasis Type="Italic">A. thaliana</Emphasis> centromeres by heterologous CENH3 requires high similarity to the endogenous protein

The centromere is an essential chromosomal component assembling the kinetochore for chromosome attachment to the spindle microtubules and for directing the chromosome segregation during nuclear division. Kinetochore assembly requires deposition of the centromeric histone H3 variant (CENH3) into centromeric nucleosomes. CENH3 has a variable N-terminal and a more conserved C-terminal part, including the loop1 region of the histone fold domain, which is considered to be critical for centromere targeting. To investigate the structural requirements for centromere targeting, constructs for EYFP-tagged CENH3 of A. lyrata, A. arenosa, Capsella bursa-pastoris, Zea mays and Luzula nivea (the latter with holocentric chromosomes) were transformed into A. thaliana. Except for LnCENH3, all recombinant CENH3 proteins targeted A. thaliana centromeres, but the more distantly related the heterologous protein is, the lower is the efficiency of targeting. Alignment of CENH3 sequences revealed that the tested species share only three amino acids at loop1 region: threonine2, arginine12 and alanine15. These three amino acids were substituted by asparagine, proline and valine encoding sequences within a recombinant EYFP-AtCENH3 construct via PCR mutagenesis prior to transformation of A. thaliana. After transformation, immunostaining of root tip nuclei with anti-GFP antibodies yielded only diffuse signals, indicating that the original three amino acids are necessary but not sufficient for targeting A. thaliana centromeres.     

Altered Mating-Type Identity in the Fungus Podospora Anserina Leads to Selfish Nuclei, Uniparental Progeny, and Haploid Meiosis

In wild-type crosses of the filamentous ascomycete Podospora anserina, after fertilization, only nuclei of opposite mating type can form dikaryons that undergo karyogamy and meiosis, producing biparental progeny. To determine the role played by the mating type in these steps, the four mat genes were mutagenized in vitro and introduced into a strain deleted for its mat locus. Genetic and cytological analyses of these mutant strains, crossed to each other and to wild type, showed that mating-type information is required for recognition of nuclear identity during the early steps of sexual reproduction. In crosses with strains carrying a mating-type mutation, two unusual developmental patterns were observed: monokaryotic cells, resulting in haploid meiosis, and uniparental dikaryotic cells providing, after karyogamy and meiosis, a uniparental progeny. Altered mating-type identity leads to selfish behavior of the mutant nucleus: it migrates alone or paired, ignoring its wild-type partner in all mutant X wild-type crosses. This behavior is nucleus-autonomous because, in the same cytoplasm, the wild-type nuclei form only biparental dikaryons. In P. anserina, mat genes are thus required to ensure a biparental dikaryotic state but appear dispensable for later stages, such as meiosis and sporulation.     

Loading of the centromeric histone H3 variant during meiosis–how does it differ from mitosis?

In eukaryotic phyla studied so far, the essential centromeric histone H3 variant (CENH3) is loaded to centromeric nucleosomes after S-phase (except for yeast) but before mitotic segregation (except for metazoan). While the C-terminal part of CENH3 seems to be sufficient for mitotic centromere function in plants, meiotic centromeres neither load nor tolerate impaired CENH3 molecules. However, details about CENH3 deposition in meiocytes are unknown (except for Drosophila). Therefore, we quantified fluorescence signals after the immunostaining of CENH3 along meiotic and mitotic nuclear division cycles of rye, a monocotyledonous plant. One peak of fluorescence intensity appeared in the early meiotic prophase of pollen mother cells and a second one during interkinesis, both followed by a decrease of CENH3. Then, the next loading occurred in the male gametophyte before its first mitotic division. These data indicate that CENH3 loading differs between mitotic and meiotic nuclei. Contrary to the situation in mitotic cycles, CENH3 deposition is biphasic during meiosis and apparently linked with a quality check, a removal of impaired CENH3 molecules, and a general loss of CENH3 after each loading phase. These steps ensure an endowment of centromeres with a sufficient amount of correct CENH3 molecules as a prerequisite for centromere maintenance during mitotic cycles of the microgametophyte and the progeny. From a comparison with data available for Drosophila, we hypothesise that the post-divisional mitotic CENH3 loading in metazoans is evolutionarily derived from the post-divisional meiotic loading phase, while the pre-divisional first meiotic loading has been conserved among eukaryotes.     

Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize

Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.     

Centromeric histone H3 protein: from basic study to plant breeding applications

Centromere is the defining unit of a chromosome where kinetochore complex assembles and facilitates chromosome segregation. Centromeres contain unique repetitive sequences and are enriched with transposons and retrotransposons. Although how centromere is determined is still not clearly understood, binding of a key protein, namely, the Centromeric Histone H3 (CENH3) to centromeric repetitive DNA sequences has been found to be critical for the specification of centromere. Hence, centromeres are said to be epigenetically specified by CENH3. Despite considerable variation in size and sequence, CENH3 protein shows significant conservation of structure and function. CENH3 disruption or overexpression shows severe defects in spindle fiber attachment and ultimately leads to embryo lethality. Basic studies on complementation of CENH3 in Arabidopsis thaliana have led to the development of a novel method of haploid production through selective elimination of one set of parental chromosomes in the zygote. These findings have also shed new light on selective loss of chromosomes in interspecific crosses of Hordeum vulgare × H. bulbosum. Here, we briefly review unique features of CENH3 and discuss the new plant breeding opportunities that have emerged from the study of CENH3.     

Hfq variant with altered RNA binding functions

The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS–lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq–RNA interaction but has probably no effect on the folding and the oligomerization of the protein.     

Rapid attainment of a doubled haploid line from transgenic maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) plants by means of anther culture

Summary We present a strategy for establishing a transgenic doubled haploid maize line from heterozygous transgenic material by means of anther culture. Compared to conventional inbreeding, the in vitro androgenesis technique enables a faster generation of virtually fully homozygous lines. Since the androgenic response is highly genotype-dependent, we crossed transgenic, non-androgenic plants carrying a herbicide resistance marker gene (pat, encoding for phosphinothricin acetyl transferase) with a highly androgenic genotype. The transgenic progenies were used as donor plants for anther culture. One transgenic and three non-transgenic doubled haploid lines have been established within approximately 1 yr. The homozygosity of all four doubled haploid lines was tested by analysis of simple sequence repeat (SSR) markers at 19 different loci. Polymorphisms were found between the lines but not within the lines indicating the homozygous nature of the entire plant genome gained by anther culture. Southern blot analysis revealed that the transgenic donor plants and their doubled haploid progeny exhibited the same integration pattern of the pat gene. No segregation of the herbicide resistance trait has been observed among the progeny of the transgenic doubled haploid line.     

Chromosome Dynamics Visualized with an Anti-Centromeric Histone H3 Antibody in Allium

Due to the ease with which chromosomes can be observed, the Allium species, and onion in particular, have been familiar materials employed in cytogenetic experiments in biology. In this study, centromeric histone H3 (CENH3)-coding cDNAs were identified in four Allium species (onion, welsh onion, garlic and garlic chives) and cloned. Anti-CENH3 antibody was then raised against a deduced amino acid sequence of CENH3 of welsh onion. The antibody recognized all CENH3 orthologs of the Allium species tested. Immunostaining with the antibody enabled clear visualization of chromosome behavior during mitosis in the species. Furthermore, three-dimensional (3D) observation of mitotic cell division was achieved by subjecting root sections to immunohistochemical techniques. The 3D dynamics of the cells and position of cell-cycle marker proteins (CENH3 and α-tubulin) were clearly revealed by immunohistochemical staining with the antibodies. The immunohistochemical analysis made it possible to establish an overview of the location of dividing cells in the root tissues. This breakthrough in technique, in addition to the two centromeric DNA sequences isolated from welsh onion by chromatin immuno-precipitation using the antibody, should lead to a better understanding of plant cell division. A phylogenetic analysis of Allium CENH3s together with the previously reported plant CENH3s showed two separate clades for monocot species tested. One clade was made from CENH3s of the Allium species with those of Poaceae species, and the other from CENH3s of a holocentric species (Luzula nivea). These data may imply functional differences of CENH3s between holocentric and monocentric species. Centromeric localization of DNA sequences isolated from welsh onion by chromatin immuno-precipitation (ChIP) using the antibody was confirmed by fluorescence in situ hybridization and ChIP-quantitative PCR.     

Molecular and functional dissection of the maize B chromosome centromere

The centromere of the maize (Zea mays) B chromosome contains several megabases of a B-specific repeat (ZmBs), a 156-bp satellite repeat (CentC), and centromere-specific retrotransposons (CRM elements). Here, we demonstrate that only a small fraction of the ZmBs repeats interacts with CENH3, the histone H3 variant specific to centromeres. CentC, which marks the CENH3-associated chromatin in maize A centromeres, is restricted to an approximately 700-kb domain within the larger context of the ZmBs repeats. The breakpoints of five B centromere misdivision derivatives are mapped within this domain. In addition, the fraction of this domain remaining after misdivision correlates well with the quantity of CENH3 on the centromere. Thus, the functional boundaries of the B centromere are mapped to a relatively small CentC- and CRM-rich region that is embedded within multimegabase arrays of the ZmBs repeat. Our results demonstrate that the amount of CENH3 at the B centromere can be varied, but with decreasing amounts, the function of the centromere becomes impaired.     

CENH3 interacts with the centromeric retrotransposon <Emphasis Type="Italic">cereba</Emphasis> and GC-rich satellites and locates to centromeric substructures in barley

The chromosomal location of centromere-specific histone H3 (CENH3) is the assembly site for the kinetochore complex of active centromeres. Chromatin immunoprecipitation data indicated that CENH3 interacts in barley with cereba, a centromeric retroelement (CR)-like element conserved among cereal centromeres and barley-specific GC-rich centromeric satellite sequences. Anti-CENH3 signals on extended chromatin fibers always colocalized with the centromeric sequences but did not encompass the entire area covered by such centromeric repeats. This indicates that the CENH3 protein is bound only to a fraction of the centromeric repeats. At mitotic metaphase, CENH3, histone H3, and serine 10 phosphorylated histone H3 predominated within distinct structural subdomains of the centromere, as demonstrated by immunogold labeling for high resolution scanning electron microscopy.     

Only Missense Mutations Affecting the DNA Binding Domain of P53 Influence Outcomes in Patients with Breast Carcinoma

The presence of a TP53 gene mutation can influence tumour response to some treatments, especially in breast cancer. In this study, we analysed p53 mRNA expression, LOH at 17p13 and TP53 mutations from exons 2 to 11 in 206 patients with breast carcinoma and correlated the results with disease-free and overall survival. The observed mutations were classified according to their type and location in the three protein domains (transactivation domain, DNA binding domain, oligomerization domain) and correlated with disease-free and overall survival. In our population, neither p53 mRNA expression nor LOH correlated with outcome. Concerning TP53 mutations, 27% of tumours were mutated (53/197) and the presence of a mutation in the TP53 gene was associated with worse overall survival (p = 0.0026) but not with disease-free survival (p = 0.0697), with median survival of 80 months and 78 months, respectively. When alterations were segregated into mutation categories and locations, and related to survival, tumours harbouring mutations other than missense mutations in the DNA binding domain of P53 had the same survival profiles as wild-type tumours. Concerning missense mutations in the DNA binding domain, median disease-free and overall survival was 23 months and 35 months, respectively (p = 0.0021 and p<0.0001, respectively), compared with 78 and 80 months in mutated tumours overall. This work shows that disease-free and overall survival in patients with a frameshift mutation of TP53 or missense mutation in the oligomerization domain are the same as those in wild-type TP53 patients.     

The Rapidly Evolving Centromere-Specific Histone Has Stringent Functional Requirements in Arabidopsis thaliana

Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone-fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including Saccharomyces cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain–CENH3 histone-fold domain chimera rescued viability of cenh3-1, but CENH3''s lacking a tail domain were nonfunctional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP–CENH3 from the sister species A. arenosa functionally replaces A. thaliana CENH3. GFP–CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in A. thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.CENTROMERES are essential for chromosome inheritance, because they nucleate kinetochores, the protein complexes on eukaryotic chromosomes that attach to spindle microtubules. Despite the essential requirement for centromeres in chromosome segregation, their DNA sequences and the sequences of kinetochore proteins are highly variable. Kinetochores in Saccharomyces cerevisiae and related budding yeasts assemble on small, unique centromere DNAs (125 bp in S. cerevisiae) (Meraldi et al. 2006). Centromere DNAs in the fission yeast Schizosaccharomyces pombe are larger, consisting of a central core sequence of 4–5 kb, which binds kinetochore proteins, flanked by large inverted repeats whose heterochromatic nature is important for centromere function (the total size of the S. pombe centromere DNA is 35–110 kb). At the other extreme from small yeast centromeres are holocentric organisms, such as Caenorhabditis elegans, in which kinetochore proteins bind along the entire length of mitotic chromosomes (Dernburg 2001). Most plants and animals have extremely large centromere DNA tracts consisting of megabases of simple tandem repeats. The repeat sequence evolves extremely rapidly, and only a small fraction of the repeat array is likely to be bound by kinetochore proteins. Furthermore, kinetochores can be nucleated by noncentromeric DNA sequences in plant and animal cells (Amor and Choo 2002; Nagaki et al. 2004; Nasuda et al. 2005; Heun et al. 2006; Wade et al. 2009). Despite these findings, the maintenance of massive centromere repeat arrays in both animal and plant taxa suggests that repeats are a central feature of centromere biology in these organisms.Although centromere DNAs are extremely diverse, all eukaryote kinetochores contain the centromere-specific histone H3 variant CENH3 (originally described as CENP-A in human) (Henikoff and Dalal 2005; Black and Bassett 2008). CENH3 replaces conventional H3 specifically in a subset of centromere nucleosomes. It is essential for kinetochore function in all eukaryotes where this requirement has been tested. Conventional histones are among the most conserved proteins in eukaryote genomes. In contrast, CENH3 is rapidly evolving. The C-terminal histone-fold domain, which complexes with other histones to form the globular nucleosome core, can be aligned with conventional H3''s but evolves rapidly and shows signatures of adaptive evolution in some residues (Malik and Henikoff 2001; Talbert et al. 2002; Cooper and Henikoff 2004). The N-terminal tail domain of conventional histone H3 protrudes from the nucleosome core and is not resolved in the structure solved by X-ray crystallography (Luger et al. 1997). In CENH3, the tail domain evolves so rapidly that its sequence can barely be aligned between closely related species.Experiments in yeast and in animals have delineated functionally important regions within CENH3. S. cerevisiae kinetochores contain only a single CENH3/Cse4p nucleosome (Furuyama and Biggins 2007). In S. cerevisiae Cse4p, amino acid residues required for normal function are distributed throughout the histone-fold domain (Keith et al. 1999). The N-terminal tail of Cse4p contains an essential region termed the END domain, but overexpression of a Cse4p lacking the tail altogether can rescue a cse4 deletion mutant (Chen et al. 2000; Morey et al. 2004). In Drosophila melanogaster cells, CENH3/Cid from the distantly related D. bipectinata did not localize to kinetochores unless a specific region of the histone-fold domain, loop 1, was swapped with the corresponding region from D. melanogaster CENH3/Cid (Vermaak et al. 2002). In human, the histone-fold domain is important for centromere targeting (Sullivan et al. 1994). The functionally important region within the histone-fold domain was further defined by inserting loop 1 and the α-2 helix from CENH3/CENP-A (termed the CENP-A targeting domain, or CATD) into conventional H3 (Black et al. 2004). H3 containing the CATD acquires several functions of CENP-A when expressed in human cells. It localizes to kinetochores, binds the kinetochore protein CENP-N, has a rigid secondary structure when assembled into nucleosomes, and can restore normal chromosome segregation in cells depleted for CENP-A using RNA interference (RNAi) (Black et al. 2004, 2007a,b; Carroll et al. 2009).Despite these extensive studies, questions about structure–function relationships within CENH3 remain. CENH3 function may differ between small yeast centromeres and the large tandem repeat centromeres of animals and plants, particularly because larger centromere DNAs are likely to contain many more CENH3 nucleosomes and may require a higher level of organization. Experiments in D. melanogaster and in human cells have used RNAi to downregulate the endogenous protein, and a conditional knockout has been made in chicken DT-40 cells (Blower and Karpen 2001; Goshima et al. 2003; Regnier et al. 2005; Black et al. 2007b). These experiments are challenging because CENH3 is very stable. If preexisting CENH3 is partitioned equally between duplicated sister centromeres, its amount will be approximately halved at each cell division. Therefore the protein may persist for many cell divisions after induction of RNAi, as shown by Western blots indicating that ∼10% of endogenous CENH3 remains in human cells subjected to two rounds of RNAi (Black et al. 2007b).We have chosen to study CENH3 in the model plant A. thaliana, which combines facile genetics and transgenesis with centromere DNA structure that is similar to most plants and animals (megabases of tandem repeats with a repeating unit of 178 bp) (Murata et al. 1994; Copenhaver et al. 1999). Although Drosophila and mouse CENH3 knockout mutants have been characterized (Howman et al. 2000; Blower et al. 2006), a large-scale structure–function analysis of CENH3 has not been attempted in these organisms. A cenh3 null mutant in A. thaliana allows us to completely replace the endogenous protein with transgenic variants (Ravi and Chan 2010). Here we report four major conclusions regarding CENH3 function in A. thaliana: (1) CENH3 function requires an N-terminal histone tail domain, although either the CENH3 tail or the H3 tail can support mitotic chromosome segregation. (2) Inserting the CENP-A targeting domain of CENH3 into H3 does not confer CENH3 function. (3) Complementation of cenh3 by heterologous CENH3 requires that the species of origin be closely related to A. thaliana. (4) Localization of a heterologous CENH3 protein to kinetochores in the presence of native CENH3 does not necessarily indicate that it can complement a cenh3 mutant. Overall, our results indicate that requirements for CENH3 function in A. thaliana are more stringent that those obtained in human cells. They underscore the usefulness of comparative studies of centromere function using genetically tractable experimental organisms.