WT ospti1a

EV EV 18 264 6

HAstrepII-OsPti1a

OsPti1a-HAstrepII

OsPti1a

CBB

WB: anti-HA antibody

BA

C

OsPti1a-HAstrepII

18 264 6

EV

ospti1aWT

EV HAstrepII-OsPti1a

EV

ospti1aWT

EV

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.

S M S M

OsPti1a

CBB

OsPti1aWT ΔN-OsPti1a

Transiently expression assay in N. benthamiana

WB:anti-OsPti1a antibody

A

C

Supplemental Figure 2. Transient expression analysis of OsPti1a mutants in N. benthamiana.(A) The cellular localization of OsPti1a expressed after agroinfiltrating Nicotiana benthamiana. 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 agroinfiltrating N. 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).

OsPti1aWT OsPti1aC6A OsPti1aC7A OsPti1aC6A/C7A

MSCFACCGMSCFAACGMSCFACAGMSCFAAAG

B

OsPti1

aW

T

OsPti1

aC6A

OsPti1

aC7A

OsPti1

aC6A

/C7A

CBB

OsPti1a

WB: anti-OsPti1a antibody

MS MS MS MS

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).

WB: anti-OsPti1a antibody

10 min

180 min

60 min

17 18 19 20 21 23 25 27 2922 24 26 2816

440 158 66kDa

Frac. No.

OsPti1a*

OsPti1a*

OsPti1a*

0 min

17 18 19 20 21 23 25 27 2922 24 26 2816

440 158 66kDa

Frac. No.