Studies On The Mechanism Of OsSHOC1 And OsPTD In Regulating Meiosis Crossover Formation In Rice

Plant meiocytes halve the chromosome number through meiotic division,which provides a mechanism for guaranteeing the constant ploidy after fusion of male and female gametes.Moreover,repairing meiotic DNA double strand breaks(DSBs)via the homologous recombination pathway,can promote the exchange of genetic material between parents and increase the genetic diversity,which is the foundation of plant evolution to adapt to environmental changes.crossovers(COs),the product of homologous recombination repair,play an important role in ensuring the correct separation of homologous chromosomes in the first meiotic division(meiosis I)as well as increasing genetic material exchange.In most eukaryotes,a set of conserved proteins that are collectively termed ZMM proteins(named after molecular zipper 1 [ZIP1],ZIP2,ZIP3,ZIP4,Mut S homologue 4 [MSH4],MSH5,meiotic recombination 3[MER3] and sporulation 16 [SPO16] in Saccharomyces cerevisiae)are essential for the formation of the majority of meiotic crossovers(COs).Recent reports indicated that ZIP2 acts together with SPO16 and ZIP4 to control interference-sensitive(class I)CO formation through recognizing and stabilizing early recombination intermediates in budding yeast.However,whether this mechanism is conserved in plants is not clear.We isolated a mutant of OsSHOC1(SHORTAGE OF CHIASMATA 1),the homolog of ZIP2 in rice,by radiation mutagenesis of 9522(O.sativa ssp.japonica).Meanwhile,the CRISPR-Cas9 technology was used to obtain the knock-out mutants of its interacting protein Os PTD(PARTING DANCERS;SPO16 ortholog).We characterize the biological function of OsSHOC1 and Os PTD,and their relationships with other ZMM proteins through the phenotype identification and gene function analysis.The main results we got are as follows:1.Disruption of OsSHOC1 caused a reduction in total COs to ～83% of that in wild type.The number and distribution of COs in Osshoc1-2 single mutant and Osshoc1-2Osgen1 double mutant indicated that OsSHOC1 affects class I CO formation.FISH(Fluorescent in situ hybridization)results showed that the early telomere bouquet and later homologous chromosomes paring and synapsis were completed normally in Osshoc1-2 male meiocytes.γH2AX(Phosphorylation of H2 A histone family member X)foci are used as an indirect marker of DSBs.Os COM1(Completion of meiosis 1),Os RAD51C(RAD51[Radiation-sensitive 51] paralog)and Os DMC1(DNA meiotic recombinase 1)are involving in early steps of meiotic DSBs repair.Immunolocalization assays showed that there were no significant differences in the foci number of γH2AX,Os COM1,Os RAD51 C and Os DMC1 between the wild-type and Osshoc1-2 male meiotic cells,indicating that OsSHOC1 has no effect on the DSBs formation and early meiotic recombination repair steps.2.Immunolocalization assays showed that XPF(Xeroderma pigmentosa complementation group F)-like protein,OsSHOC1,displayed an extensive colocalization with its interacting ERCC1(Excision repair cross complementation group1)-like protein Os PTD on the prophase I chromosomes.Furthermore,the CO frequency in Osshoc1-2 Osptd1 double mutant decreased to the same level as that in Osshoc1-2,Osptd-1 single mutants.This suggests that the OsSHOC1 and Os PTD regulate rice class I CO formation by forming an XPF-ERCC1-like complex in vivo.3.The abnormal chromosomal localizations of Os HEI10(Human enhancer of invasion 10;ZIP3 ortholog),Os ZIP4 and Os MER3 proteins were detected in Osshoc1-2 and Osptd-1 male meiocytes.Additionally,CO frequency in Osshoc1-2 Oshei10 and Osshoc1-2 Oszip4 double mutants decreased significantly compared with that in Oshei10 and Oszip4 single mutants,while the reduced level of CO frequency was similar to that in Osshoc1-2,Osptd-1 and Osshoc1-2 Osptd-1 mutants.These results suggest that OsSHOC1 and Os PTD function in the same pathway together with Os HEI10,Os ZIP4 and Os MER3,and regulate class I CO formation by affecting ZMM proteins recruitment.4.In consistent with these results,mutations in Os HEI10 and Os ZIP4 genes also affect the normal chromosomal localization of OsSHOC1 and Os PTD proteins.Further studies showed that OsSHOC1 can interact directly with Os ZIP4,and Os PTD can interact directly with Os HEI10.Bi FC(bimolecular fluorescence complementation)and Y3H(Yeast Three-Hybrid)showed that different combinations of heterotrimers could be formed between OsSHOC1,Os PTD,Os HEI10 and Os ZIP4.In addition,we found that OsSHOC1 and Os ZIP4 are also able to interact with Os MSH5,and Os HEI10 interacts with Os ZIP4.In conclusion,we propose that OsSHOC1,Os PTD,Os ZIP4or/and Os HEI10 may form a multiprotein complex(conserved ZZS[ZIP2-ZIP4-SPO16]-like complex or heterotetramer)to promote class I CO formation.In this study,we performed a more comprehensive characterization of the functions of OsSHOC1 and Os PTD proteins in controlling rice meiotic CO formation using cytology,genetics,biochemistry methods.Especially,the studies on the proteinprotein interaction and chromosomal localization dependence between rice ZMM proteins provide insights for understanding how ZMM proteins regulate rice CO formation,and the functional conservation and differentiation of ZMM proteins in different species.