Arabidopsis mitochondrial single-stranded DNA-binding proteins SSB1 and SSB2 are essential regulators of mtDNA replication and homologous recombination

Faithful DNA replication is one of the most essential processes in almost all living organisms.However,the proteins responsible for organellar DNA replication are still largely unknown in plants.Here,we show that the two mitochondriontargeted single-stranded DNA-binding(SSB)proteins SSB1 and SSB2 directly interact with each other and act as key factors for mitochondrial DNA(mt DNA)maintenance,as their single or double loss-of-function mutants exhibit severe germination delay and growth retardation.The mt DNA levels in mutants lacking SSB1 and/or SSB2 function were two-to four-fold higher than in the wild-type(WT),revealing a negative role for SSB1/2 in regulating mt DNA replication.Genetic analysis indicated that SSB1 functions upstream of mitochondrial DNA POLYMERASE IA(POLIA)or POLIB in mt DNA replication,as mutation in either gene restored the high mt DNA copy number of the ssb1-1 mutant back to WT levels.In addition,SSB1 and SSB2 also participate in mitochondrial genome maintenance by influencing mt DNA homologous recombination(HR).Additional genetic analysis suggested that SSB1 functions upstream of ORGANELLAR SINGLE-STRANDED DNABINDING PROTEIN1(OSB1)during mt DNA replication,while SSB1 may act downstream of OSB1and MUTS HOMOLOG1 for mt DNA HR.Overall,our results yield new insights into the roles of the plant mitochondrion-targeted SSB proteins and OSB1 in maintaining mt DNA stability via affecting DNA replication and DNA HR.