Generating Novel Male Sterile Tomatoes by Editing Respiratory Burst Oxidase Homolog Genes

Xiaojuan Dai,Hongtao Xie,Minglei Song,Lianghui Zhao,Wei Huang,Zhaobo Lang,Xiaomu Niu,Changle Ma,Chenglan Liu,Huanan Han,Xuesong Cao

doi:10.3389/fpls.2021.817101

Abstract

Hybrid breeding of tomatoes (Solanum lycopersicum), an important vegetable crop, is an effective way to improve yield and enhance disease and stress resistance. However, the efficiency of tomato hybridization is hindered by self-fertilization, which can be overcome using male sterile lines. It has been reported that reactive oxygen species (ROS) act as a key regulator for anther development, mediated by RBOH (Respiratory Burst Oxidase Homolog) genes. Here, two tomato anther-expressed genes, LeRBOH (Solyc01g099620) and LeRBOHE (Solyc07g042460), were selected to cultivate novel tomato male sterile strains. By using a CRISPR/Cas9 system with a two-sgRNA module, the lerboh, lerbohe, and lerboh lerbohe mutant lines were generated, among which the lerbohe and lerboh lerbohe mutants displayed complete male sterility but could accept wild-type pollens and produce fruits normally. Further analysis uncovered significantly decreased ROS levels and abnormal programmed cell death in lerboh lerbohe anthers, indicating a key role of ROS metabolism in tomato pollen development. Taken together, our work demonstrates a successful application of gene editing via CRISPR/Cas9 in generating male sterile tomatoes and afforded helpful information for understanding how RBOH genes regulating tomato reproduction process.

Highlights

Tomato (Solanum lycopersicum) is a highly cultivated and consumed vegetable around the world
It has been reported that LeRBOHE and LeRBOH show a temporal expression pattern during anther development (Yu et al, 2017)
Two sgRNAs, with spacer sequences targeting the first exon of LeRBOH and LeRBOHE, respectively, were separated by tRNA and driven by the Arabidopsis U6 promoter, while SpCas9 was driven by a CaMV35S promoter (Figure 1A)

Summary

INTRODUCTION

Tomato (Solanum lycopersicum) is a highly cultivated and consumed vegetable around the world. The mitochondrial protein Wa352 inhibits the ROS scavenging function of COX11, promoting PCD in the tapetum and subsequently leading to pollen abortion (Luo et al, 2013; Wang et al, 2018). Another rice male sterile mutant dtc exhibits decreased ROS accumulation in the anthers and a delayed PCD in hypertrophic tapetum (Yi et al, 2016). As the qPCR results describing, LeRBOH displayed a anther-specific high expression while LeRBOHE exhibited the highest expression in anther and stem, indicating they may play a role in regulating anther development Whether these two RBOH genes regulate pollen fertility remains unknown. Our work revealed mechanism by which RBOHs regulate development of tomato anthers, and successfully established a complete male sterile strain for tomato hybrid breeding

MATERIALS AND METHODS

RESULTS

DISCUSSION

DATA AVAILABILITY STATEMENT

CONCLUSION