Assessment of the roles of OsSPO11-2 and OsSPO11-4 in rice meiosis using CRISPR/Cas9 mutagenesis.

Meiotic recombination is initiated by the programmed formation of chromosomal DNA double-strand breaks (DSBs) catalyzed by the transesterase SPO11 complex during the prophase of meiosis. In Arabidopsis, in contrast to yeast and mammals, DSBs are presumably catalyzed by two distinct SPO11 proteins, AtSPO11-1 and AtSPO11-2, together with the M-TOPOVIB. Five OsSPO11 genes have been identified in rice. To clarify the roles of these SPO11 paralogs, we used CRISPR/Cas9 mutagenesis to induce null mutants in OsSPO11-1, OsSPO11-2 and OsSPO11-4. The resulting mutations occurred at high frequency (80%) and proved to be uniformly biallelic in T-DNA transformants. Similar to Osspo11-1, biallelic mutations in OsSPO11-2 first exon led to complete panicle sterility, and consequently, none of these primary transformants produced seeds. Conversely, all the Osspo11-4 biallelic mutants were fertile. Then, to generate segregating Osspo11-2 mutant lines, we developed a successful alternative strategy based on dual intron targeting. Similar to Osspo11-1, the pollen mother cells of Osspo11-2 T1 progeny plants, showed an absence of bivalent formation at metaphase I, aberrant segregation of homologous chromosomes and formation of nonviable tetrads. In contrast, the chromosome behavior in Osspo11-4 male meiocytes was undistinguishable to that in the wild-type. Immunostaining of prophase pollen-mother-cells with antibodies directed against the strand invasion recombinase OsDMC1 confirmed the presence of an average of 114 foci and 101 foci colocalizing with chromosome axes per WT and Osspo11-4 cell, respectively. As expected, we observed very few DMC1 foci (19 on average) in the sterile Osspso11-1 mutant but surprisingly 85 foci in the sterile Osspo11-2 mutant, indicative of a divergent function between these two SPO11 genes in rice. Altogether, this study demonstrates that whereas OsSPO11-1 and OsSPO11-2 are the likely orthologs of Arabidopsis AtSPO11-1 and AtSPO11-2, the additional OsSPO11-4 gene has no major role in wild-type rice meiosis.