Point Mutations in Centromeric Histone Induce Post-zygotic Incompatibility and Uniparental Inheritance

The centromeric histone 3 variant (CENH3, aka CENP-A) is essential for the segregation of sister chromatids during mitosis and meiosis. To better define CENH3 functional constraints, we complemented a null allele in Arabidopsis with a variety of mutant alleles, each inducing a single amino acid change in conserved residues of the histone fold domain. Many of these transgenic missense lines displayed wild-type growth and fertility on self-pollination, but exhibited frequent post-zygotic death and uniparental inheritance when crossed with wild-type plants. The failure of centromeres marked by these missense mutation in the histone fold domain of CENH3 reproduces the genome elimination syndromes described with chimeric CENH3 and CENH3 from diverged species. Additionally, evidence that a single point mutation is sufficient to generate a haploid inducer provide a simple one-step method for the identification of non-transgenic haploid inducers in existing mutagenized collections of crop species. As proof of the extreme simplicity of this approach to create haploid-inducing lines, we performed an in silico search for previously identified point mutations in CENH3 and identified an Arabidopsis line carrying the A86V substitution within the histone fold domain. This A87V non-transgenic line, while fully fertile on self-pollination, produced postzygotic death and uniparental haploids when crossed to wild type.     

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.

     

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.     

MUN (MERISTEM UNSTRUCTURED), encoding a SPC24 homolog of NDC80 kinetochore complex,affects development through cell division in Arabidopsis thaliana

Kinetochore, a protein super‐complex on the centromere of chromosomes, mediates chromosome segregation during cell division by providing attachment sites for spindle microtubules. The NDC80 complex, composed of four proteins, NDC80, NUF2, SPC24 and SPC25, is localized at the outer kinetochore and connects spindle fibers to the kinetochore. Although it is conserved across species, functional studies of this complex are rare in Arabidopsis. Here, we characterize a recessive mutant, meristem unstructured‐1 (mun‐1), exhibiting an abnormal phenotype with unstructured shoot apical meristem caused by ectopic expression of the WUSCHEL gene in unexpected tissues. mun‐1 is a weak allele because of the insertion of T‐DNA in the promoter region of the SPC24 homolog. The mutant exhibits stunted growth, embryo arrest, DNA aneuploidy, and defects in chromosome segregation with a low cell division rate. Null mutants of MUN from TALEN and CRISPR/Cas9‐mediated mutagenesis showed zygotic embryonic lethality similar to nuf2‐1; however, the null mutations were fully transmissible via pollen and ovules. Interactions among the components of the NDC80 complex were confirmed in a yeast two‐hybrid assay and in planta co‐immunoprecipitation. MUN is co‐localized at the centromere with HTR12/CENH3, which is a centromere‐specific histone variant, but MUN is not required to recruit HTR12/CENH3 to the kinetochore. Our results support that MUN is a functional homolog of SPC24 in Arabidopsis, which is required for proper cell division. In addition, we report the ectopic generations of stem cell niches by the malfunction of kinetochore components.     

The deposition of CENH3 in maize is stringently regulated

The centromere, as an essential element to mediate chromosome segregation, is epigenetically determined by CENH3‐containing nucleosomes as a functional marker; therefore the accurate deposition of CENH3 is crucial for chromosome transmission. We characterized the deposition of CENH3 in maize by over‐expression and mutational analysis. Our results revealed that over‐expressing CENH3 in callus is lethal while over‐expressing GFP‐CENH3 and CENH3‐YFP in callus and plants is not and can be partly deposited normally. Different mutations of GFP‐CENH3 demonstrated that CENH3‐Thr4 in the N‐terminus was needed for the deposition as a positive phosphorylation site and the last five amino acids in the C‐terminus are necessary for deposition. The C‐terminal tail of CENH3 is confirmed to be responsible for the interaction of CENH3 and histone H4, which indicates that CENH3 maintains deposition in centromeres via interacting with H4 to form stable nucleosomes. For GFP‐CENH3 and CENH3‐YFP, the fused tags at the termini probably affect the structure of CENH3 and reduce its interaction with other proteins, which in turn could decrease proper deposition. Taken together, multiple amino acids or motifs were shown to play essential roles in CENH3 deposition, which is suggested to be affected by numerous factors in maize.     

Combining next‐generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M₂) family derived from a heterozygous (M₁) parent were identified using whole‐genome shotgun (WGS) sequencing of a small number of (M₂) individuals with mutant and wild‐type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self‐pollinated (M₃) offspring. Finally, we filtered for segregating EMS‐induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M₂) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non‐synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.     

In vitro production of haploid and doubled haploid plants from pollinated ovaries of maize (Zea mays)

Haploid induction has potential application for maize breeding. This paper reports that maize haploid plants have been induced by in vitro culture of pollinated ovaries. From a total of 26,400 cultured ovaries, 24 haploid plants were obtained and two of them were doubled after colchicine treatment. The maximum frequency of gynogenesis was 0.17% at 19.5 h post-pollination (HPP). The results showed that HPP was an important factor affecting plant induction from ovaries. Regenerated diploid R₀ plants were then subjected to genetic analysis using SSR molecular markers. One R₀ plant, whose progeny revealed a high level of homogeneity for several agro-morphological traits, was homozygous at 20 loci tested, with 11 showing paternal and 9 maternal banding pattern. This demonstrates that it is feasible to induce maize haploid plants by in vitro culture of pollinated ovaries.     

Mutation by DNA shuffling of 5‐enolpyruvylshikimate‐3‐phosphate synthase from Malus domestica for improved glyphosate resistance

A new 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate‐tolerant plants. However, wild‐type MdEPSPS is not suitable for the development of transgenic glyphosate‐tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate‐resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site‐directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single‐site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate‐resistant crops.     

A thermodynamic approach to the conformational preferences of the 180–195 segment derived from the human prion protein α2‐helix

On consideration that intrinsic structural weakness could affect the segment spanning the α2‐helical residues 173–195 of the PrP, we have investigated the conformational stabilities of some synthetic Ala‐scanned analogs of the peptide derived from the 180–195 C‐terminal sequence, using a novel approach whose theoretical basis originates from protein thermodynamics. Even though a quantitative comparison among peptides could not be assessed to rank them according to the effect caused by single amino acid substitution, as a general trend, all peptides invariably showed an appreciable preference for an α‐type organization, consistently with the fact that the wild‐type sequence is organized as an α‐helix in the native protein. Moreover, the substitution of whatever single amino acid in the wild‐type sequence reduced the gap between the α‐ and the β‐propensity, invariably enhancing the latter, but in any case this gap was larger than that evaluated for the full‐length α2‐helix‐derived peptide. It appears that the low β‐conformation propensity of the 180–195 region depends on the simultaneous presence of all of the Ala‐scanned residues, indirectly confirming that the N‐terminal 173–179 segment could play a major role in determining the chameleon conformational behavior of the entire 173–195 region in the PrP. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development

Multilocular silique is a desirable agricultural trait with great potential for the development of high‐yield varieties of Brassica. To date, no spontaneous or induced multilocular mutants have been reported in Brassica napus, which likely reflects its allotetraploid nature and the extremely low probability of the simultaneous random mutagenesis of multiple gene copies with functional redundancy. Here, we present evidence for the efficient knockout of rapeseed homologues of CLAVATA3 (CLV3) for a secreted peptide and its related receptors CLV1 and CLV2 in the CLV signalling pathway using the CRISPR/Cas9 system and achieved stable transmission of the mutations across three generations. Each BnCLV gene has two copies located in two subgenomes. The multilocular phenotype can be recovered only in knockout mutations of both copies of each BnCLV gene, illustrating that the simultaneous alteration of multiple gene copies by CRISPR/Cas9 mutagenesis has great potential in generating agronomically important mutations in rapeseed. The mutagenesis efficiency varied widely from 0% to 48.65% in T₀ with different single‐guide RNAs (sgRNAs), indicating that the appropriate selection of the sgRNA is important for effectively generating indels in rapeseed. The double mutation of BnCLV3 produced more leaves and multilocular siliques with a significantly higher number of seeds per silique and a higher seed weight than the wild‐type and single mutant plants, potentially contributing to increased seed production. We also assessed the efficiency of the horizontal transfer of Cas9/gRNA cassettes by pollination. Our findings reveal the potential for plant breeding strategies to improve yield traits in currently cultivated rapeseed varieties.     

Enhanced production of aureofuscin by over-expression of AURJ3M, positive regulator of aureofuscin biosynthesis in Streptomyces aureofuscus

Aims: The production of aureofuscin is very low in the wild‐type strain. We attempt to increase the production of aureofuscin by over‐expression of a controlling gene in the wild‐type strain. Methods and Results: The aurj3M gene was PCR‐amplified from Streptomyces aureofuscus SYAU0709, ligated into vector pMD19 and sequenced. The predicted translation of the 579‐bp cloned fragment was 97% similar to pimM from Streptomyces natalensis, which has an N‐terminal PAS domain and a LuxR‐type C‐terminal helix–turn–helix. Recombinant bacterial strains were constructed by transforming SYAU0709 with an expression plasmid (pBJJ3M) that contained aurj3M, thereby increasing the number of aurj3M gene copies. Conclusions: Bioassays for the antibiotic compound aureofuscin indicated that the recombinant bacteria had greater antifungal activity than the wild‐type strain. Specifically, the recombinant strain produced approx. 600% more aureofuscin, as quantified by high‐performance liquid chromatography analysis. Significance and Impact of the Study: To our knowledge, this approach has not been attempted in S. aureofuscus before and few genes in the aureofuscin pathway have been cloned and characterized.     

Design,selection, and characterization of a split chorismate mutase

Split proteins are versatile tools for detecting protein–protein interactions and studying protein folding. Here, we report a new, particularly small split enzyme, engineered from a thermostable chorismate mutase (CM). Upon dissecting the helical‐bundle CM from Methanococcus jannaschii into a short N‐terminal helix and a 3‐helix segment and attaching an antiparallel leucine zipper dimerization domain to the individual fragments, we obtained a weakly active heterodimeric mutase. Using combinatorial mutagenesis and in vivo selection, we optimized the short linker sequences connecting the leucine zipper to the enzyme domain. One of the selected CMs was characterized in detail. It spontaneously assembles from the separately inactive fragments and exhibits wild‐type like CM activity. Owing to the availability of a well characterized selection system, the simple 4‐helix bundle topology, and the small size of the N‐terminal helix, the heterodimeric CM could be a valuable scaffold for enzyme engineering efforts and as a split sensor for specifically oriented protein–protein interactions.     

A centromeric DNA sequence colocalized with a centromere-specific histone H3 in tobacco

Centromeres play an important role in segregating chromosomes into daughter cells, and centromeric DNA assembles specific proteins to form a complex referred to as the kinetochore. Among these proteins, centromere-specific histone H3 (CENH3) is one of the most characterized and found to be located only on active centromeres. We isolated four different CENH3-coding complementary DNAs (cDNAs), two from Nicotiana tabaccum and one each from the ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis and raised an antibody against N-terminal amino acid sequences deduced from the cDNAs. Immunostaining with the antibody revealed the preferential centromere localization, indicating that the cDNAs cloned in this study encode authentic tobacco CENH3. A tobacco centromeric DNA sequence (Nt2-7) was also identified by chromatin immunoprecipitation cloning using the antibody. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Knockout of two BnaMAX1 homologs by CRISPR/Cas9‐targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L.)

Plant height and branch number are essential components of rapeseed plant architecture and are directly correlated with its yield. Presently, improvement of plant architecture is a major challenge in rapeseed breeding. In this study, we first verified that the two rapeseed BnaMAX1 genes had redundant functions resembling those of Arabidopsis MAX1, which regulates plant height and axillary bud outgrowth. Therefore, we designed two sgRNAs to edit these BnaMAX1 homologs using the CRISPR/Cas9 system. The T₀ plants were edited very efficiently (56.30%–67.38%) at the BnaMAX1 target sites resulting in homozygous, heterozygous, bi‐allelic and chimeric mutations. Transmission tests revealed that the mutations were passed on to the T₁ and T₂ progeny. We also obtained transgene‐free lines created by the CRISPR/Cas9 editing, and no mutations were detected in potential off‐target sites. Notably, simultaneous knockout of all four BnaMAX1 alleles resulted in semi‐dwarf and increased branching phenotypes with more siliques, contributing to increased yield per plant relative to wild type. Therefore, these semi‐dwarf and increased branching characteristics have the potential to help construct a rapeseed ideotype. Significantly, the editing resources obtained in our study provide desirable germplasm for further breeding of high yield in rapeseed.     

High‐efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system

The base‐editing technique using CRISPR/nCas9 (Cas9 nickase) or dCas9 (deactivated Cas9) fused with cytidine deaminase is a powerful tool to create point mutations. In this study, a novel G. hirsutum‐Base Editor 3 (GhBE3) base‐editing system has been developed to create single‐base mutations in the allotetraploid genome of cotton (Gossypium hirsutum). A cytidine deaminase sequence (APOBEC) fused with nCas9 and uracil glycosylase inhibitor (UGI) was inserted into our CRISPR/Cas9 plasmid (pRGEB32‐GhU6.7). Three target sites were chosen for two target genes, GhCLA and GhPEBP, to test the efficiency and accuracy of GhBE3. The editing efficiency ranged from 26.67 to 57.78% at the three target sites. Targeted deep sequencing revealed that the C→T substitution efficiency within an ‘editing window’, approximately six‐nucleotide windows of ?17 to ?12 bp from the PAM sequence, was up to 18.63% of the total sequences. The 27 most likely off‐target sites predicted by CRISPR‐P and Cas‐OFFinder tools were analysed by targeted deep sequencing, and it was found that rare C→T substitutions (average < 0.1%) were detected in the editing windows of these sites. Furthermore, whole‐genome sequencing analyses on two GhCLA‐edited and one wild‐type plants with about 100× depth showed that no bona fide off‐target mutations were detectable from 1500 predicted potential off‐target sites across the genome. In addition, the edited bases were inherited to T1 progeny. These results demonstrate that GhBE3 has high specificity and accuracy for the generation of targeted point mutations in allotetraploid cotton.     

Specific molecular marker of the genes controlling linolenic acid content in rapeseed

In rapeseed, which is an agronomically important oilseed, variation in the linolenic acid content of the oil has been obtained through chemical mutagenesis treatment. Conventional breeding of this quantitative trait, however requires specific molecular markers. By means of biochemical experiments, we have established that the induced variation in linolenic acid content is associated with the fad3 gene encoding the microsomal ¹⁵ desaturase. Using a pair of primers specific to this gene and a doubled haploid progeny derived from a low linolenic x high linolenic acid F₁hybrid, we have identified a polymorphism of the fad3 alleles between the low- and the high-linolenic acid genotypes. The structure exon/intron of the fad3 DNA sequence seems to be very similar to that of the Arabidopsis fad3 gene. The choice of the primer pair allows specific amplification of one of the two rapeseed fad3 genes. The value and contribution of specific markers to conventional plant breeding is discussed.     

Conditional allelic replacement applied to genes encoding the histone variant H3.3 in the mouse

Post‐translational modifications to residues in core histones convey epigenetic information. Their function can be evaluated in amino acid substitution mutants, although to date this method has not been used in mice. To this end, we have evaluated gene targeting vectors designed for Cre recombinase‐mediated conditional allelic replacement at the two unlinked genes encoding the histone variant H3.3. The conditional alleles consist of an uninterrupted wild‐type H3.3 coding sequence upstream of a desired alternative or proxy coding sequence. The arrangement of two loxP sites allows Cre‐mediated replacement of the wild‐type coding sequence with the proxy. To demonstrate proof of principle, at each locus we replaced the wild‐type coding sequence with a fluorescent reporter. This produced null alleles that will be useful to analyse the effects of H3.3 deficiency in development. Each targeting vector can readily be retrofitted with a proxy coding sequence encoding a modified H3.3 protein. Such vectors will allow for the conditional substitution of specific residues in order to dissect the roles of H3.3 post‐translational modifications in development and disease. genesis, 51:142?146, 2013. © 2013 Wiley Periodicals, Inc.