Accurate and adequate spatiotemporal expression and localization of RPW8.2 is key to activation of resistance at the host-pathogen interface

Abstract

Numerous fungal and oomycete pathogens penetrate the plant cell wall and extract nutrition from the host cells by a feeding structure called the haustorium. We recently revealed that the Arabidopsis resistance protein RPW8.2 is specifically targeted to the extrahaustorial membrane (EHM) for activation of haustorium-targeted resistance to powdery mildew pathogens. Consistent with its EHM-localization, RPW8.2 contains a putative transmembrane (TM) domain at its N-terminus. Here, we show that translational fusion of YFP to the N-terminus of RPW8.2 results in localization of YFP-RPW8.2 to both the plasma membrane and the EHM, and loss of RPW8.2's defense function. We also show that deletion of the TM domain results in mis-localization of the RPW8.2-YFP fusion protein and extremely low levels of accumulation. These results indicate that an intact N-terminal TM domain is necessary for EHM-specific localization and defense function of RPW8.2. In addition, we show that when expressed from the strong constitutive 35S viral promoter, RPW8.2 accumulates at low levels in the EHM insufficient to activate resistance, highlighting the importance of strong spatiotemporal expression of RPW8.2 from its native promoter. Taken together, our results indicate that accurate and adequate spatiotemporal expression and localization of RPW8.2 is key to activation of resistance at the host-pathogen interface.

In order to establish successful colonization on plant hosts, a haustorium-forming fungus such as powdery mildew must conquer two spatio-temporally interconnected layers of host resistance: pre-invasion (penetration) resistance and post-invasion resistance.¹ Pre-invasion resistance protects plants from non-adapted pathogens by blocking their entry into the host cell.^2–4 One common induced cellular defense response at this resistance level is the deposition of defense chemicals, including callose (β-1,3-glucan) at the site of penetration, resulting in cell wall apposition, a subcellular structure also known as a papilla.^5–7 It has been reported that a syntaxin encoded by PENETRATION 1 (PEN1) is required for the timely assembly of the papilla,⁸ which is consistent with PEN1's role in pre-invasion resistance.² Once the fungus penetrates the plant cell wall, it will have to overcome the second layer of resistance, i.e., post-invasion resistance, to develop a functional haustorium in close contact with the host cell cytoplasm for successful colonization. Hypersensitive response (HR) manifested as rapid collapse of the invaded cell is often associated with post-invasion resistance.^9–11 Another cellular defense response to haustorial invasion is the formation of an encasement of the haustorial complex (EHC).^12–16 Like the papilla, the EHC is also enriched for callose and thought to be formed via extension from the papilla by rim-growth.¹⁷

We have recently reported that RPW8.2-mediated broad-spectrum powdery mildew resistance is associated with both HR and an enhancement of EHC formation.¹⁸ Most strikingly, we found that the RPW8.2-YFP fusion protein expressed from its native promoter (NP) is specifically targeted to the extrahaustorial membrane (EHM), suggesting that RPW8.2 functions at the host-pathogen interface to activate post-invasion resistance. How RPW8.2 is targeted to the EHM and directs host defense to the host-pathogen interface remains to be an open question.

YFP-RPW8.2 is Non Functional and is Localized to Both the Plasma Membrane and the Extrahaustorial Membrane

The RPW8.2 protein, as other members of the small RPW8 protein family, contain a predicated N-terminal transmembrane domain (TM) (amino acid 1 to 22) or signal peptide.^19,20 To investigate the role of the putative TM domain, we translationally fused YFP to the N-terminus of RPW8.2 and the NP::YFP-RPW8.2 construct was introduced into the mildew-susceptible, RPW8.2-null Col-0 background. We found that the majority (>75%) of transgenic lines expressing NP::YFP-RPW8.2 exhibited a wavy leaf phenotype which had not been observed in plants expressing NP::RPW8.2-YFP (Fig. 1A and B). Unlike Col-0 lines transgenic for NP::RPW8.2-YFP (of which, ∼5–10% showed spontaneous HR-like cell death), none of the Col-0 lines expressing NP::YFP-RPW8.2 developed HR-like lesions visible to the naked eye. We then evaluated the disease reaction phenotypes of these NP::YFP-RPW8.2 transgenic lines in response to infection by Golovinomyces cichoracearum (Gc) UCSC1, the causal agent of powdery mildew diseases in Arabidopsis and many other plant species.²¹ Except for a few (4/35) transgenic lines that showed some slightly reduced susceptibility (Fig. 1A), we found the majority were as susceptible to Gc UCSC1 as Col-0 (data not shown). These results suggest that unlike RPW8.2-YFP which largely retains the function of the native RPW8.2 protein,¹⁸ YFP-RPW8.2 is largely nonfunctional in defense. We performed trypan blue staining with Arabidopsis leaves prior to inoculation and 3 days post-inoculation (dpi) of Gc UCSC1 to see if there is any cell death in the lines transgenic for NP::YFP-RPW8.2. We found that there were some small clusters of cell death on both uninoculated and inoculated wavy leaves, and the cell death was not tightly associated with fungal invasion (Fig. 1C and D). A close microscopic examination of the spontaneous cell death lesions revealed that some surrounding epidermal and mesophyll cells had irregular shapes and sizes, resulting in contraction of the neighboring areas. This may explain the curly, wavy leaf phenotype.

Figure 1.

Leaf phenotypes of transgenic plants expressing different RPW8.2 constructs. (A) Representative powdery mildew-infected leaves of independent Col-gl lines expressing the indicated DN A constructs. Pictures were taken at 7 or 10 days post-inoculation (dpi) with Golovinomyces cichoracearum UC SC1. (B) Representative wavy leaves from four independent Col-gl lines expressing YFP-RPW8.2 and a normal leaf from a Col-gl line expressing YFP. (C and D) Cell death in Col-gl lines expressing YFP-RPW8.2 prior to inoculation (C) or at 3 dpi (D). Leaf sections from respective plants were stained with trypan blue. Dead plant cells and vascular veins, and fungal mycelia were stained blue. Scale bar, 100 µm.

We then questioned if YFP-RPW8.2 is correctly targeted to the EHM induced by Gc UCSC1. Interestingly, unlike RPW8.2-YFP, which is highly specifically targeted to the EHM (Fig. 2A; Wang et al., 2009), fluorescent signals from YFP-RPW8.2 was found to be distributed in both the EHM and the cytoplasm or plasma membrane (PM) (Fig. 2B). To make sure if YFP-RPW8.2 is indeed localized at the PM, we treated a leaf section with the endomembrane dye FM4-64 and performed cytoplasmolysis. As shown in Figure 2C, YFP-RPW8.2 was nicely co-localized with the FM4-64 stained PM, indicating that YFP-RPW8.2 is not only targeted to the EHM, but also distributed at the PM. Apparently, this decrease in localization specificity could result in a reduced efficacy of YFP-RPW8.2 in triggering EHM-directed defense against powdery mildew compared to RPW8.2-YFP or the native RPW8.2 protein. However, given the seemingly preferential EHM-localization of YFP-RPW8.2 versus its PM-localization in the same cells (Fig. 2B), one would not expect a nearly complete loss of resistance in plants expressing YFP-RPW8.2. Therefore, it is possible that, in addition to a compromised EHM-targeting specificity, YFP fusion at the N-terminus of RPW8.2 may result in (i) change of RPW8.2's membrane orientation/association due to masking the N-terminal domain as a potential signal peptide for co-translational translocation at the endoplamic reticulum, or (ii) alteration of the protein conformation that renders RPW8.2 nonfunctional, or (iii) disruption of RPW8.2's interaction with other proteins, which is required for its defense function, via the N-terminal domain.

Figure 2.

Laser scanning confocal microscopic images showing subcellular localization of different YFP-tagged RPW8.2 proteins. (A) RPW8.2-YFP (green) expressed from the native promoter is specifically localized to the extrahaustorial membrane encasing the haustorium (stained red by 0.5% propidium iodide). (B) YFP-RPW8.2 expressed from the native promoter is localized to both the plasma membrane and the extrahaustorial membrane. The arrow indicates the fungal penetration site. (C) Cytoplasmolysis of YFP-RPW8.2 expressing cells showing YFP-RPW8.2 is indeed localized to the plasma membrane stained red by the fluorescent dye FM4-64. (D and E) Low-level accumulation of Δ30RPW8.2-YFP expressed from the native promoter in punctate spots (arrows). (F and G) Typical low-level accumulation of RPW8.2-YFP expressed from the 35S promoter in unchallenged epidermal cells (F) or in a representative challenged epidermal cell at 2 dpi (G). The arrow indicates a haustorium decorated with RPW8.2-YFP in punctate spots presumably at the extrahaustorial membrane. All pictures are Z-stack of 15–25 images. Scale bars, 10 µm for all except D (50 µm).

Adequate Expression and EHM-Accumulation of RPW8.2 is Required for Activation of Resistance

To test if overexpression of RPW8.2 could improve resistance, we expressed both YFP-RPW8.2 and RPW8.2-YFP from the strong constitutive viral 35S promoter. Quite counterintuitively, we found that both YFP-RPW8.2 and RPW8.2-YFP were expressed at very low levels prior to and after Gc UCSC1 infection with protein localization patterns similar to those expressed from the native promoter. For example, RPW8.2-YFP expressed from the 35S promoter was found in punctate spots along cytoplasmic streams in unchallenged epidermal cells of some transgenic lines (Fig. 2F). After powdery mildew infection, RPW8.2-YFP was mostly found in punctate spots presumably at the EHM (Fig. 2G). Not surprisingly, all transgenic lines (>15 examined) expressing either of the two constructs from the 35S promoter were generally susceptible to the pathogen (data not shown). Based on these observations, we concluded that an adequate dosage of the functional, EHM-localized RPW8.2 protein is essential for inducing interface-focused resistance to powdery mildew. Our results also confirmed our previous findings at the protein level that the native promoters of RPW8.1 and RPW8.2 are required for salicylic acid-dependent feedback amplification of RPW8.1 and RPW8.2 in cells invaded by powdery mildew.²²

The N-Terminus of RPW8.2 is Indispensable for its EHM Localization and Resistance Function

To further clarify the importance of the TM domain of RPW8.2, we translationally fused two RPW8.2 mutants in which the N-terminal TM domain was truncated from amino acids 2 to 22 (Δ22RPW8.2) or from 2 to 30 (Δ30RPW8.2) with YFP at the C-terminus of RPW8.2. We individually introduced these two constructs into Col-0 and found that all Col-0 transgenic lines (>15) expressing Δ22RPW8.2-YFP or Δ30RPW8.2-YFP from the RPW8.2 native promoter were fully susceptible to Gc UCSC1 (Fig. 1A). Examination of infected leaves by confocal microscopy showed that these fusion proteins were rarely detectable in uninoculated or infected leaves. In a few mesophyll cells where YFP signals were detectable, we observed some fluorescent spots in the size of ∼3 µm as shown in Figure 2D and E. We have never detected any YFP signals in the EHM, indicating a complete loss of EHM-localization and accumulation for these two TM-truncated RPW8.2 fusion proteins. Based on these results, we speculate that the N-terminal TM domain of RPW8.2 is not only important for the membrane anchorage of RPW8.2 for EHM-targeting but also plays a role for RPW8.2 accumulation by stabilizing the protein and/or contributing to the function of RPW8.2 in triggering the SA-dependent feedback amplification of RPW8.2.

Taken together, our results support a conclusion that adequate, spatially correct and temporally amplifiable RPW8.2 is critical to RPW8.2-mediated, EHM-focused resistance against haustorial invasion from powdery mildew pathogens.

Addendum to: Wang W, Wen Y, Berkey R, Xiao S. Specific targeting of the Arabidopsis resistance protein RPW8.2 to the interfacial membrane encasing the fungal Haustorium renders broad-spectrum resistance to powdery mildew. Plant Cell. 2009;21:2898–2913. doi: 10.1105/tpc.109.067587.