Characterisation of novel synaptonemal complex initiation protein ASYNAPTIC5 in Arabidopsis thaliana

The synaptonemal complex (SC) is a structurally conserved meiosis-specific tripartite protein ultrastructure that regulates crossover (CO) frequency and positioning across homologous chromosomes in Arabidopsis thaliana. CO assurance, the mechanism by which each homologous chromosome pair receives at least one CO, is associated with positive CO interference, whereby two COs are spaced further apart than by chance, as well as the prevention of clustered COs generated by negative CO interference. CO patterning is regulated by the chromosome axis and SC, composed of lateral element proteins ASY1, ASY3 and ASY4, AAA+ ATPase PCH2, central element proteins SCEP1 and SCEP2, and transverse filament protein ZYP1. Here, ASYNAPTIC5 (ASY5) has been identified as a novel SC central region protein necessary for SC initiation and CO interference, generating ~194.6 variably-sized axis-associated foci at leptotene which migrate to the SC throughout meiotic prophase I. In asy5 heterozygous mutants, E3 ligase HEI10-marked CO sites are more frequent and cluster relative to the wild-type despite retained ZYP1-defined synapsis. In asy5 homozygous mutants, chiasmata number is reduced, and HEI10 localisation behaviour is changed relative to the wild-type, while ZYP1 loads only as ~28.2/33.0 axis-associated foci. Additionally, yeast-2-hybrid reveals strong protein-protein interactions between ASY5 and itself, SCEP1, and ZYP1. Analysis of asy5 double mutant lines reveals additive reductions in chiasmata in asy1/asy5 and asy5/pch2, alongside changes in CO positioning by an additive increase in univalent chromosome frequency in asy3/asy5, suggesting that ASY5 functions concomitantly with the chromosome axis to regulate CO patterning. Fluorescent Tagged Lines also reveal that both heterozygous and homozygous asy5 mutants exhibit impaired positive CO interference and clustering of COs by negative CO interference, like previous observations in asy4 and serine/threonine kinase atm mutants. Furthermore, overexpression of HEI10 in asy5 heterozygous mutants additively increases HEI10-marked CO frequency, while failing to restore CO assurance in asy5 homozygous mutants.