Male‐sterile maize plants produced by targeted mutagenesis of the cytochrome P450‐like gene (MS26) using a re‐designed I–CreI homing endonuclease

Abstract

SUMMARYThe I-CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double-strand breaks and enhancing DNA recombination reactions in maize cells. The DNA-binding properties of this protein were re-designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single-chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium-mediated transformation, the cleavage resulted in mutations at a co-delivered extra-chromosomal ms26-site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26-site in 5.8% of transgenic T 0 plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of doublestrand break repair generated by non-homologous end joining. One of 21 mutagenized T 0 plants carried two mutated alleles of the MS26 gene. As expected, the bi-allelic mutant T 0 plant and the T 1 progeny homozygous for the ms26 mutant alleles were male-sterile. This paper described the second maize chromosomal locus (liguless-1 being the first one) mutagenized by a re-designed I-CreI-based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.

IRREGULAR POLLEN EXINE1 Is a Novel Factor in Anther Cuticle and Pollen Exine Formation

“…In addition, MS26 may hydroxylate C16 and C18 fatty acids at the v-carbon position (Dobritsa et al, 2009;Li et al, 2010;Djukanovic et al, 2013). To sum up, we speculate that the major function of IPE1 in anther development is as follows: v-hydroxy C16/C18 fatty acids formed by MS26 can be converted into C16/C18 diacids by IPE1 in the tapetum and long-chain v-aldehyde dehydrogenases, which will further enrich the lipid metabolic pathway in anthers, especially the formation of dicarboxylic acids.…”

“…Aliphatic materials containing wax, cutin, and sporopollenin share one common catalytic pathway in the tapetum, and they will be transported to the surface of anthers and microspores . In maize, knowledge about the development of the anther cuticle and pollen exine is limited (Cigan et al, 2001;Skibbe and Schnable, 2005;Djukanovic et al, 2013). In this study, we compared three male-sterile mutants of maize (ipe1, ms26, and ms45) in which the phenotypes of pollen exine and anther cuticle were all defective (Fig.…”

“…Microspores show early vacuolation and are aborted after the tetrad stage. MS26 probably encodes a cytochrome P450 monooxygenase (Djukanovic et al, 2013) that is homologous to CYP704B1 in Arabidopsis (Arabidopsis thaliana), CYP704B2 in rice, and BnMS1 and BnMS2 in Brassica napus, the mutation of which results in defective pollen exine. CYP704B1 and CYP704B2 have a similar function in the v-hydroxylation of C16 and C18 fatty acids (Dobritsa et al, 2009;Li et al, 2010;Yi et al, 2010).…”

Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

“…Sequencing data were in agreement with the qPCR results, confirming the high frequency of mutations at all three sites (Table III). As expected, T0 plants containing biallelic mutations at either Ms26 or Ms45 were male sterile (Cigan et al, 2001;Djukanovic et al, 2013). Plants were also screened by Cas9 and gRNA-specific PCR (Supplemental Table S3) for the presence of stably integrated copies of Cas9 and gRNA DNA expression cassettes.…”

“…To ensure good U6 polymerase III expression and not introduce a mismatch within the gRNA spacer, all target sequences contained a G at their 59 end. Two of these locations, one near liguleless1 (LIG1) and another within the 5th exon of the maize fertility gene Ms26, were previously mutated in maize by customdesigned homing endonucleases named LIG3:4 (Gao et al, 2010) and Ems26 (Djukanovic et al, 2013), respectively. To compare the efficiency of generating DSBs by Cas9-gRNA with that of these homing endonucleases, maize immature embryos were bombarded with DNA vectors containing constitutively expressed meganucleases (LIG3:4 and Ems26) or Cas9 components (S. pyogenes Cas9 and gRNA) and developmental genes (maize OVULE DEVELOPMENTAL PROTEIN2 [ODP2] and maize WUSCHEL [WUS]) and then analyzed by amplicon deep sequencing for the presence of mutations at the target sites.…”