A comprehensive mutational analysis of the Arabidopsis resistance protein RPW8.2 reveals key amino acids for defense activation and protein targeting

Abstract

The Arabidopsis thaliana resistance to powdery mildew8.2 (RPW8.2) protein is specifically targeted to the extrahaustorial membrane (EHM) encasing the haustorium, or fungal feeding structure, where RPW8.2 activates broad-spectrum resistance against powdery mildew pathogens. How RPW8.2 activates defenses at a precise subcellular locale is not known. Here, we report a comprehensive mutational analysis in which more than 100 RPW8.2 mutants were functionally evaluated for their defense and trafficking properties. We show that three amino acid residues (i.e., threonine-64, valine-68, and aspartic acid-116) are critical for RPW8.2-mediated cell death and resistance to powdery mildew (Golovinomyces cichoracearum UCSC1). Also, we reveal that two arginine (R)- or lysine (K)-enriched short motifs (i.e., R/K-R/K-x-R/K) make up the likely core EHM-targeting signals, which, together with the N-terminal transmembrane domain, define a minimal sequence of 60 amino acids that is necessary and sufficient for EHM localization. In addition, some RPW8.2 mutants localize to the nucleus and/or to a potentially novel membrane that wraps around plastids or plastid-derived stromules. Results from this study not only reveal critical amino acid elements in RPW8.2 that enable haustorium-targeted trafficking and defense, but also provide evidence for the existence of a specific, EHM-oriented membrane trafficking pathway in leaf epidermal cells invaded by powdery mildew.

Figure 1.

Schematic Illustration of RPW8.2 Mutants Constructed and Tested. RPW8.2 mutant genes were in-frame fused with YFP and tested in Arabidopsis by stable expression. For site-specific mutagenesis, only the functionally important mutants are highlighted above the schematic RPW8.2 sequence; the remaining mutants are indicated by gray bars underneath. For NAAIRS replacement, five of the 29 constructs show significant reduction in EHM targeting (gray bar). For deletion analysis, constructs in black show EHM localization; constructs in gray show no or little EHM localization. Internal deletions are shown by dashed line flanked by two dark circles. The two shaded regions delimited by the mutational analyses are critical for EHM localization of RPW8.2. Stars indicate the functionally informative breaking points. The black arrow indicates the minimal sequence construct in the bottom containing 60 amino acids. TMS-R82 andTMA-R82, RPW8.2-YFP constructs in which the TMD is replaced by that of SYP122 or ACBP2. WT, wild type.

Figure 2.

Cell Death and Disease Reaction Phenotypes of Site-Directed RPW8.2 Mutants. (A) Representative leaves of Col-gl lines transgenic for 35S:YFP or P_RPW8.2:RPW8.2-YFP (line R2Y4) as control. Plants were inoculated with Gc UCSC1 and pictures taken at 7 d postinoculation (dpi). (B) to (O) Representative mildew-infected leaves at 7 or 10 d postinoculation or uninfected plants expressing the indicated YFP-tagged RPW8.2 mutants from the RPW8.2 promoter in comparison with Col-gl or Col-gl transgenic for 35S:YFP. Arrowheads indicate spontaneous HR-like cell death lesions; arrows indicate big necrotic lesions resulting from merge of small lesions.

Figure 3.

The N-Terminal TMD Is Required for EHM-Specific Localization of RPW8.2. Col-gl transgenic lines expressing each of the four N-terminal deletion RPW8.2 mutants tagged with YFP were inoculated with Gc UCSC1. Infected leaves were collected at 2 d postinoculation, stained with PI, and subjected to confocal microscopy. The YFP signal is pseudo-colored in green and PI-stained structures in red. Images are representative Z-stack projections of 15 to 65 optical sections. h, haustorium; n, nucleus; p, penetration site. Bars = 10 μm. (A) Mutant R82^∆5-12 in some cells showed reduced EHM localization as reflected by small puncta and aggregates at or peripheral to the EHM, particularly at the haustorial neck region (arrow). (B) R82^∆5-12 as protein aggregate (arrow) was seen around the penetration site (top panel) and/or surrounding the haustorial neck (low panel). (C) R82^∆5-12 was found as small puncta in a fireworks-like domain centering in the penetration and as small puncta or diffuse signal at the EHM in the same cell (inset). Note that when viewed horizontally, the R82^∆5-12–labeled fireworks-like domain seemed to be a thin layer aligning to the cell wall (bottom panel). The haustorial neck region (arrow) was also labeled by R82^∆5-12. (D) Mutant R82^∆5-14 was also found in the fireworks-like domain in some cells, while it was also found at the EHM in other cells (inset). (E) Mutant R82^∆5-15 was found in puncta not apparently associated with the EHM, indicating it is incapable of EHM targeting. (F) Mutant R82^∆5-18 was exclusively found as varied sized puncta in the nucleus.

Figure 4.

The C-Terminal Portion Is Required for Suppression of Nuclear Localization of RPW8.2. YFP signal from tagged RPW8.2 mutant proteins is pseudo-colored green, PI-stained structures are red, and DAPI-stained nuclei are blue. Images are representative Z-stack projections of 15 to 45 optical sections. h, haustorium; n, nucleus. Bars = 10 μm. (A) Nuclear localization of R82^∆138-174. (B) EHM localization of R82^∆138-174. (C) Epidermal cells expressing YFP alone as control to show that both nuclei and the haustorium were stained red by PI. (D) EHM and nuclear localization of R82^Δ120-174. (E) EHM and nuclear localization of R82^∆113-174. (F) Nuclear localization and weak EHM targeting of R82^Δ112-174. (G) Exclusive nuclear localization of R82^Δ111-174. (H) Nuclear localization of R82^Δ110-174. Note both the haustorium and nucleus were stained blue by DAPI. (I) Nuclear localization of R82^Δ88-174.

Figure 5.

RPW8.2 Variants Are Targeted to the PSM. YFP signal from tagged RPW8.2 mutant proteins is pseudo-colored green, PI-stained structures are red, and autofluorescent chloroplasts or plastids are blue. Images are representative Z-stack projections of 15 to 45 optical sections. h, haustorium. Bars = 10 μm. (A) R82^∆120-174 was observed in the PSM tightly associated with and connecting individual plastids (asterisks). Note that there are bulges or lumps close to the encased plastids or in the middle of the membrane strand. (B) and (C) R82^∆43-97 was found in the PSM as puncta at or peripheral to the EHM. (C) R82^∆43-97 was targeted at the EHM as shown by diffuse YFP signal in the EHM. (D) to (G) Localization of mutant proteins R82^∆43-97 (D), R82^∆43-115 (E), R82^∆43-135 (F), and R82^∆43-141 (G) in epidermal cells containing a haustorium. Note the big dots/patches (arrows) in some cells. (H) R82^∆65-93 was found in ring structures (asterisks) associated with chloroplasts in mesophyll cells. (I) R82^{∆65-93+∆138-174} was occasionally found both in a PSM (asterisks) and the EHM and the tubules that connect these two compartments. (J) R82^{∆65-93+∆138-174} was observed in the PSM similarly as R82^∆120-174. The bulged portion is indicated by arrows. (K) and (L) Colocalization analysis of YFP-tagged R82^{∆65-93+∆138-174} expressed from the RPW8.2 promoter and a stromule marker (a plastid-targeting [PT] signal in fusion with mCherry; Nelson et al., 2007) expressed from the 35S promoter in stable Arabidopsis Col-0 plants. While some R82^{∆65-93+∆138-174} signal seemed to colocalize with PT-mCherry in thin stromule strands (arrows in [K]), most of it was found in the periphery of PT-mCherry labeled plastids or stromule protrusions (arrows in [L]).

Figure 6.

NAAIRS Scanning Identified Short Amino Acid Motifs in RPW8.2 Important for EHM Targeting. YFP signal from tagged RPW8.2 mutant proteins is pseudo-colored green, PI-stained structures are red, and autofluorescent chloroplasts or plastids are blue. Images are representative Z-stack projections of 15 to 45 optical sections. h, haustorium; p, penetration site. Bars = 10 μm. (A) The RPW8.2 protein sequence and positions of the 29 NAAIRS replacement and potential functional motifs and residues. Short lines underneath the amino acid sequences indicate positions of NAAIRS replacements; green lines indicated mutants with more or less normal EHM localization; red lines indicate mutants largely defective in EHM targeting; black lines indicates mutants without detectable protein accumulation. Two R/K-R/K-x-R/K motifs critical for EHM targeting are shaded pink; two putative NLSs are shaded green; two putative NESs are shaded purple. (B) The NAAIRS replacement mutant R82^N20-25 was only rarely observed in the EHM. In most cases, it was found as varied sized puncta peripheral to the haustorium. (C) R82^N20-25 was occasionally observed in ring structures surrounding chloroplasts in the mesophyll cells. (D) and (E) R82^N26-31 is largely defective in EHM targeting, forming protein aggregates (arrows) unrelated to the haustorium (D) and/or small puncta at or peripheral to the EHM (E). (F) and (G) R82^N95-100 is largely defective in EHM targeting, forming big protein aggregates (arrows) and small puncta at or peripheral to the EHM. (H) R82^{N20-25+N95-100} is defective in EHM targeting, forming small puncta in the cytoplasm and protein aggregates (arrows) at the penetration site where the papilla was stained red by PI. (I) An optical section from (H) with bright field showing the presence of the haustorium. Arrows indicate protein aggregates. (J) R82^{N20-25+N95-100} was occasionally observed in ring structures surrounding chloroplasts in the mesophyll cells. (K) R82^{N26-31+N95-100} is defective in EHM targeting, forming small puncta in the cytoplasm and around the haustorium, and big aggregates (arrow) at the penetration site. (L) An optical section from (K) with bright field showing the presence of the haustorium.

Figure 7.

Site-Directed Mutagenesis at the Two R/K-R/K-x-R/K–Containing Motifs Provided Further Evidence for Their Role in EHM Targeting. YFP signal from tagged RPW8.2 mutant proteins is pseudo-colored green, and PI-stained structures are red. Images are representative Z-stack projections of 15 to 45 optical sections. h, haustorium. Bars = 10 μm. (A) R82^R28A is largely defective in EHM targeting, forming small puncta and big aggregates (arrows) at or peripheral to the EHM. (B) R82^F98A is largely defective in EHM targeting, forming small puncta and patches at or peripheral to the EHM. (C) R82^R24A+R95A is largely defective in EHM targeting, forming small puncta and big aggregates (arrows) at or peripheral to the EHM. (D) R82^R24A+K97A is also largely defective in EHM targeting. YFP signal was mainly detected in the portion of the EHM around the haustorial neck (arrow). Weak YFP signal was also observed in a ring structure surrounding the penetration site and possibly in nearby PM (asterisks). (E) R82^K26A+R95A is defective in EHM targeting as shown by formation of varied sized puncta in the cytoplasm unrelated to the EHM.

Figure 8.

Minimal Sequence Requirement of RPW8.2 for EHM Targeting and a Model for Intracellular Trafficking Pathways in Epidermal Cells Invaded by Haustoria of Powdery Mildew. YFP signal from the tagged RPW8.2 mutant protein is pseudo-colored green, PI-stained structures are red, and autofluorescent chloroplasts are blue. Images are representative Z-stack projections of 15 to 45 optical sections. h, haustorium; n, nucleus. Bars = 10 μm. (A) to (C) R82^{∆43-93+∆113-174} is capable of reaching the EHM as mostly diffuse signal (A) or both diffuse and small punctate signal ([B] and [C]). Insets are single optical sections. Note weak YFP signal was also detected in ring structures surrounding the nucleus and plastids (arrows). (D) R82^{∆43-93+∆113-174} was detected in the PSM 20 min after treatment with 10 µM abscisic acid. Note the YFP-positive stromule-like membrane filaments (arrows) extended from plastids into the nucleus. (E) R82^{∆43-93+∆113-174} was detected in punctate rings surrounding plastids (asterisks) labeled by a stromule marker PT-mCherry (Nelson et al., 2007) (pseudo-colored red). (F) A cartoon illustrating vesicle trafficking in a leaf epidermal cell invaded by a haustorium of powdery mildew. During the biogenesis of the EHM, major trafficking at the trans-Golgi network is oriented toward the EHM (1), while vesicle trafficking to PM (2) and other organelles such as the nucleus remain active. Vesicles loaded with protein cargos containing EHM targeting motifs such as RPW8.2 are specifically targeted to the EHM, while vesicles containing proteins without a strong sorting signal may passively enter the major EHM-oriented trafficking pathway (1). A common feature(s) may be shared between the EHM and the PSM, which may provide a trafficking cue for RPW8.2’s localization. The PSM may further serve as a trafficking highway for RPW8.2 to reach the EHM (3). In addition, RPW8.2 contains NLSs and export signals; consequently, some RPW8.2 mutant proteins are nuclear localized. This raises a possibility that a small portion of RPW8.2 may be nuclear localized for defense activation (4) and suggests that balanced trafficking forces may be required to ensure EHM targeting of RPW8.2. It has been proposed that some PM-localized proteins may be translocalized to the EHM via the endocytic vesicle trafficking pathway (Lu et al., 2012) (5); however, definitive evidence remains to be provided.