A conserved RWP-RK transcription factor VSR1 controls gametic differentiation in volvocine algae

Volvocine green algae are a model for understanding the evolution of mating types and sexes. They are facultatively sexual, with gametic differentiation occurring in response to nitrogen starvation (-N) in most genera, and to sex inducer hormone (SI) in Volvox . The conserved RWP RK family transcription factor (TF) MID is encoded by the minus mating type ( MT ) locus or male sex-determining region (SDR) of heterothallic volvocine species and dominantly determines minus or male gametic differentiation. However, the factor(s) responsible for establishing the default plus or female differentiation programs have remained elusive. We performed a phylo transcriptomic screen for autosomal RWP-RK TFs induced during gametogenesis in unicellular isogamous Chlamydomonas reinhardtii (Chlamydomonas) and in multicellular oogamous Volvox carteri (Volvox) and identified a single conserved ortho-group we named Volvocine Sex Regulator 1 (VSR1). Chlamydomonas vsr1 mutants of either mating type failed to mate and could not induce expression of key mating-type-specific genes. Similarly, Volvox vsr1 mutants in either sex could initiate sexual embryogenesis, but the presumptive eggs or androgonidia (sperm packet precursors) were infertile and unable to express key sex-specific genes. Yeast two-hybrid assays identified a conserved domain in VSR1 capable of self-interaction or interaction with the conserved N terminal domain of MID. In vivo co-immunoprecipitation experiments demonstrated association of VSR1 and MID in both Chlamydomonas and Volvox. These data support a new model for volvocine sexual differentiation where VSR1 homodimers activate expression of plus /female gamete-specific-genes, but when MID is present MID-VSR1 heterodimers are preferentially formed and activate minus /male gamete-specific-genes. Significance Statement Sex and recombination are conserved features of eukaryotic life cycles, but sex determination mechanisms are diverse, and are poorly understood in most major taxa. Our study identified a long-sought regulator of sexual differentiation in volvocine green algae—the RWP-RK family transcription factor (TF) VSR1— leading to the first complete paradigm for mating type or sex determination in this lineage. Our results support a model where gametically expressed VSR1 homodimerizes and activates plus /female specific genes. When the dominant sex-linked minus /male RWP-RK family TF MID is present MID-VSR1 heterodimers are preferentially formed and activate minus /male genes. The widespread association of RWP-RK TFs with gamete differentiation in the green lineage suggests that a similar paradigm may operate throughout the plant kingdom.