The Plant Membrane-Associated REMORIN1.3 Accumulates in Discrete Perihaustorial Domains and Enhances Susceptibility to Phytophthora infestans

Abstract

Filamentous pathogens such as the oomycete Phytophthora infestans infect plants by developing specialized structures termed haustoria inside the host cells. Haustoria are thought to enable the secretion of effector proteins into the plant cells. Haustorium biogenesis, therefore, is critical for pathogen accommodation in the host tissue. Haustoria are enveloped by a specialized host-derived membrane, the extrahaustorial membrane (EHM), which is distinct from the plant plasma membrane. The mechanisms underlying the biogenesis of the EHM are unknown. Remarkably, several plasma membrane-localized proteins are excluded from the EHM, but the remorin REM1.3 accumulates around P. infestans haustoria. Here, we used overexpression, colocalization with reporter proteins, and superresolution microscopy in cells infected by P. infestans to reveal discrete EHM domains labeled by REM1.3 and the P. infestans effector AVRblb2. Moreover, SYNAPTOTAGMIN1, another previously identified perihaustorial protein, localized to subdomains that are mainly not labeled by REM1.3 and AVRblb2. Functional characterization of REM1.3 revealed that it is a susceptibility factor that promotes infection by P. infestans. This activity, and REM1.3 recruitment to the EHM, require the REM1.3 membrane-binding domain. Our results implicate REM1.3 membrane microdomains in plant susceptibility to an oomycete pathogen.

Figure 1.

REM1.3 localizes at the PM and the EHM in cells infected by P. infestans. Coexpression of RFP:REM1.3 and GFP (A), RFP:REM1.3 and GFP:HaRXL17 (B), and YFP:REM1.3 and RFP:AVRblb2 (C) by A. tumefaciens-mediated transient transformation under the control of the CaMV 35S promoter in haustoriated cells discriminates between host subcellular compartments surrounding haustoria. GFP, HaRXL17, and AVRblb2 label the cytoplasm and nucleus, the tonoplast, and the EHM and PM, respectively. Colocalization is only observed between REM1.3 and AVRblb2. Images show single optical sections. The fluorescence plots show relative fluorescence along the dotted line connecting points a and b. Arrowheads point to the tips of haustoria. A.U., Arbitrary units; Cyt., cytoplasm; Ton., tonoplast. Bars = 7.5 μm.

Figure 2.

REM1.3 localizes around a subpopulation of noncallosic haustoria during P. infestans infection. A, Representative image of a stable 35S-YFP:REM1.3 N. benthamiana transgenic plant inoculated by P. infestans strain 88069 expressing RFP (88069td). Haustoria are indicated with closed arrowheads when surrounded by YFP:REM1.3 and with open arrowheads otherwise. B, Frequency of the colocalization of YFP:REM1.3 with RFP:AVRblb2 around P. infestans haustoria. YFP:REM1.3 with RFP:AVRblb2 constructs were codelivered into N. benthamiana leaves by A. tumefaciens-mediated transformation. C, P. infestans 88069td-inoculated leaves expressing YFP:REM1.3 and stained for callose. Haustoria surrounded by YFP:REM1.3 (closed arrowheads) never showed a callose neck band (open arrowheads). Images show single optical sections. The frequency of observations is indicated.

Figure 3.

REM1.3 colocalizes with the P. infestans RXLR effector AVRblb2 in domains around haustoria. A, Coexpression of EHM markers by A. tumefaciens-mediated transient transformation under the control of the CaMV 35S promoter in haustoriated cells reveals perihaustorial membrane domains. Left, YFP:REM1.3 and RFP:AVRblb2 show nearly full colocalization around haustoria, with all domains strongly labeled by YFP:REM1.3 (closed arrowheads) showing intense RFP fluorescence. Middle, SYT1 strongly labels domains that are only weakly labeled by RFP:AVRblb2 (open arrowheads). Right, SYT1 labels domains around haustoria that are not or weakly labeled by YFP:REM1.3 (open arrowheads). B, Correlation between the RFP and YFP fluorescence signals around haustoria in cells coexpressing RFP:AVRblb2 and YFP:REM1.3. The fluorescence is measured along the dotted line connecting points a and b, as shown in the inset. The average Pearson correlation coefficient for RFP and YFP fluorescence signals along six different perihaustorial membranes is 0.79. A.U., Arbitrary units. C, Quantification of fluorescence correlation for perihaustorial markers highlights the existence of at least two types of domains around haustoria. Pearson correlation coefficients were calculated in cells coexpressing free GFP+RFP:REM1.3, YFP:REM1.3+RFP:AVRblb2, GFP:SYT1+RFP:AVRblb2, and GFP:SYT1+RFP:REM1.3. Only cells in which REM1.3 accumulated around haustoria were considered. Significant differences of the means were assessed using Welch’s t test (***P < 0.001). Measurements were performed over at least two independent inoculation events.

Figure 4.

Validation of the occurrence of subdomains at the EHM using superresolution microscopy. YFP:REM1.3 and RFP:AVRblb2 show perfect localization at the EHM mainly at some foci (top row), while GFP:SYT1 labels different microdomains compared with RFP:REM1.3 (middle row). Consistently, RFP:AVRblb2 and GFP:SYT1 also label different domains across the EHM (bottom row). Recombinant constructs were delivered using A. tumefaciens-mediated transformation. Images were obtained at 3 dpi using superresolution SIM. Images shown are maximal projections of 31, 30, and 27 frames with 0.11, 0.11, and 0.12 μm steps for the top, middle, and bottom rows, respectively.

Figure 5.

REM1.3 overexpression increases susceptibility to P. infestans in N. benthamiana. A, Validation of YFP:REM1.3 overexpression in N. benthamiana transgenic plants by anti-remorin western blot in two independent 35S-YFP:REM1.3 lines (OX1.4 and OX2.2) compared with wild-type plants (WT). B, Type and frequency of symptoms caused by P. infestans 88069 at 5 dpi on overexpression and wild-type plants as a percentage of 40 infection foci over three independent experiments including three independent overexpression lines. C, Representative images of symptoms caused by P. infestans 88069 on N. benthamiana overexpression and wild-type plants at 5 dpi. D, Quantification of P. infestans 88069td growth in N. benthamiana lines by measurement of RFP fluorescence. Representative fluorescence images show P. infestans 88069td growth in overexpression and wild-type plants at 4 dpi. Bars = 5 mm. Histograms show relative fluorescence intensity, calculated as the mean pixel intensity over a 0.655-cm² image centered on the lesion and expressed as a percentage of the intensity measured on wild-type plants. Three to six images were analyzed per N. benthamiana line, and error bars show sd. E, N. benthamiana leaf infiltrated with A. tumefaciens carrying either 35S-GFP or 35S-YFP:REM1.3 (left and right, respectively) and inoculated with P. infestans 88069 24 h later. Images were taken and the size of lesions measured at 5 dpi. F, Relative P. infestans lesion size on half-leaves infiltrated with 35S-GFP and 35S-YFP:REM1.3. Significance was assayed using Student’s t test (***P < 0.01) over 12 lesions in three independent experiments. G, Total proteins extracted from half-leaves infiltrated with 35S-GFP and 35S-YFP:REM1.3 and probed by anti-GFP western blots showing similar expression levels for GFP and YFP:REM1.3 constructs.

Figure 6.

Silencing of REM1.3 enhances resistance to P. infestans in N. benthamiana. A, Validation of the silencing of REM1.3 orthologs in N. benthamiana by anti-REM western blot. The REM1.3 protein amount was estimated based on the western-blot signal. B, Type and frequency of symptoms caused by P. infestans 88069 at 6 dpi as a percentage of at least 12 infection foci over three independent experiments. C, Representative images of symptoms caused by P. infestans 88069 on N. benthamiana at 7 dpi. D, Top, representative fluorescence images showing P. infestans 88069td growth at 4 dpi. Bars = 5 mm. Bottom, quantification of P. infestans 88069td growth in N. benthamiana lines by measurement of RFP fluorescence. Histograms show relative fluorescence intensity calculated as for Figure 5. Three to six images were analyzed per N. benthamiana line, and error bars show sd. e.v., Plants infiltrated with the pTV00 empty vector; VIGS, plants infiltrated with the REM1.3 VIGS silencing construct; WT, wild-type plants.

Figure 7.

The REM1.3 ortholog promotes susceptibility to P. infestans in tomato. A, Symptoms caused by P. infestans 88069 at 4 dpi on tomato plants overexpressing a tomato REM1.3 ortholog (SE), empty vector-transformed plants (e.v.), and wild-type (WT) and REM1.3 antisense (AS) plants. B, Box plot showing the distribution of the relative sizes of lesions at 4 dpi on tomato plants with different levels of REM1.3. At least 48 infection foci were measured per line over three independent experiments. The significance of differences compared with the wild type was assessed by Student’s t test (***P < 0.01). Overexpression and silencing of REM were verified by western-blot analysis of individual plants (Supplemental Fig. S3).

Figure 8.

The REM1.3 membrane-binding domain is required for perihaustorial targeting. Confocal micrographs show the subcellular localization of YFP fusions with wild-type REM1.3, REM1.3 lacking the C-terminal membrane anchor domain (ΔCA), and REM1.3 with mutated C-terminal membrane anchor domain (*) in uninfected cells (A) and cells infected by P. infestans 88069td (B). The tips of haustoria are shown by closed arrowheads when surrounded by YFP labeling and with open arrowheads otherwise. Constructs controlled by the 35S promoter were delivered using A. tumefaciens-mediated transformation. Images shown are single optical plane sections except for uninfected cells expressing YFP:REM1.3^ΔCA and YFP:REM1.3*, which correspond to maximal projections of 32 frames with 1 µm steps.

Figure 9.

The REM1.3 membrane anchor is required for the promotion of susceptibility to P. infestans. A, Symptoms caused by P. infestans 88069 at 5 dpi on leaves transiently overexpressing YFP fusions with wild-type REM1.3 on one half and free GFP or REM1.3 lacking the C-terminal membrane anchor domain (ΔCA) or REM1.3 with mutated C-terminal membrane anchor domain (*) on the other half. B, Box plot showing relative sizes of the lesions over 12 to 54 infection foci in three independent experiments. Significance of differences compared with GFP-expressing leaves was assessed by Student’s t test (***P < 0.01). Constructs controlled by the 35S promoter were delivered using A. tumefaciens-mediated transformation; their expression was verified by detection of fluorescence.