Supplemental Information

Cell Host & Microbe, Volume 13

Supplemental Information

An OsCEBiP/OsCERK1-OsRacGEF1-OsRac1

Module Is an Essential Early Component

of Chitin-Induced Rice Immunity

Akira Akamatsu, Hann Lin Wong, Masayuki Fujiwara, Jun Okuda, Keita Nishide, Kazumi Uno, Keiko Imai, Kenji Umemura, Tsutomu Kawasaki, Yoji Kawano, and Ko Shimamoto

1

Figure S1. Sphingolipid Induces OsRac1 Activation in Rice Protoplasts, Related to Figure 1

(A) A schematic representation of Raichu-OsRac1 bound to GTP or GDP. (B) Diagrams of Raichu-OsRac1 and the positions of the DN and CA mutations. (C) OsRac1 activation during MTI. Time lapse imaging of Raichu-OsRac1-WT expressed in rice protoplasts. After sphingolipid elicitor (SE) treatment, emission ratios were measured at 3-min intervals. Eight colors from red to blue represent this ratio. (D) The emission ratio (Venus/CFP) of Raichu-OsRac1-WT. Green, 3.0 μg/ml SE treatment; Red, W5 buffer treatment. (n=5) Bars are SE. (E) The average FRET ratio of rice protoplasts transformed with Raichu-OsRac1-WT, -CA, or -DN. The ratios were measured 30-60 min after SE treatment. Values marked by the same letters are not significantly different from each other. (P < 0.01; n > 20). Bars are SE.

2

Figure S2. Identification of OsRacGEF1 as an OsRac1-Interacting Protein in Yeast, Related to Figure 2

(A) A phylogenetic tree was constructed with GENETYX software. There are 11 OsRacGEF genes in rice and 14 AtRopGEF genes in Arabidopsis. (B) A schematic representation of OsRacGEF1. C1-C3 represent the subdomains of the conserved PRONE domain in the RacGEF family. (C) Growth of yeast colonies on plates lacking histidine (His(-)) and containing 100 mM 3-aminotriazole (3-AT) indicates a positive interaction between OsRac1 D125N and OsRacGEF1.

(D) Effects of C- and N-terminal tag fusions on OsRacGEF1 function. Induction of the defense-related gene PAL1 was measured using qPCR. Values marked by the same letters are not significantly different from each other. (P < 0.01; n > 3). Bars are SE. (E) Localization of OsCERK1 in rice protoplasts. Rice protoplasts were cotransformed with the fluorescent constructs OsCERK1-GFP (green) and CFP-HDEL (magenta). Expression of these genes was driven by the OsCERK1 promoter of OsCERK1 (left) and the CaMV 35S (35S) promoter (right). Bars are 5 μm. (F) Intramolecular interaction of OsRacGEF1. Growth of yeast colonies on plates lacking histidine

3

(His(-)) indicates a positive interaction between the C-terminal region and the PRONE domain. (G) Interaction of OsRac1-WT with the OsRacGEF family in yeast two-hybrid assays using split-ubiquitin hybrid system. Growth of yeast colonies on minimal medium containing 5-fluoroorotic acid (FOA; 0.1%).

Figure S3. OsRacGEF1 Expression in Transgenic Plants, Related to Figure 3

(A) Constructs for OsRacGEF1 RNAi and OsRacGEF1 S549A overexpression (OX) in transgenic cells and plants. C-ter., C-terminal region of OsRacGEF1 was used as trigger region for RNAi silencing. The construct for OsRacGEF1 OX carries the coding sequence (CDS) of the OsRacGEF1 S549A mutant. (B) RT-PCR analysis of OsRacGEF genes mRNA expression. The wild type (WT) is a nontransgenic control. Hpt, mRNA of the hygromycin resistance gene; mRNA of rice actin used as control. (C) qPCR of OsRacGEF1 in OsRacGEF1 S549A OX plants; ＃8, ＃19, ＃21 are independent OsRacGEF1 S549A OX lines. Relative expression (WT=1). (**P < 0.01; n=3). Bars are SE.

4

Figure S4. Interactions between OsRacGEF1 and PRRs, Related to Figure 4

(A and B）Venus-OsRacGEF1 and OsCERK1-FLAG or XA21-FLAG were transiently coexpressed in rice protoplasts. Co-IP was carried out with anti-GFP (IP: α-GFP), and the proteins were analyzed by western blotting with anti-GFP antibody (IB: α-FLAG). (B) Proteins were extracted after treatment with 10 μg/ml of chitin. (C) Yeast two-hybrid assays of OsRacGEF1 deletion mutants and the cytoplasmic domain (CD) of OsFLS2. Yeast growth on selective plates without histidine (His (-)) indicates a positive interaction. (D) myc-OsRac1, Venus-OsRacGEF1 and OsCERK1-FLAG were transiently coexpressed in rice protoplasts. CoIP was carried out with anti-myc (IP: α-myc), and the proteins were analyzed by western blotting with anti-myc antibody (IB: α-GFP and α-FLAG).

5