Additional file 1 of OsWRKY62 and OsWRKY76 Interact with Importin α1s for Negative Regulation of Defensive Responses in Rice Nucleus
Creators: name: Xu, Xiaohui
name: Wang, Han
name: Liu, Jiqin
name: Han, Shuying
name: Lin, Miaomiao
name: Guo, Zejian
name: Chen, Xujun
Publisher: name: figshare
Year: 2022
Subjects: subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Environmental Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Plant Biology
subject: Virology

description: Additional file 1.Fig. S1. Analysis of OsWRKY62 and OsWRKY76 interacting with OsIMα1 in yeast. (A) Analysis of OsWRKY76 (W76.1) and its deletion mutants, and OsIMα∆IBB1 auto-activation. (B) Schematic diagrams of OsWRKY62.1 (W62.1) and its deletion mutants. (C) Analysis of OsWRKY62 and its mutants interacting with OsIMα∆IBB1. Yeast cells with serial dilutions were incubated in synthetic dropout medium lacking Leu and Trp (left) or Leu, Trp, His, and Ade (right) and photographed 3 d after plating. Yeast cells harboring AD-T with BD-53 or BD-Lam vectors were used as the positive or negative control, respectively. Fig. S2. Phylogenetic analysis of importin αs. Importin αs from Oryza sativa (Os), Lycopersicon esculentum (Le), and Arabidopsis thaliana (At) were compared. Multiple sequence alignments of amino acid sequences were generated using ClustalW in MEGA7.0. The sequence alignments obtained were used as input for the neighbor-joining method using MEGA7.0 to construct the phylogenetic tree. For phylogenetic tree construction, a bootstrap method with 1,000 replications was used for test of phylogeny. Scale bar indicates 0.2 amino acid substitution per site. Fig. S3. OsIMΔIBBα1a interacting with OsIMβ1 and increased OsWRKY62.1-GFP nuclear localization through overexpressing OsIMα1a. (A) BiFC visualizations of IMα1a and IMα∆IBB1a interacting with IMβ1. IMβ1 was fused in frame with YFP N-terminal region (YFPN) and IMα1a and IMα∆IBB1a were fused with YFP C-terminal region (YFPC). The plasmids indicated were introduced into N. benthamiana leaves through agroinfiltration method. Red fluorescence (dsREDNLS) shows nuclear localization. From left panels to right: YFP images (YFP), dsRED images (RED), and combined YFP and RED in the bright field (Merged). (B) Sheaths from three-week-old 35S::OsWRKY62.1-GFP (35S:: W62.1-GFP) and 35S::OsWRKY62.1-GFP/CDU::IMα1a (genetic cross progeny) plants were used. DAPI for nuclear staining. From top panels to bottom: DAPI, GFP, DIC, and the bright field image combined the fluorescent images (Merged). Fig. S4. Simulated structures of AvrPib and the WRKY domain of OsWRKY62.1. (A) The structure of AvrPib was from Zhang et al. (2018). The positive-charged amino acids of AvrPib are shown in red in the structure. (B) The structure of W62WD is simulated based on AtWRKY1WD (PDB code: 2AYD) using homology-modeling by SWISS-MODEL server. Fig. S5. Information of OsIMα1 knockout mutants. (A) Knockout mutant of OsIMα1a (imα1aKO). (B) Knockout mutant of OsIMα1b (imα1bKO). The sequences of the target sites are shown in green and the inserted nucleotides are in blue. Fig. S6. OsIMα1 positively regulated resistance against rice blast fungus. (A) Three-week-old transgenic and wild-type (ZH17) plants were inoculated with M. oryzae SZ (5 × 105 spores/mL) by foliar spraying. Photographs were taken six days after the inoculation. Bar = 2 cm. (B) P-values were calculated by one-tailed Student's t-test. Prefix CDU for OsIMα1a and OsIMα1b overexpressing plants and suffix KO for the knockout lines. Fig. S7. Induction of OsIMα1 expression. (A) Induction of OsIMα1a and OsIMα1b expression by flg22 (1 µM) or chitin (200 µg/mL) treatment. (B) Induction of OsIMα1a and OsIMα1b expression by M. oryzae SZ. Transcriptional levels of OsWRKY62 (C) and OsWRKY76 (D) in their overexpression plants. (E) Expression of OsIMα1a and OsIMα1b in OsWRKY62 and OsWRKY76 overexpressing and knockout plants. Fig. S8. Analysis of OsWRKY62.1 localization. Sheaths of 35S::W62.1-dsREDNLS and 35S::W62.1-dsREDNES rice plants were used for fluorescence observation. DAPI for nucleus staining. From top panels to bottom: DAPI, RED, DIC, and the bright field image combined the fluorescent images (Merged). Bar = 20 µm. Table S1 Primers used in this study.