Immunogenic assessment of plant‐produced human papillomavirus type 16 L1/L2 chimaeras

Cervical cancer is caused by infection with human papillomaviruses (HPV) and is a global concern, particularly in developing countries, which have ~80% of the burden. HPV L1 virus‐like particle (VLP) type–restricted vaccines prevent new infections and associated disease. However, their high cost has limited their application, and cytological screening programmes are still required to detect malignant lesions associated with the nonvaccine types. Thus, there is an urgent need for cheap second‐generation HPV vaccines that protect against multiple types. The objective of this study was to express novel HPV‐16 L1‐based chimaeras, containing cross‐protective epitopes from the L2 minor capsid protein, in tobacco plants. These L1/L2 chimaeras contained epitope sequences derived from HPV‐16 L2 amino acid 108–120, 56–81 or 17–36 substituted into the C‐terminal helix 4 (h4) region of L1 from amino acid 414. All chimaeras were expressed in Nicotiana benthamiana via an Agrobacterium‐mediated transient system and targeted to chloroplasts. The chimaeras were highly expressed with yields of ~1.2 g/kg plant tissue; however, they assembled differently, indicating that the length and nature of the L2 epitope affect VLP assembly. The chimaera containing L2 amino acids 108–120 was the most successful candidate vaccine. It assembled into small VLPs and elicited anti‐L1 and anti‐L2 responses in mice, and antisera neutralized homologous HPV‐16 and heterologous HPV‐52 pseudovirions. The other chimaeras predominantly assembled into capsomeres and other aggregates and elicited weaker humoral immune responses, demonstrating the importance of VLP assembly for the immunogenicity of candidate vaccines.     

Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12

The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at ?12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.     

Effect of nucleocapsid on multimerization of <Emphasis Type="Italic">gypsy</Emphasis> structural protein GAG

The structural protein (Gag) of Drosophila retrovirus gypsy contains capsid and nucleocapsid domains. Gag forms virus-like particles in a bacterial cell; furthermore, its capsid alone is able to form aggregates. However, aggregates assembled from the capsid vary in size and are less organized than particles formed by a full-length Gag. The nucleocapsid determines the organization and structure of the particles, which is ensured by the amino acid residues at its N-terminal (a nucleocapsid proximal part). The assembly of the particle occurs in the presence of any RNAs or single-stranded DNA oligonucleotides.     

Oligomerization and higher‐order assembly contribute to sub‐cellular localization of a bacterial scaffold

In Caulobacter crescentus, the PopZ polar scaffold protein supports asymmetric cell division by recruiting distinct sets of binding partners to opposite cell poles. To understand how polar organizing centres are established by PopZ, we investigated a set of mutated PopZ proteins for defects in sub‐cellular localization and recruitment activity. We identified a domain within the C‐terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N‐terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation. Mutations that inhibited dynamic PopZ localization inhibited the recruitment of other factors to cell poles. Mutations in the C‐terminal domain also blocked discrete steps in the assembly of higher‐order structures. Biophysical analysis of purified wild type and assembly defective mutant proteins indicates that PopZ self‐associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. The final six amino acids of PopZ are necessary for connecting the hexamers into filaments, and these structures are important for sub‐cellular localization. Thus, PopZ undergoes multiple orders of self‐assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter.     

Molecular cloning,expression and characterisation of Afp4, an antifreeze protein from Glaciozyma antarctica

Antifreeze proteins (AFPs) are proteins with affinity towards ice and contribute to the survival of psychrophiles in subzero environment. Limited studies have been conducted on how AFPs from psychrophilic yeasts interact with ice. In this study, we describe the functional properties of an antifreeze protein from a psychrophilic Antarctic yeast, Glaciozyma antarctica. A cDNA encoding the antifreeze protein, AFP4, from G. antarctica PI12 was amplified from the mRNA extracted from cells grown at 4 °C. Sequence characterisation of Afp4 showed high similarity to fungal AFPs from Leucosporidium sp. AY30, LeIBP (93 %). The 786-bp cDNA encodes a 261-amino-acid protein with a theoretical pI of 4.4. Attempts to produce the recombinant Afp4 in Escherichia coli resulted in the formation of inclusion bodies (IB). The IB were subsequently denatured and refolded by dilution. Gel filtration confirmed that the refolded recombinant Afp4 is monomeric with molecular mass of ~25 kDa. Thermal hysteresis (TH) and recrystallisation inhibition assays confirmed the function of Afp4 as an antifreeze protein. In the presence of Afp4, ice crystals were modified into hexagonal shapes with TH values of 0.08 °C and smaller ice grains were observed compared with solutions without AFP. Structural analyses via homology modelling showed that Afp4 folds into β-helices with three distinct faces: a, b and c. Superimposition analyses predicted the b-face as the ice-binding surface of Afp4, whereby the mechanism of interaction is driven by hydrophobic interactions and the flatness of surface. This study may contribute towards an understanding of AFPs from psychrophilic yeasts.     

The tail of a chaperonin: the C-terminal region of Esctierichia coli GroEL protein

The active form of the KSP60 molecular chaperone of Escherichia coli, GroEL, is a pair of seven-membered rings. We have used site-directed mutagenesis to construct forms of the 547-amino-acid monomer truncated at the C-terminus. We show here that forms that are 520 amino acids long or longer are close to being fully functional. Removing one further amino acid, however, results in a protein, GroEL519, which retains little function. This truncated form is metabolically stable but is not recovered from the cell in particle form. When synthesized at high levels, it prevents the normal assembly of GroEL547 present in the same cell. When synthesized at low levels, it can be included, probably at low molar ratios, in particles formed by assembly-competent forms of GroEL. This can be seen as partial complementation of the temperature-sensitive mutant groEL44. We conclude that amino acid 520 is cruical for particle assembly. GroEL516 has in vivo properties similar to those of GroEL519, but the still shorter form, GroEL504, appears to be inactive.     

A chromosome‐level genome assembly of the parasitoid wasp Pteromalus puparum

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short‐read, PacBio long‐read and Hi‐C scaffolding technologies was used to develop a high‐quality chromosome‐level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome‐level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338 Mb with a contig N50 of 38.7 kb and a scaffold N50 of 1.16 Mb. Hi‐C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8 Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single‐Copy Orthologs) gene set, supporting a high‐quality assembly and annotation. In total, 40.1% (135.6 Mb) of the assembly is composed of repetitive sequences, and 14,946 protein‐coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase‐related proteins and kynurenine–oxoglutarate transaminases, which have amplified in the P. puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P. puparum. These examples illustrate how chromosome‐level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.     

Self‐assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids

The morphology of structures formed by the self‐assembly of short N‐terminal t‐butyloxycarbonyl (Boc) and C‐terminal methyl ester (OMe) protected and Boc‐deprotected hydrophobic peptide esters was investigated. We have observed that Boc‐protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc‐Ile‐Ile‐OMe, Boc‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe and Boc‐Trp‐Ile‐Ile‐OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc‐Leu‐Ile‐Ile‐OMe and H‐Leu‐Ile‐Ile‐OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self‐assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well‐defined tertiary structures, upon removal of the Boc group, only H‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe had the ability to adopt β‐structure. Our results indicate that hydrophobic interaction is a very important determinant for self‐assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self‐assembly. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Oryza sativa Lysine‐Histidine‐type Transporter 1 functions in root uptake and root‐to‐shoot allocation of amino acids in rice

In agricultural soils, amino acids can represent vital nitrogen (N) sources for crop growth and yield. However, the molecular mechanisms underlying amino acid uptake and allocation are poorly understood in crop plants. This study shows that rice (Oryza sativa L.) roots can acquire aspartate at soil concentration, and that japonica subspecies take up this acidic amino acid 1.5‐fold more efficiently than indica subspecies. Genetic association analyses with 68 representative japonica or indica germplasms identified rice Lysine‐Histidine‐type Transporter 1 (OsLHT1) as a candidate gene associated with the aspartate uptake trait. When expressed in yeast, OsLHT1 supported cell growth on a broad spectrum of amino acids, and effectively transported aspartate, asparagine and glutamate. OsLHT1 is localized throughout the rice root, including root hairs, epidermis, cortex and stele, and to the leaf vasculature. Knockout of OsLHT1 in japonica resulted in reduced root uptake of amino acids. Furthermore, in ¹⁵N‐amino acid‐fed mutants versus wild‐type, a higher percentage of ¹⁵N remained in roots instead of being allocated to the shoot. ¹⁵N‐ammonium uptake and subsequently the delivery of root‐synthesized amino acids to Oslht1 shoots were also significantly decreased, which was accompanied by reduced shoot growth. These results together provide evidence that OsLHT1 functions in both root uptake and root to shoot allocation of a broad spectrum of amino acids in rice.     

Small rubber particle proteins from Taraxacum brevicorniculatum promote stress tolerance and influence the size and distribution of lipid droplets and artificial poly(cis‐1,4‐isoprene) bodies

Natural rubber biosynthesis occurs on rubber particles, i.e. organelles resembling small lipid droplets localized in the laticifers of latex‐containing plant species, such as Hevea brasiliensis and Taraxacum brevicorniculatum. The latter expresses five small rubber particle protein (SRPP) isoforms named TbSRPP1–5, the most abundant proteins in rubber particles. These proteins maintain particle stability and are therefore necessary for rubber biosynthesis. TbSRPP1–5 were transiently expressed in Nicotiana benthamiana protoplasts and the proteins were found to be localized on lipid droplets and in the endoplasmic reticulum, with TbSRPP1 and TbSRPP3 also present in the cytosol. Bimolecular fluorescence complementation confirmed pairwise interactions between all proteins except TbSRPP2. The corresponding genes showed diverse expression profiles in young T. brevicorniculatum plants exposed to abiotic stress, and all except TbSRPP4 and TbSRPP5 were upregulated. Young Arabidopsis thaliana plants that overexpressed TbSRPP2 and TbSRPP3 tolerated drought stress better than wild‐type plants. Furthermore, we used rubber particle extracts and standards to investigate the affinity of the TbSRPPs for different phospholipids, revealing a preference for negatively charged head groups and 18:2/16:0 fatty acid chains. This finding may explain the effect of TbSRPP3–5 on the dispersity of artificial poly(cis‐1,4‐isoprene) bodies and on the lipid droplet distribution we observed in N. benthamiana leaves. Our data provide insight into the assembly of TbSRPPs on rubber particles, their role in rubber particle structure, and the link between rubber biosynthesis and lipid droplet‐associated stress responses, suggesting that SRPPs form the basis of evolutionarily conserved intracellular complexes in plants.     

Identification of the amino acid residues and domains in the cysteine‐rich protein of Chinese wheat mosaic virus that are important for RNA silencing suppression and subcellular localization

Cysteine‐rich proteins (CRPs) encoded by some plant viruses in diverse genera function as RNA silencing suppressors. Within the N‐terminal portion of CRPs encoded by furoviruses, there are six conserved cysteine residues and a Cys–Gly–X–X–His motif (Cys, cysteine; Gly, glycine; His, histidine; X, any amino acid residue) with unknown function. The central domains contain coiled‐coil heptad amino acid repeats that usually mediate protein dimerization. Here, we present evidence that the conserved cysteine residues and Cys–Gly–X–X–His motif in the CRP of Chinese wheat mosaic virus (CWMV) are critical for protein stability and silencing suppression activity. Mutation of a leucine residue in the third coiled‐coil heptad impaired CWMV CRP activity for suppression of local silencing, but not for the promotion of cell‐to‐cell movement of Potato virus X (PVX). In planta and in vitro analysis of wild‐type and mutant proteins indicated that the ability of the CRP to self‐interact was correlated with its suppression activity. Deletion of up to 40 amino acids at the C‐terminus did not abolish suppression activity, but disrupted the association of CRP with endoplasmic reticulum (ER), and reduced its activity in the enhancement of PVX symptom severity. Interestingly, a short region in the C‐terminal domain, predicted to form an amphipathic α‐helical structure, was responsible for the association of CWMV CRP with ER. Overall, our results demonstrate that the N‐terminal and central regions are the functional domains for suppression activity, whereas the C‐terminal region primarily functions to target CWMV CRP to the ER.     

Centromere localization and function of Mis18 requires Yippee‐like domain‐mediated oligomerization

Mis18 is a key regulator responsible for the centromere localization of the CENP‐A chaperone Scm3 in Schizosaccharomyces pombe and HJURP in humans, which establishes CENP‐A chromatin that defines centromeres. The molecular and structural determinants of Mis18 centromere targeting remain elusive. Here, by combining structural, biochemical, and yeast genetic studies, we show that the oligomerization of S. pombe Mis18, mediated via its conserved N‐terminal Yippee‐like domain, is crucial for its centromere localization and function. The crystal structure of the N‐terminal Yippee‐like domain reveals a fold containing a cradle‐shaped pocket that is implicated in protein/nucleic acid binding, which we show is required for Mis18 function. While the N‐terminal Yippee‐like domain forms a homodimer in vitro and in vivo, full‐length Mis18, including the C‐terminal α‐helical domain, forms a homotetramer in vitro. We also show that the Yippee‐like domains of human Mis18α/Mis18β interact to form a heterodimer, implying a conserved structural theme for Mis18 regulation.     

Analysis of Tomato spotted wilt virus NSs protein indicates the importance of the N‐terminal domain for avirulence and RNA silencing suppression

Recently, Tomato spotted wilt virus (TSWV) nonstructural protein NSs has been identified unambiguously as an avirulence (Avr) determinant for Tomato spotted wilt (Tsw)‐based resistance. The observation that NSs from two natural resistance‐breaking isolates had lost RNA silencing suppressor (RSS) activity and Avr suggested a link between the two functions. To test this, a large set of NSs mutants was generated by alanine substitutions in NSs from resistance‐inducing wild‐type strains (NSs^RI), amino acid reversions in NSs from resistance‐breaking strains (NSs^RB), domain deletions and swapping. Testing these mutants for their ability to suppress green fluorescent protein (GFP) silencing and to trigger a Tsw‐mediated hypersensitive response (HR) revealed that the two functions can be separated. Changes in the N‐terminal domain were found to be detrimental for both activities and indicated the importance of this domain, additionally supported by domain swapping between NSs^RI and NSs^RB. Swapping domains between the closely related Tospovirus Groundnut ringspot virus (GRSV) NSs and TSWV NSs^RI showed that Avr functionality could not simply be transferred between species. Although deletion of the C‐terminal domain rendered NSs completely dysfunctional, only a few single‐amino‐acid mutations in the C‐terminus affected both functions. Mutation of a GW/WG motif (position 17/18) rendered NSs completely dysfunctional for RSS and Avr activity, and indicated a putative interaction between NSs and Argonaute 1 (AGO1), and its importance in TSWV virulence and viral counter defence against RNA interference.     

Bidirectional solid phase synthesis of a model oligoglycine bolaamphiphile and purification by rapid self‐assembly

We utilised a simple bidirectional (N→C and C→N) solid phase synthesis strategy entailing conventional solid phase peptide synthesis and fragment condensation with a water‐soluble carbodiimide to synthesise a model anionic glycylglycine bolaamphiphile containing a suberic acid linker moiety, namely N,N′‐suberoyldiglycylglycine. The synthetic suberoyldiglycylglycine was purified using its inherent ability to rapidly self‐assemble in an aqueous acidic solution (0.1% trifluoroacetic acid). Monitoring of the rapid assembly process corroborated our visual observation and confirmed packing‐directed self‐assembly rather than non‐specific aggregation or precipitation. The progress of suberoyldiglycylglycine self‐assembly was observed to be via the formation of oligomers in the solution, which then self‐assembled to form layered β‐sheet type macrostructures. Within 24 h, nanotubes grew from these macrostructures and eventually combined to formed microtubes, which we isolated after 5–7 days. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin

Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N‐terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin‐like substrate specificity was purified from the bacterial culture supernatant. The N‐terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg¹⁵¹? Ser¹⁵² bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.

     

Intein‐mediated protein assembly in transgenic wheat: production of active barnase and acetolactate synthase from split genes

Engineering traits by the assembly of non‐functional gene products is a promising tool for modern plant biotechnology. In this article, we describe the establishment of male sterility and herbicide resistance in wheat (Triticum aestivum) by complementing inactive precursor protein fragments through a split intein system. N‐ and C‐terminal fragments of a barnase gene from Bacillus amyloliquifaciens were fused to intein sequences from the Synechocystis sp. gene DnaB and delivered into the wheat genome via biolistic particle bombardment. Both barnase fragments were expressed under the control of a tapetum‐specific promoter. High efficiency of the split barnase system was achieved by introducing GGGGS linkers between the fusion domains of the assembled protein. Depending on the vector version that was transformed, up to 51% of primary transformed plants produced sterile pollen. In the F₁ progeny, the male‐sterile phenotype segregated with both barnase gene fragments. Expression of the cytotoxic barnase in the tapetum did not apparently affect the vegetative phenotype and remained stable under increased temperatures. In addition, the reconstitution of sulphonylurea resistance was achieved by DnaE intein‐mediated assembly of a mutated acetolactate synthase (ALS) protein from rice. The impacts of the technical advances revealed in this study on the concepts for trait control, transgene containment and hybrid breeding are discussed.     

Functional domain analysis of the Mycoplasma pneumoniae co‐chaperone TopJ

Colonization of conducting airways of humans by the prokaryote Mycoplasma pneumoniae is mediated by a differentiated terminal organelle important in cytadherence, gliding motility and cell division. TopJ is a predicted J‐domain co‐chaperone also having domains unique to mycoplasma terminal organelle proteins and is essential for terminal organelle function, as well as stabilization of protein P24, which is required for normal initiation of terminal organelle formation. J‐domains activate the ATPase of DnaK chaperones, facilitating peptide binding and proper protein folding. We performed mutational analysis of the predicted J‐domain, central acidic and proline‐rich (APR) domain, and C‐terminal domain of TopJ and assessed the phenotypic consequences when introduced into an M. pneumoniae topJ mutant. A TopJ derivative with amino acid substitutions in the canonical J‐domain histidine–proline–aspartic acid motif restored P24 levels but not normal motility, morphology or cytadherence, consistent with a J‐domain co‐chaperone function. In contrast, TopJ derivatives having APR or C‐terminal domain deletions were less stable and failed to restore P24, but resulted in normal morphology, intermediate gliding motility and cytadherence levels exceeding that of wild‐type cells. Results from immunofluorescence microscopy suggest that both the APR and C‐terminal domains, but not the histidine–proline–aspartic acid motif, are critical for TopJ localization to the terminal organelle.     

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

The wild‐type HIV‐1 capsid protein (CA) self‐assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV‐1. Mass‐per‐length values of CA assemblies determined by dark‐field transmission electron microscopy indicate a variety of structures, ranging from single‐wall tubes to multiwall tubes that approximate solid rods. Two‐dimensional (2D) solid state ¹³C? ¹³C and ¹⁵N? ¹³C NMR spectra of uniformly ¹⁵N,¹³C‐labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3‐¹³C₂‐glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N‐terminal (NTD) and C‐terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self‐assembly of full‐length CA into tubes. 2D ¹H‐¹³C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the α‐helices of NTD and CTD and from the N‐ and C‐terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.     

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.