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To investigate the molecular mechanism of the  ADVENTITIOUS ROOTING RELATED OXYGENASE 1  ( ARRO-1 ) gene in adventitious rooting of tree peony, the  PsARRO-1  gene was isolated from  Paeonia ostii . Subsequently, a yeast two-hybrid (Y2H) cDNA library using the split-ubiquitin yeast membrane system was constructed, the insert fragments lengths of pPR3-N membrane Y2H library were mainly concentrated between 500 and 3,000 bp, and the library capacity reached 1.3 × 10 7  CFUs, with an efficiency of recombinant of 100%, which met the requirements for subsequent screening of PsARRO-1-interacting proteins. Finally, 15 candidate PsARRO-1-interacting proteins were screened out by pBT3SUC-ARRO, among them potassium transporter 2, cytochrome b5 isoform E, and high mobility group B protein 3 (HMGB3) might be involved in the formation of roots. Both direct Y2H and bimolecular fluorescence complementation assays confirmed that PsARRO-1 interacted with PsHMGB3, which shared high identity with EgHMGB3 and MiHMGB3. The expression of  PsHMGB3  was significantly up-regulated in the root outgrowth stage, and the overexpression of  PsHMGB3  in  Arabidopsis  resulted in lengthened primary roots, more lateral roots. In short, the Y2H library provides a large number of useful resources for functional genomics research on roots of tree peony.

Construction of a membrane yeast two-hybrid library and its application in identification of PsARRO-1-interacting proteins in tree peony