Pdest32

To construct the 35S::LcERF056-GFP expression plasmid, the CDS of the LcERF056 was inserted into the binary vector p163-GFP. Both 35S::GFP and the recombinant vectors were transiently expressed in Arabidopsis protoplasts, as previously described [38 (link)]. Then the protoplasts were incubated in dark for at least 16 h. The GFP fluorescence was observed using a confocal laser scanning microscope (Zeiss LSM700).
Saccharomyces cerevisiae yeast strain AH109 and vector pDEST32 (Invitrogen, Carlsbad, CA) were used for studying the transactivation of LcERF056. The CDS of LcERF056 was cloned into pDEST32 vector, and the constructed or empty plasmids were transformed into the yeast strain AH109 as per the manufacturer’s instructions (Clontech, Palo Alto, CA). The transformants were cultured on SD/−Leu and SD/−Leu/−His/−Ade + 3-Amino-1, 2, 4-triazole (3-AT) solid medium. β-Galactosidase filter-lifted test was measured according to the Yeast Protocols Handbook (Clontech).

The entry clone containing FYVE3 cDNA was recombined with the destination vector pDEST22 (Thermo Fisher Scientific) via the LR Clonase II reaction. The resulting pDEST22-FYVE3 and pDEST32 expression vectors for bait proteins fused to SAR1B, SAR1C, ATG8A, ATG8E, ATG8F, ATG8I, ATG18A and FYVE2 (Kim et al., 2022 (link)) were used for transformation of the AH109 strain and Y187 strain through the Yeastmaker Yeast Transformation System 2 (Clontech, Mountain View, CA, USA). To determine protein interaction, yeast colonies isolated from Trp- and Leu- deficient medium were transferred to Trp-, Leu-, and His- deficient medium containing 0 and 1 mM 3-amino-1,2,4-triazole.