Fertility restoration of maize CMS‐C altered by a single amino acid substitution within the Rf4 bHLH transcription factor

• Published in: The Plant journal : for cell and molecular biology Vol. 101; no. 1; pp. 101 - 111
• Main Authors: Jaqueth, Jennifer S., Hou, Zhenglin, Zheng, Peizhong, Ren, Ruihua, Nagel, Bruce A., Cutter, Gary, Niu, Xiaomu, Vollbrecht, Erik, Greene, Thomas W., Kumpatla, Siva P.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.01.2020
• Subjects: Agricultural production; Amino acid substitution; Amino acids; bHLH transcription factor; Chromosome 8; Chromosomes; Cloning; Corn; CRISPR; CRISPR‐Cas9; Cytoplasmic male sterility; C‐Type cytoplasmic male sterility; Dimers; Fertility; Gene expression; Helix-loop-helix proteins (basic); Homology; Localization; Male sterility; Mate selection; Mitochondria; Recombinants; Restoration; Restorer of fertility; structural modeling; Substitutes; Transcription factors; Zea mays; structural modeling; Zea mays; C-Type cytoplasmic male sterility; bHLH transcription factor; Restorer of fertility; CRISPR-Cas9
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.14521

Summary Type C cytoplasmic male sterility (CMS‐C) is the most commonly used form of CMS in maize hybrid seed production. Restorer of fertility 4 (Rf4), the major fertility restorer gene of CMS‐C, is located on chromosome 8S. To positionally clone Rf4, a large F3 population derived from a cross between a non‐restorer and restorer (n = 5104) was screened for recombinants and then phenotyped for tassel fertility, resulting in a final map‐based cloning interval of 12 kb. Within this 12‐kb interval, the only likely candidate for Rf4 was GRMZM2G021276, a basic helix−loop−helix (bHLH) transcription factor with tassel‐specific expression. The Rf4 gene product contains a nuclear localization signal and is likely to not interact directly with the mitochondria. Sequence analysis of Rf4 revealed four encoded amino acid substitutions between restoring and non‐restoring inbreds, however only one substitution, F187Y, was within the highly conserved bHLH domain. The hypothesis that Rf4 restoration is altered by a single amino acid was tested by using clustered regularly interspaced short palindromic repeat (CRISPR)‐CRISPR associated protein 9 (Cas9) homology directed repair (HDR) to create isogenic lines that varied for the F187Y substitution. In a population of these CRISPR‐Cas9 edited plants (n = 780) that was phenotyped for tassel fertility, plants containing F187 were completely fertile, indicating fertility restoration, and plants containing Y187 were sterile, indicating lack of fertility restoration. Structural modeling shows that this amino acid residue 187 is located within the four helix bundle core, a critical region for stabilizing dimer conformation and affecting interaction partner selection. Significance Statement Using positional cloning, we determined that Rf4, the major fertility restorer for CMS‐C, is a bHLH transcription factor also annotated as Ms23, an essential early acting factor in tapetal development, suggesting the CMS restoring ability of this gene is a secondary function. We identified and confirmed, with CRISPR‐Cas9 HDR editing, the restoration casual amino acid substitution, at the center of the bHLH dimer interface.