ospti1a

1. A B 4 6 18 26 EV EV OsPti1a -HAstrepII EV EV HAstrepII -OsPti1a WT ospti1a WT ospti1a ospti1a WT C OsPti1a -HAstrepII HAstrepII -OsPti1a EV EV 4 6 18 26 OsPti1a CBB WB: anti-HA antibody Supplemental Figure 1. Complementation test of HAstrepII-OsPti1a or OsPti1a-HAstrepII in the ospti1a mutant background. In (A) and (B), transgenic plants expressing HAstrepII-OsPti1a, OsPti1a-HAstrepII, or empty vector (EV) in the ospti1a mutant were grown under same conditions, respectively. The photograph shows the T0 transgenic plants in the ospti1a background and WT. The numbers indicate independent transgenic plant lines. (C) Immunoblot analysis of total proteins extracted from leaves of these transgenic plants. Lower panels indicate CBB staining of the membrane after immunoblotting.

2. A Transiently expression assay in N. benthamiana OsPti1aWT ΔN-OsPti1a S M S M MSCFACCG MSCFAACG MSCFACAG MSCFAAAG OsPti1aWT OsPti1aC6A OsPti1aC7A OsPti1aC6A/C7A OsPti1a CBB WB:anti-OsPti1a antibody B OsPti1aC6A/C7A OsPti1aC7A OsPti1aC6A OsPti1aWT S M S M S M S M OsPti1a C CBB WB: anti-OsPti1a antibody Supplemental Figure 2. Transient expression analysis of OsPti1a mutants in N. benthamiana. (A) The cellular localization of OsPti1a expressed after agroinfiltratingNicotianabenthamiana. N. benthamiana leaves were infiltrated with Rhizobium radiobacter EHA105 containing OsPti1a or ΔN-OsPti1a cDNA. Leaves were harvested 2 days after agroinfiltration. Total proteins were extracted and fractionated into soluble (S) and microsomal (M) fractions. OsPti1a proteins were detected by an anti-OsPti1a antibody. (B) Point mutations in the N-terminal sequence of OsPti1a. (C) Immunoblot analysis of cellular fractions of the mutated OsPti1a proteins expressed after agroinfiltratingN. benthamiana. Mutated variants of OsPti1a were transiently expressed in N. benthamiana. Three days after inoculation, proteins were extracted, fractionated, and subjected to immunoblot analysis using an anti-OsPti1a antibody (upper panel). After immunoblotting, the PVDF membrane was stained with CBB (lower panel).

3. kDa 440 158 66 kDa 440 158 66 19 16 17 18 20 21 22 23 24 25 26 27 28 29 Frac. No. 19 16 17 18 20 21 22 23 24 25 26 27 28 29 Frac. No. 0 min 10 min * OsPti1a * 60 min OsPti1a * 180 min OsPti1a WB: anti-OsPti1aantibody Supplemental Figure 3. The size of the OsPti1a complex does not change in response to chitin treatment. Gel filtration fractions of protein extracts from WT suspension cultured cells after chitin treatment were subjected to immunoblot analyses (left panel) using an anti-OsPti1a antibody. The arrows and asterisks indicate the positions of OsPti1a and cross-reacting bands, respectively. The fraction numbers and molecular masses (kDa) are indicated at the top of the figure. After immunoblotting, the PVDF membrane was stained with CBB (right panels).