CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex

Naho Kanda,Atsushi Toyoda,Jun Abe,Machiko Ichikawa,Ayaka Ono,Yuki Tsuchikane,Hiroyuki Sekimoto,Asao Fujiyama,Tomoaki Nishiyama

doi:10.1038/s41598-017-18251-8

Abstract

Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt+) and mating-type minus (mt−). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt+ and mt− cells, respectively. PR-IP mediates the release of protoplasts from mt− cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt− cells of the C. psl. complex. When the knockout mt− cells were mixed with wild-type mt+ cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.

Highlights

Sexual reproduction occurs in a wide variety of organisms, including plants, animals, and fungi
We focused on two genes named CpRLK1 and CpRLP1 because CpRLK1 encodes a receptor-like protein kinase (RLK), whereas CpRLP1 encodes a leucine-rich repeat (LRR) receptor-like protein (RLP)
We revealed the phenotypes of CpRLP1 knockout mutants and evaluated the role of CpRLP1 in successful mating of the C. psl. complex

Summary

Introduction

Sexual reproduction occurs in a wide variety of organisms, including plants, animals, and fungi. To study the molecular mechanism of sexual partner recognition in charophycean algae, we focused on the Closterium peracerosum-strigosum-littorale complex Complex is one of the most widely studied unicellular charophycean algae in terms of sexual reproduction[10,11]. Sexual reproduction between mt+ and mt− cells is regulated by two sex pheromones, namely protoplast-release-inducing protein (PR-IP) and PR-IP Inducer. PR-IP is produced by mt+ cells and induces protoplast release from mt− cells. We recently proposed a possible sexual reproduction mechanism in the C. psl. A cDNA microarray analysis revealed 88 pheromone-inducible, conjugation-related and/or sex-specific genes[15]. We analyzed transformants expressing antisense CpRLP1 RNA and knockout lines generated by the newly established CRISPR/Cas[9] system to determine the physiological function of CpRLP1. We conclude that CpRLP1 regulates the timing of protoplast release, which is required for cell fusion, possibly through inhibiting the action of PR-IP

Methods

Results

Conclusion