Homologues of the RPW8 Resistance Protein Are Localized to the Extrahaustorial Membrane that Is Likely Synthesized De Novo

Abstract

Upon penetration of the host cell wall, the powdery mildew fungus develops a feeding structure named the haustorium in the invaded host cell. Concomitant with haustorial biogenesis, the extrahaustorial membrane (EHM) is formed to separate the haustorium from the host cell cytoplasm. The Arabidopsis resistance protein RPW8.2 is specifically targeted to the EHM where it activates haustorium-targeted resistance against powdery mildew. RPW8.2 belongs to a small family with six members in Arabidopsis (Arabidopsis thaliana). Whether Homologs of RPW8 (HR) 1 to HR4 are also localized to the EHM and contribute to resistance has not been determined. Here, we report that overexpression of HR1, HR2, or HR3 led to enhanced resistance to powdery mildew, while genetic depletion of HR2 or HR3 resulted in enhanced susceptibility, indicating that these RPW8 homologs contribute to basal resistance. Interestingly, we found that N-terminally YFP-tagged HR1 to HR3 are also EHM-localized. This suggests that EHM-targeting is an ancestral feature of the RPW8 family. Indeed, two RPW8 homologs from Brassica oleracea tested also exhibit EHM-localization. Domain swapping analysis between HR3 and RPW8.2 suggests that sequence diversification in the N-terminal 146 amino acids of RPW8.2 probably functionally distinguishes it from other family members. Moreover, we found that N-terminally YFP-tagged HR3 is also localized to the plasma membrane and the fungal penetration site (the papilla) in addition to the EHM. Using this unique feature of YFP-HR3, we obtained preliminary evidence to suggest that the EHM is unlikely derived from invagination of the plasma membrane, rather it may be mainly synthesized de novo.

Figure 1.

RPW8 homologs contribute to basal resistance to powdery mildew. A, Disease reaction phenotypes of representative leaves of T3 Col-gl lines overexpressing (i.e. 35S plus NP) HR1, HR2, HR3, or HR4 infected with Gc UCSC1. Col-gl and Col-nahG were used as control. Pictures were taken at 12 dpi. B, RT-PCR analysis of representative overexpression lines and Col-gl using gene-specific primers (top panel). UBC21 was used as control (bottom panel). cDNA was synthesized using total RNA prepared from uninfected plants. PCR was done with 24 (UBC21) or 30 (other genes) cycles. ImageJ (NIH) was used to estimate band intensities. C, Quantitative assay of disease susceptibility. Asterisks indicate significance at P < 0.01 compared with Col-gl based on Student’s t test. Data represent means ± se (n = 4) from one of three independent experiments. D, RT-PCR analysis of gene knockout or knockdown T-DNA insertion lines. Top panels (left to right): HR1, HR2, or HR3 using gene-specific primers within Exon 1 upstream of all T-DNA insertions. Bottom panels (all): UBC21. Infected leaves at 3 dpi were used for RNA extraction and cDNA synthesis. PCR was done with 22 (UBC21) or 30 (other genes) cycles. ImageJ was used to estimate band intensities. E, Representative infected leaves or plants of indicated mutant and control lines at 12 dpi. F, Quantitative assay of disease susceptibility at 12 dpi. Data represent means ± se (n = 4) from one of three independent experiments. Student’s t test was used to examine if the difference relative to Col-0 is statistically significant (* P < 0.05; ** P < 0.01).

Figure 2.

Expression of HR3-YFP results in necrotic cell death. A, Representative plants (five weeks old) or infected leaves (12 dpi) expressing indicated DNA constructs under control of their respective NPs in Col-0. More than 30 T1 lines for each construct were examined. Note that there was no obvious altered growth and defense phenotypes in any of the transgenic lines compared with Col-0. B, Representative T1 plants transgenic for NP::HR3-YFP with varied degrees of stunted growth and spontaneous cell death (red arrows). C, A group of T1 plants transgenic for NP::HR3-YFP at 10 dpi with Gc UCSC1 showing varied degree of resistance (indicated by red arrowheads) and susceptibility (indicated by yellow arrowheads). D, Representative T1 transgenic plants (upper panel) and total number of T1 plants with spontaneous leaf necrotic death (arrows) or without (arrowheads; lower panel) for the indicated DNA constructs.

Figure 3.

Sequence and function diversification of RPW8.2 from HR3. A, Protein sequence alignment between HR3 and RPW8.2. The blue arrowhead indicates the position (amino acid 147) in HR3 for recombination with the C terminus of RPW8.2, which is indicated by a red arrowhead. The green arrowhead indicates Asp-116 in RPW8.2, which is conserved in HR3. The black arrow indicates Gly-186, which is mutated to Asp in the HR3^ΔCt26/G186D mutant. The two core EHM-targeting motifs are boxed in red. B, Schematic illustration of the chimeric R82n-HR3c and HR3n-R82c genes in fusion with YFP driven either by the RPW8.2 or HR3 promoter. HR3n = HR3^1-147, R82n = RPW8.2^1-146, HR3c = HR3^148-213, and R82c = RPW8.2^147-174. C, Representative images showing expression and localization of the indicated fusion proteins. Note that HR3n-R82c-YFP was undetectable. Bar, 20 μm. D, Phenotypes of the indicated genotypes. Representative leaves from infected plants at 12 dpi were shown to reflect the functionality of the chimeric genes. Plants transgenic for HR3p::HR3n-R82c-YFP were also susceptible (not shown). *, Haustoria.

Figure 4.

Subcellular localization of YFP-HR1, YFP-HR2, YFP-HR3, and YFP-HR4. Except for YFP-HR4, which was transiently expressed in leaves of N. benthamiana, the rest were stably expressed in Arabidopsis Col-0 (At Col-0). A, Representative images showing cytoplasmic and likely plasma membrane localization of indicated fusion proteins expressed from the 35S promoter in leaves of Arabidopsis transgenic plants (for HR1, HR2 and HR3) or agroinfiltrated N. benthamiana plants (for HR4). B, A representative image showing colocalization of YFP-HR3 with AtPIP2A-mCherry in N. benthamiana epidermal cells when transiently coexpressed. C, Representative images showing likely EHM-localization of indicated fusion proteins expressed from the 35S promoter at 2 dpi with Gc UCSC1. D, EHM-localization of YFP-HR3 expressed from the HR3 NP in At Col-0 at 2 dpi with Gc UCSC1. Bar, 20 μm in A; 50 μm in B; 10 μm in C and D. H, Haustorium; Nb, N. benthamiana.

Figure 5.

YFP-HR3 is localized to the plasma membrane, the EHM, and the papilla. A, Representative image showing that YFP-HR3 is localized to the PM (arrow), the EHM (labeled by RPW8.2-RFP), and the papilla (arrowhead). Inset is a close-up view of the transverse section of a fungal penetration site where both YFP-HR3 and callose focally accumulate. Note the heterogeneous distribution of YFP-HR3. B, Representative isolated haustorial complex with an intact EHM labeled by both YFP-HR3 and RPW8.2-RFP. C, PM-localization of GFP-SYP131 in the epidermal cells containing haustoria (arrows). D, Maximum projection of a Z-stack image Z-stack image showing YFP-RPW8.2 is localized to the EHM but not in the papilla where callose is deposited (arrow). Bar, 10 μm in A, B, and D; 50 μm in C. Cp, Chloroplast.

Figure 6.

Brassica RPW8 homologs are also localized to the EHM and the papilla. A and B, Representative powdery mildew-infected T1 plants expressing BoHRa-YFP (A) or BoHRb-YFP (B) from the RPW8.2 promoter (upper panel) and total number of T1 transgenic plants from each indicated category (lower panel). Arrows indicate plants with spontaneous cell death and resistance to powdery mildew (disease reaction score, DR = 0 to 1; Xiao et al., 2005); arrowheads indicate plants displaying enhanced mildew resistance without spontaneous cell death (DR = 1 to 2); asterisks indicate plants susceptible to powdery mildew (DR = 2 to 3 or 3). Pictures were taken at 9 dpi. C and D, Representative images showing accumulation of BoHRa-YFP (C) or YFP-BoHRa (D) in the papilla. E, Representative image showing EHM-localization of YFP-BoHRa. Bar, 10 μm in C to E.

Figure 7.

The EHM is physically separable from the PM and the papilla. Gc UCSC-infected leaves expressing YFP-HR3 (from 35S) and RPW8.2-RFP (from the RPW8.2 promoter) were subjected to plasmolysis (0.5 M NaCl). A, Representative confocal image taken at 10 min after treatment. Note that the cytoplasm with YFP-HR3-labeled PM was shrunk around the seemingly unaffected haustorium labeled by both YFP-HR3 and RPW8.2-RFP. B and C, Representative confocal images taken at 30 min after treatment showing that the cytoplasm with YFP-HR3-labeled PM was nearly (B) or completely (C) detached from the haustorial complex with an intact EHM labeled by YFP-HR3 and RPW8.2-RFP. Note that YFP-HR3 localized to the papilla stained by PI (arrows in C) was also unaffected. Bar, 10 μm in A to C. C, Cytoplasm; H, haustorium.

Figure 8.

Papilla-accumulation of YFP-HR3 is BFA sensitive while its targeting to the EHM is not. A, Surface-applied FM4-64 (20 μM) in 0.002% Silwet L-77 stained the PM and the papilla in 20 min (image on the left) but did not stain the EHM labeled by YFP-HR3 in 45 min (image on the right). B, When vacuum-infiltrated into leaf tissue (see “Materials and Methods”), FM4-64 could stain the EHM (arrow) labeled by RPW8.2-YFP as well as the haustorial membrane lobes. Notably, the haustorial neck is often more strongly stained (arrowhead). C, BFA inhibits focal accumulation of YFP-HR3 and callose to the papilla. Quantification of relative fluorescence intensity for YFP-HR3 and Sirofluor-stained callose at the papilla in confocal images. Data represent means ± se (n = 6) from one of two independent experiments. Student’s t test was used to compare the differences between treatments (* indicates P < 0.01, ** indicates P < 0.001). D, Representative images showing localization of YFP-HR3 to the PM, the papilla, and the EHM at 45 hpi in epidermal cells infiltrated with 1% DMSO buffer (upper panel) or 300 μM BFA (lower panel) at approximately 6 hpi from the base of the leaf. The EHM was labeled by RPW8.2-RFP and the callose was stained with Sirofluor. Note that both YFP-HR3 and callose were diminished in the papilla due to BFA treatment, but YFP-HR3 and RPW8.2-RFP at the EHM were unaffected. Bar, 10 μm in A to E. H, Haustorium; p, papilla.