Distinct Cell-Autonomous Functions of RETINOBLASTOMA-RELATED in Arabidopsis Stem Cells Revealed by the Brother of Brainbow Clonal Analysis System

Abstract

Mutations that cause lethality in the gametophyte phase pose a major challenge for studying postfertilization gene function. When both male and female haploid cells require a functional gene copy, null alleles cause developmental arrest before the formation of the zygote, precluding further investigation. The Arabidopsis thaliana Rb homolog RETINOBLASTOMA-RELATED (RBR) has an important function in the stem cell niche, but its requirement in both male and female gametophytes has prevented full loss-of-function studies. To circumvent this obstacle, we designed a clonal deletion system named BOB (Brother of Brainbow) in which null mutant sectors marked by double fluorescence are generated in a fully complemented wild-type background. In this system, both copies of a complementing RBR transgene are eliminated by tissue-specific and inducible CRE expression, and homozygous mutant clones can be distinguished visually. Since mutant sectors can be produced in a homozygous, rather than a heterozygous, background, this system facilitates clonal deletion analysis not only for gametophytic lethal alleles but also for any type of mutation. Using the BOB system, we show that RBR has unique cell-autonomous functions in different cell types within the root stem cell niche.