Synthetic Rf proteins targeted to cleave orf279 for CMS/Rf system in hybrid wheat breeding

Cytoplasmic male sterility (CMS) technology utilise a mitochondrial gene that causes pollen abortion and male sterility in plant hybrid breeding. In turn, plant fertility in the F1 generation is restored by the application of Rf (Restorer-of-fertility) genes, which suppress the CMS expression and often originate from the pentatricopeptide repeat protein (PPR) family. CMS-Rf system could be used to develop hybrid wheat. Recently, orf279 has been determined as the genetic cause of CMS in wheat plants carrying Triticum timopheevii CMS and the Rf1 and Rf3 restorer genes have been sequenced. However, seed set analyses have shown that F1 fertility restoration conferred by both genes is incomplete. RNA Electrophoretic Mobility Shift Assay (REMSA) indicated that the binding affinity of Rf1 and Rf3 proteins to their RNA targets is weaker compared to other well-characterised PPR proteins. Thus, it has been hypothesised that the poor RNA-binding of Rf1 and Rf3 could be the reason for incomplete fertility restoration. My studies aim to improve the binding affinity of Rf1 and Rf3 to their RNA targets. I will analyse the binding site of Rf1 and Rf3 and design synthetic Rf proteins with the best amino acid combination that matched each nucleotide target using Golden Gate Type IIS assembly system. The synthetic proteins will be tested in vivo using transient transformation of wheat protoplasts. The outcome of this project will be beneficial to hybrid wheat breeding programs using CMS system.