Bimolecular Fluorescence Complementation of ONAC Interactions

Bimolecular fluorescence complementation assay for determining the ONAC096-ONAC096, ONAC066-ONAC066, and ONAC096-ONAC066 interactions was carried out as previously described (Liu et al., 2019 (link)). Coding sequences of ONAC066 and ONAC096 were amplified with gene-specific primers (Supplementary Table 1) and inserted into p2YC, yielding p2YC-ONAC066 and p2YC-ONAC096, or cloned into p2YN, generating p2YN-ONAC066 and p2YN-ONAC096. Agrobacteria harboring different indicated pairs of fused or empty plasmids were infiltrated into leaves of N. benthamiana plants expressing a red nuclear marker protein RFP-H2B (Chakrabarty et al., 2007 (link)) as previously described (Liu et al., 2019 (link)). At 48 h after agroinfiltration, YFP and RFP signals were detected and photographed under a Zeiss LSM780 confocal laser scanning microscope (Zeiss, Oberkochen, Germany).