A single nucleotide substitution in the coding region of Ogura male sterile gene, orf138, determines effectiveness of a fertility restorer gene, Rfo, in radish

Abstract

Cytoplasmic male sterility (CMS) observed in many plants leads defect in the production of functional pollen, while the expression of CMS is suppressed by a fertility restorer gene in the nuclear genome. Ogura CMS of radish is induced by a mitochondrial orf138, and a fertility restorer gene, Rfo, encodes a P-type PPR protein, ORF687, acting at the translational level. But, the exact function of ORF687 is still unclear. We found a Japanese variety showing male sterility even in the presence of Rfo. We examined the pollen fertility, Rfo expression, and orf138 mRNA in progenies of this variety. The progeny with Type H orf138 and Rfo showed male sterility when their orf138 mRNA was unprocessed within the coding region. By contrast, all progeny with Type A orf138 were fertile though orf138 mRNA remained unprocessed in the coding region, demonstrating that ORF687 functions on Type A but not on Type H. In silico analysis suggested a specific binding site of ORF687 in the coding region, not the 5′ untranslated region estimated previously, of Type A. A single nucleotide substitution in the putative binding site diminishes affinity of ORF687 in Type H and is most likely the cause of the ineffectiveness of ORF687. Furthermore, fertility restoration by RNA processing at a novel site in some progeny plants indicated a new and the third fertility restorer gene, Rfs, for orf138. This study clarified that direct ORF687 binding to the coding region of orf138 is essential for fertility restoration by Rfo.

Intraspecific variations of the cytoplasmic male sterility genes orf108 and orf117 in Brassica maurorum and Moricandia arvensis, and the specificity of the mRNA processing

“…These data suggest that nucleotide substitutions at the cleavage site of CMS genes inhibit the suppression induced by the specific Rf gene. We recently found a similar relationship between the CMS gene (orf138) and the Rf genes in Ogura CMS of radish (Yamagishi et al 2021). ORF687 encoded by Rfo binds to orf138 mRNA, but a single nucleotide substitution of orf138 in the binding site diminished the effect of the Rfo gene by drastically decreasing the binding affinity of ORF687, and the male sterility phenotype was expressed despite the presence of Rfo.…”

Mechanism and Utilization of Ogura Cytoplasmic Male Sterility in Cruciferae Crops

“…A new restorer gene of Ogura CMS, Rft, which was found in the Tomioka population of Japanese wild radish, was also a homolog of Rfo and had 19 SNPs compared with Rfo [ 50 ]. Yamagishi et al (2021) confirmed that Ogura CMS had a new fertility restorer gene, Rfs , which could restore the fertility of individuals by processing orf138 mRNA and was different from Rft [ 54 ].…”

“…An allele sequence analysis of the fertility restorer gene revealed four substituted amino acids (the 118th, 153rd, 170th and 171st amino acids) in the second and third repeats of the PPR, suggesting an essential role in the fertility restoration of these domains formed by these repeats in the Rfo gene-encoding protein ORF687 [ 123 ]. Yamagishi et al (2021) and Imai et al (2002) also considered that the 118th and 153rd amino acids in ORF687 play a crucial role in fertility restoration [ 54 , 183 ]. By comparing the transcript sequences of rfo / rfo and Rfo / Rfo homozygote plants, it was found that four amino acids (the 176th to 179th amino acids) of the Rfo gene were essential, and the deletion of these four amino acids in the central region of the Rfo gene-encoding protein reduced fertility restoration ability [ 182 ].…”

“…In the interaction of a nuclear fertility restorer gene and a mitochondrial CMS gene, Uyttewaal et al [ 57 ] proposed that ORF687 could associate with the 5’ untranslated region (5’ UTR) of orf138 mRNA. Yamagishi et al (2021) found that direct ORF687 binding to the coding region of orf138 , not the 5’ UTR inferred previously, was essential for fertility restoration by Rfo [ 54 ]. It was also found that either a single nucleotide substitution (the 61st nucleotide) in orf138 or two amino acid substitutions (the 118th and 153rd amino acids) in ORF687 will prevent the Rfo gene from restoring the fertility of Ogura CMS materials with the orf138 gene [ 54 ].…”

“…Yamagishi et al (2021) found that direct ORF687 binding to the coding region of orf138 , not the 5’ UTR inferred previously, was essential for fertility restoration by Rfo [ 54 ]. It was also found that either a single nucleotide substitution (the 61st nucleotide) in orf138 or two amino acid substitutions (the 118th and 153rd amino acids) in ORF687 will prevent the Rfo gene from restoring the fertility of Ogura CMS materials with the orf138 gene [ 54 ]. Wang et al (2021) demonstrated that PPR-B bound within the coding sequence of orf138 and found that the GTAAAGTTAGTGTAATA sequence of the orf138 transcript was the PPR-B binding site [ 184 ].…”