RPW8.2-YFP Is Targeted to the Periphery of Haustoria of Other Pathogens.
(A) and (B) RPW8.2-YFP is localized at the periphery of haustoria (H) of Bgh in epidermal cells of eds1-2 plants expressing NP:RPW8.2-YFP. The image in (A) shows a small haustorium targeted by RPW8.2-YFP. The image in (B) shows a bigger haustorium half-encased by RPW8.2-YFP–labeled EHM. The pictures were taken at 28 HAI. Bars =10 μm.
(C) and (D) RPW8.2-YFP is localized at the periphery of haustoria (H) of H. arabidopsidis Noco2 in epidermal cells of Col-0 plants expressing NP:RPW8.2-YFP. The inset in (C) is a single optical section of the same image shown. The oomycete haustoria (H) are recognizable under differential interference contrast microscopy (D). The images were collected at 4 DAI. Bars = 10 μm.

RPW8.2-YFP Localization to the EHM Is Revealed by Immunogold Labeling Using Anti-GFP (Which Also Detects YFP) and TEM.
(A) A lower-magnification image of a haustorium (H) in a cell of a transgenic Col-0 line expressing RPW8.2-YFP. Note the encasement of the haustorial complex (EHC). Bar = 1 μm.
(B) A close-up of the squared section in (A) showing that the immunogold particles (10 nm) were specifically localized at the EHM (arrowheads) near a haustorial lobe (L). Note a few particles (gray arrows) likely representing RPW8.2-YFP vesicles in the EHC region. Bar = 0.2 μm.
(C) and (D) Sections of a haustorium in a wild-type Col-0 cell at the same magnification as in (A) and (B), respectively.

RPW8 Enhances the Formation of the EHC and Accumulation of H₂O₂ in the HC.
Plants of indicated genotypes, including Col-0 lines transgenic for NP:RPW8.2-YFP (R2Y4) or RPW8.1 and RPW8.2 (S5), were inoculated with G. cichoracearum UCSC1 and subject to trypan blue (TB)/aniline blue (AB)/3,3′-diaminobenzidine (DAB) staining to visualize dying/dead host cells and fungal structures (TB), callose deposition (AB), and H₂O₂ accumulation (DAB), respectively.
(A) Representative leaf sections showing three types of invaded cells: cells containing a haustorium with (black arrows) or without the EHC (green arrows) and cells undergoing HR (red arrow) at 5 DAI. Mycelia were removed to aid visualization of haustoria. Bars = 50 μm.
(B) Single representative cells containing a shrunken haustorium with the EHC (top panel) or a healthy haustorium without EHC (bottom panel). Note the callose (stained blue by AB) deposition in the EHC and at the penetration site (i.e., the papilla; arrow). Bars = 20 μm.
(C) and (D) Types and frequencies of host-pathogen interactions at 42 and 120 HAI. White, light-gray, and dark-gray bars indicate frequencies of living cells containing a haustorium without an EHC, living cells containing a haustorium with an EHC, and cells undergoing HR, respectively. Data are means ± se, calculated from three duplicated experiments. Asterisks indicate values significantly different from that of Col-0 (C) or S5 (D) (P < 0.01; n = 3, Student's t test).
(E) Perturbation of the integrity of the EHC by pmr4-1 results in more frequent activation of cell death in the RPW8 background at 48 HAI (top panel; also see [D]), which is preceded by H₂O₂ release from the HC into the cytoplasm (reflected by reddish brown precipitates; bottom panel) at 36 HAI. Bars = 10 μm.

RPW8.2 Is Targeted to the Periphery of the Fungal Haustorium.
Except as noted, Col-0 lines expressing RPW8.2-YFP from the native RPW8.2 promoter (NP) were inoculated with G. cichoracearum UCSC1, directly examined under a Zeiss epifluorescence microscope ([A] and [B]), or stained with PI to visualize the fungal structures and imaged by CLSM at 20 to 42 HAI. Bars = 50 μm in (A) and (B) and 5 μm in (C) to (E).
(A) Lack of RPW8.2-YFP expression in inoculated leaf epidermal cells from 0 to 15 HAI.
(B) Detection of RPW8.2-YFP expression as sac-like fluorescent objects in inoculated leaf epidermal cells at 20 HAI.
(C) Precise localization of RPW8.2-YFP to the surface of a haustorium (H). Note that the YFP signal stops at the haustorial neck. Arrowheads indicate RPW8.2-YFP–containing vesicles.
(D) A haustorium in a host cell of Col-0 expressing 35S:YFP. Note that the localization pattern of YFP is distinct from (C).
(E) Haustoria encased by RPW8.2-YFP became shrunken (white arrow) or aborted (lack of PI staining; yellow arrows).

RPW8.2 Is Targeted to the EHM.
Leaves of Col-0 or Col-0 transgenic for NP:RPW8.2-YFP were inoculated with G. cichoracearum UCSC1 and used for haustorium isolation at 2 DAI. Isolated haustoria were stained with PI and imaged by CLSM. Bars = 5 μm.
(A) A single optical section of an isolated HC from Col-0. Note the weakly PI-stained EHM (arrowhead) and numerous lobes emanating from the main body of the haustorium.
(B) A single optical section of an isolated HC showing precise colocalization of RPW8.2-YFP with the PI-stained EHM.
(C) An isolated HC showing localization of RPW8.2-YFP to the outer surface of the HC. Note the lightly PI-positive encasement of the HC (EHC).
(D) and (E) Fluorescence recovery after photobleaching on RPW8.2-YFP–labeled EHM in planta (D) and the GFP:SIMIP-labeled PM (E). Arrowheads point to the site of photobleaching.

Targeting of RPW8.2 to the EHM Requires Function of the Actin Cytoskeleton.
(A) RPW8.2-YFP's localization to the EHM is impeded by cytochalasin E but not obviously affected by oryzalin. Four independent Col-0 lines transgenic for NP:RPW8.2-YFP were used for treatments and examined at ∼34 HAI. Data are mean ± sd, derived from one (of three) representative experiment in which >100 haustorial invasion sites per line were evaluated for each concentration. Asterisks indicate significant difference compared with buffer control (P < 0.05, n = 4, Student's t test).
(B) Genetic perturbation of the actin cytoskeleton by overexpression of ADF6 results in RPW8.2-YFP accumulation in punctate spots (arrows) associated with dying mesophyll and epidermal cells that are indicated by autofluorescent ring structures. A representative epifluorescent image of a leaf section from an uninfected R2Y4 plant transgenic for 35S:ADF6 is shown. Bar = 50 μm.
(C) A representative 3,3′-diaminobenzidine-trypan blue-stained leaf section showing production/accumulation of H₂O₂ (reddish brown) and cell death (blue) in uninfected R2Y4 plants transgenic for 35S:ADF6. Bar = 50 μm.
(D) RPW8.2-YFP's localization to the EHM is compromised by ADF6 overexpression. R2Y4 or R2Y4 overexpressing ADF5 (ADF5-OE; representing bulked samples from three independent lines) or ADF6 (ADF6-OE1 and ADF6-OE2, representing bulked samples from three independent lines with reduced plant size and three with close to normal size, respectively) were inoculated with Gc UCSC1 and examined at 2 DAI. Data are mean ± se, derived from one (of two) representative experiment in which >100 haustorial invasion sites per line were evaluated. Asterisks indicate significant difference compared with R2Y4 (P < 0.05, n = 3, Student's t test).

Both Defense Activation and EHM Localization Are Required for RPW8.2 to Confer Resistance.
The RPW8.2 alleles from the indicated accessions were translationally fused with YFP at the C terminus, and all the constructs were expressed from the promoter of RPW8.2^Ms-0 in Col-0. T2 plants are shown.
(A) Representative images showing typical localization of RPW8.2 (fused to YFP) encoded by the indicated alleles (top) at 2 DAI. The disease reaction (DR) scores (0, resistant; 1 to 2, intermediate [I]; 2 to 3 or 3, susceptible [S]; Xiao et al., 2005) are shown below the images. H, haustorium. Arrowheads indicate RPW8.2^Bg-1-YFP in punctate spots. Bars = 10 μm.
(B) Representative 5-week-old T2 plants before inoculation. Note the spontaneous cell death and reduced plant stature in plants of one representative RPW8.2^Bg-1-YFP line.
(C) Representative leaves of the transgenic lines expressing the indicated RPW8.2 allele in comparison with a leaf of Col-0 (c) inoculated with Gc UCSC1 at 8 DAI. Note the induced massive cell death and fungal growth in the same leaf expressing RPW8.2^Bg-1-YFP.