Characterization Of SUN1 And SUN2 In Rice Meiosis

Meiosis is an special type of cell division during the formation of gametes in eukaryotes.In prophase I,homologous chromosomes recognize and pair with each other to complete the assembly of the association complex,which is necessary for the formating of crossovers(COs)and the normal separation of homologous chromosomes.The formation of telomere bouquets is a conserved event in meiosis,which is closely related to chromosome movement.In recent years,some factors that were involved in the formation of telomere bouquets had been discovered in yeast and mammals,the molecular model of bouquet formation has been proposed.However,that is unknown in plants.The SUN proteins are highly conserved in the telomere aggregation model of mice and fission yeast.By aligning the protein sequences,we found two candidates in rice,named SUN1 and SUN2 respectively.Using the CRISPR-Cas9 method,we knocked out both genes encoding SUN1 and SUN2 in wild type and obtained the sun1 sun2 double mutant.Observing the chromosome behavior during meiosis,we found chromosomes could not be aggregated normally at zygotene.,We further demonstrated that this defect is closely related to the abnormal formation of telomere bouquets by fluorescence in situ hybridization experiments.Moreover,the full-length chromosome synapsis,pairing and crossover formation were also affected in the sun1 sun2 double mutant.The HEI10 signal was restricted in synapsed chromosomes at pachytene and late pachytene.However,we found these faults were not dependent on early recombination events when we detected the localization of early recombinant proteins.These results indicate that the deletion of SUN1 and SUN2 affects telomere bouquet aggregation and chromosomal contraction at zygotene and leads to multiple meiotic deficiencies.By observing the localization of SUN1 and SUN2,we found that SUN1 and SUN2 co-localize on the nuclear membrane.Those SUN proteins enriched on one side of the nuclear membrane when the chromosomes contracted into a mess at zygotene.By detecting telomere aggregation,we found the formation of telomere bouquets was accompanied by chromosome contraction into a mess.This discovery suggests that polar migration of SUN1 and SUN2 may mediate telomere aggregation and chromosomal contraction in the wild type.In addition,we confirmed that the SUN1/SUN2,WIP/SINE1 and PSS1 can establish SUN-KASH-PSS1 tandem relationship on nuclear membrane.Both AtPSS1 in Arabidopsis and PSS1 in rice have similar meiosis function,we hypothesize that the formation of this structure may be closely related to the function of SUN1 and SUN2 in meiosis.SUN1 and SUN2 have high sequence similarity in rice.By observing the mutant isolated from the sun1 sun2 double mutant,sun1 exhibits similar meiosis with the wild type,but sun2 shows weaker meiotic defect than the double mutant.Pylogenetic tree analysis showed that the relationship between SUN1 in rice and AtSUN1,AtSUN2 in Arabidopsis was close,while SUN2 in rice was closely related to SUN2 in mice and ZmSUN2 in maize.In addition,Western blot analysis showed that SUN2 had significantly more protein than SUN1 in wild type.Therefore,we conclude that the evolutionary differences between SUN1 and SUN2 may cause differences in their expression levels,resulting in different phenotypes.This is different from that in Arabidopsis or mice.In summary,we found that SUN1 and SUN2 participate in chromosome contraction and telomere aggregation in rice meiosis,promoting homologous pairing,full-length chromosome synapsis and CO formation,efficiently.In addition,SUN2 may play a more important role in this process.