Molecular Mechanism Of OsMLH1 Interacting With OsMLH3 To Regulate Meiosis In Rice

Rice is not only a major food crop in the world,but also an important material for studying meiosis mechanisms in higher plants.Meiosis is one of the key processes of generational alternation in eukaryotes,and it is the key to produce new allelic mutations during sexual reproduction.Several key events including homologous pairing,synapsis,recombination,and crossover formation in meiosis are essential for efficient exchange and normal separation of homologs.Crop breeding depends upon the action of meiotic recombination to rearrange elite traits between parental chromosomes,thereby cultivating varieties with excellent traits.Therefore,analyzing the molecular mechanism of meiotic recombination is of great significance for both basic research and practical applications.In eukaryotes,DNA mismatch repair(MMR)system is highly conservative and plays an important role during DNA replication,repair,and recombination.As one of the components of MMR complex,MutL?(MLH1-MLH3)heterodimer mainly has key functions during meiotic recombination.In yeasts,mammals and plants,MLH1 and MLH3 homologs play important roles in meiotic crossover formation.Although the function of OsMLH3 in rice has been characterized recently,the cytological basis for meiotic synapsis and crossover formation needs to be further examined,and particularly the function of OsMLH1 is still poorly understood.In this study,CRISPR-Cas9 technology was used to edit the meiotic recombination genes OsMLH1 and OsMLH3,and the generated gene-editing mutants exhibited a semi-sterile phenotype.The study found that OsMLH1 promotes the timely removal of PAIR2 and the normal loading of ZEP1 by eliminating the interlocks formed during pairing and synapsis,which further promotes to the assembly of synapsis complexs.In addition,OsMLH1-OsMLH3 heterodimer regulates the formation of interference-sensitive crossovers(class I crossovers)during meiotic recombination.The main findings are as follows:1.Osmlh1 mutants affect rice fertilityBioinformatics analysis showed that the proteins encoded by OsMLH1 and OsMLH3 are very conserved among species.Two allelic mutants,Osmlh1-1 and Osmlh1-2,were created by CRISPR-Cas9 technology.Osmlh1-1 was missing a base G in the fifth exon,and Osmlh1-2 was missing a base G in the first exon.The results of phenotypic investigation and genetic analysis showed that the Osmlh1-1 mutant exhibited a semi-sterile phenotype,which was caused by a recessive mutation in OsMLH1,and the Osmlh1-2 mutant also exhibited a semi-sterile phenotype.Genetic complementation test showed that OsMLH1 restored the fertility of the Osmlh1-1 mutant.2.OsMLH1 affects the meiosis process of microsporesQRT-PCR results showed that the transcripts of OsMLH1 were highly accumulated during meiosis in young rice panicles.GUS staining results showed that the GUS proteins driven by OsMLH1 promoter were accumulated during meiosis in anthers.Subcellular localization experiment verified that OsMLH1 proteins were localized in the nucleus.Scanning electron microscopy and transmission electron microscopy observations showed that some pollen grains in Osmlh1-1 were malformed and could not synthesize starch granules.Cytological observations showed that the meiotic chromosome behaviors of pollen mother cells in Osmlh1-1 and Osmlh1-2 were abnormal.3.OsMLH1 promotes synaptonemal complex assemblyFluorescent in situ hybridization(FISH)analysis using 5S r DNA probes and telomere probes,and immunofluorescence detection(IF)experiment with the anti-Os Cen H3 antibodies confirmed that homologous pairing was normal in Osmlh1-1.IF experiment with the anti-?H2AX antibodies indicated that the initiation of homologous recombination was normal in Osmlh1-1.Further IF experiments showed that RPA2 c and MER3 were normally loaded onto chromosomes in Osmlh1-1.IF experiments with the anti-REC8,PAIR2 and ZEP1 antibodies showed that the interlocks and non-synapsed regions on the chromosomes were observed at pachytene by REC8 signals.In the corresponding separated areas,the clearance of PAIR2 was delayed and the ZEP1 proteins were not be loaded.Thus,we deduced that OsMLH1 may participate in resolution of interlocks for the normal assembly of synaptonemal complexs.4.OsMLH1 affects the formation of interference-sensitive crossoversFISH experiment and statistical analysis of meiocytes at metaphase I in Osmlh1-1showed that the cells harboring non-associated 5S r DNA signals accounted for about 41%of the measured meiocytes,the bivalent number dropped by about 42%,and the chiasma number dropped by about 53%.Kolmogorov-Smirnov test method was used to analyze the distribution of chiasmata.The results showed that the chiasma distribution in Osmlh1-1 accorded with the predicted Poisson distribution,indicating that the crossovers in Osmlh1-1 were randomly distributed and insensitive to interference.Therefore,OsMLH1 is required for the formation of interference-sensitive crossovers(class I crossovers)during meiotic recombination.5.OsMLH1 interacts with OsMLH3Yeast two-hybrid experiment verified that the interaction between OsMLH1 and OsMLH3 was dependent on each other's C-terminal peptides.In vitro pull-down experiment verified that OsMLH1 interacted directly with OsMLH3.Split-luciferase complementation experiment verified that OsMLH1 interacted with OsMLH3 in tobaccos.6.OsMLH3 is involved in meiosisQRT-PCR results showed that OsMLH3 was highly expressed during meiosis in young rice panicles.Via CRISPR-Cas9 approach,we generated two allelic mutants and designated them as Osmlh3-1 and Osmlh3-2.The results of phenotypic investigation showed that the Osmlh3-1 and Osmlh3-2 mutants exhibited significantly reduced fertility.Cytological observations showed that the meiotic chromosome behaviors of pollen mother cells in Osmlh3-2 were abnormal.7.OsMLH1 interacts with OsHEI10Yeast two-hybrid experiment and in vitro pull-down experiment confirmed that OsMLH1 interacted with OsHEI10,and their interaction depended on the C-terminal of OsMLH1.Yeast two-hybrid experiment proved that OsHEI10 could form homodimers.However,split-luciferase complementation experiment proved that OsMLH1/OsMLH3 did not interact with Os MSH4.8.Molecular model of MutL? involved in rice meiosisBased on the existing research,we summarized the molecular mechanism of MutL?(OsMLH1-OsMLH3)heterodimer regulating synapsis and CO formation in rice: During rice meiosis,the interlocks are formed at late zygotene,and removed during middle/late pachytene.The MutL?-OsHEI10 complex regulates the formation of class I crossovers at pachytene,thereby ensuring the normal formation of bivalents.OsMLH1,OsMLH3 and OsHEI10 may form a multi-protein complex during meiosis homologous recombination,thereby promoting the formation of class I crossovers.Whether MutL? heterodimers promote crossover formation is regulated by the ubiquitinylation and/or SUMOylation of OsHEI10 is worthy of further study.This paper studies the molecular mechanism of OsMLH1 interacting with OsMLH3 to participate in the process of rice meiosis,and provides relevant genetic resources and theoretical guidance for gene recombination and heterosis fixation in crop breeding.