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. 2026 Sep 2;27(9):e70331. doi: 10.1111/mpp.70331

The ER–Golgi Lipid Exchanger PsORP1 Is Essential for Phytophthora sojae Development and Is Blocked by Oxathiapiprolin via Its OSBP‐Related Domain (ORD)

Xiaofei Liu 1,2, Chengcheng Li 2, Guangda Shao 2, Qin Peng 2, Haixia Wang 2, Jianqiang Miao 2,✉, Xili Liu 1,2,✉
PMCID: PMC13538951  PMID: 42687324

ABSTRACT

Oxysterol‐binding protein (OSBP)‐related proteins (ORPs) are indispensable eukaryotic lipid transporters and promising targets for pharmaceutical and agrochemical exploitation. Among ORP family members across kingdoms, oomycete ORP1 constitutes a phylogenetically distinct subtype that evolved independently in oomycetes, which is markedly divergent from ORP homologues in animals, plants and fungi. Oxathiapiprolin, the first commercial fungicide targeting OSBP family proteins, specifically acts on oomycete ORP1 and exhibits robust inhibitory efficacy against diverse Phytophthora pathogens and downy mildews. Nevertheless, the molecular function of oomycete ORP1 and the inhibitory mechanism of oxathiapiprolin remain poorly defined. Here, we demonstrate that PsORP1 of Phytophthora sojae specifically localises to ER–Golgi membrane contact sites (MCSs) and is indispensable for vegetative growth and asexual/sexual development of P. sojae. In vitro liposome reconstitution assays confirmed that the conserved C‐terminal OSBP‐related domain (ORD) of PsORP1 mediates canonical phosphatidylserine (PS)/phosphatidylinositol 4‐phosphate (PI4P) counter‐transport. Biochemical binding assays further verified that oxathiapiprolin directly interacts with the ORD of PsORP1 and competitively blocks PS/PI4P lipid exchange. Collectively, this study demonstrates that PsORP1 localises to ER–Golgi MCSs and mediates ORD‐dependent PS/PI4P counter‐transport, which is essential for P. sojae development, and elucidates that oxathiapiprolin directly targets the ORD to competitively block this lipid exchange, providing a structural framework for rational design of next‐generation OSBP‐targeting inhibitors against oomycetes.

Keywords: mode of action, OSBP‐related protein, oxathiapiprolin, Phytophthora sojae, PS/PI4P exchange

PsORP1 anchors ER–Golgi MCSs to mediate PS/PI4P counter‐transport, and oxathiapiprolin targets the ORD to inhibit this lipid exchange.

graphic file with name MPP-27-e70331-g002.webp

1. Introduction

Lipids play multiple essential roles in eukaryotic cells, including as structural components of membranes, energy storage molecules, and signalling regulators (Holthuis and Menon 2014). The transport of lipids from the site of synthesis at the endoplasmic reticulum (ER) to various intracellular destinations can be mediated by both vesicular and non‐vesicular transport mechanisms (Wong et al. 2019). Non‐vesicular lipid transfer, governed by lipid‐binding/transfer proteins (LTPs), primarily occurs at membrane contact sites (MCSs), which are specialised regions where two organellar membranes are closely apposed, separated by a narrow cleft of approximately 10 to 30 nm (Elbaz and Schuldiner 2011; Helle et al. 2013; Wu et al. 2018).

Oxysterol‐binding protein (OSBP)‐related proteins (ORPs) constitute an evolutionarily conserved family of LTPs essential for non‐vesicular intracellular lipid trafficking in eukaryotes and have emerged as central regulators of lipid transport at MCSs formed between the ER and other cellular membranes (Raychaudhuri and Prinz 2010; Mesmin et al. 2013; Chung et al. 2015; Kawasaki et al. 2022). In humans, this protein family includes 12 members, and seven homologues (Osh1‐Osh7) have been found in Saccharomyces cerevisiae (Olkkonen and Li 2013; Pietrangelo and Ridgway 2018). All members of this protein family share a conserved OSBP‐related domain (ORD) containing a hydrophobic pocket that accommodates specific lipids, such as cholesterol, phosphatidylserine (PS), and phosphatidylinositol polyphosphates (PIPs) (Olkkonen and Li 2013; Mesmin et al. 2013; Chung et al. 2015; Kawasaki et al. 2022). Certain long ORP isoforms also possess an FFAT motif (two phenylalanines followed by an acidic tract) that is recognised by ER‐resident vesicle‐associated membrane protein (VAMP) receptors and an N‐terminal pleckstrin homology (PH) domain for binding PIPs. Through these dual molecular interactions, ORPs can mediate the formation of molecular bridges at MCSs between the ER and PIP‐rich organelle membranes, thereby facilitating non‐vesicular lipid transfer via the ORD (Delfosse et al. 2020). A prototypical example of this mechanism is provided by OSBP, which mediates the exchange of cholesterol for phosphatidylinositol 4‐phosphate (PI4P) between the Golgi apparatus and ER. The interaction of the FFAT motif with VAMP allows the association of OSBP with the ER membrane, whereas its PH domain anchors it to the trans‐Golgi network (TGN). Through its ORD, OSBP extracts and binds cholesterol synthesised in the ER and rapidly transports it to the Golgi apparatus (Mesmin et al. 2013).

ORPs are highly promising novel therapeutic targets, as their dysfunction is implicated in multiple human diseases, including metabolic disorders, cancers, neurodegenerative diseases, and viral infections (Lin et al. 2023). OSW‐1 and ORPphilins, a natural product and its structural analogs, specifically bind to the hydrophobic pocket of OSBP and ORP4 and, by inhibiting their lipid transport function, induce selective apoptosis in cancer cells (Burgett et al. 2011; Bensen et al. 2021; Lu et al. 2025). Itraconazole, which exhibits both antifungal and anticancer activities, also binds to OSBP and ORP4, blocking OSBP‐mediated lipid transport and effectively inhibiting enterovirus replication (Strating et al. 2015). Notably, both orpinolide, obtained through natural product derivation, and oxybipins, developed via structure‐based design, employ a modified cholesterol scaffold as their core structure. This enables highly selective inhibition of OSBP and has demonstrated potential in the treatment of leukaemia and anti‐infective applications (Chen and Baskin 2025). Oxathiapiprolin was specifically engineered to combat destructive diseases caused by oomycete pathogens, including downy mildew and Phytophthora spp., and represents a breakthrough in agricultural fungicide development (Pasteris et al. 2008; Miao et al. 2016). ORP1 was initially captured as a binding partner of oxathiapiprolin via affinity chromatography in Phytophthora capsici, and subsequent genetic studies have linked numerous amino acid substitutions within its ORD domain to high‐level resistance against this fungicide (Pasteris et al. 2008; Miao et al. 2016; Chen and Baskin 2025). These lines of evidence collectively establish ORP1 as the direct molecular target of oxathiapiprolin. However, the physiological function of oomycete ORP1 and the precise inhibitory mechanism by which oxathiapiprolin impairs its activity remain largely elusive.

Here, using a Tet‐on/CRISPR‐Cas9‐mediated gene inducible expression system, we demonstrate that PsORP1 is essential for vegetative growth and asexual/sexual development of P. sojae, and specifically localises to ER–Golgi MCSs. Critically, we decipher the ORD‐domain‐dependent PS/PI4P transport machinery and demonstrate that oxathiapiprolin exerts potent fungicidal activity through competitive inhibition of this lipid exchange process, thereby revealing its molecular mode of action and bridging a critical gap in oomycete ORPs biology.

2. Results

2.1. PsORP1 Is Essential for the Survival of P. sojae

Oomycete ORP1 is a long‐chain ORP containing multiple conserved domains: an N‐terminal PH domain, a central START domain, and a C‐terminal ORD. Notably, the START domain has not been identified in any other known ORPs from animals, plants, or fungi, highlighting its evolutionary distinctiveness in oomycete ORP1. Moreover, though most eukaryotic long‐chain ORPs possess a canonical FFAT motif that mediates interactions with ER‐anchored VAPs, this motif is conspicuously absent in Phytophthora ORP1. Intriguingly, the START domain of ORP1 harbours two predicted transmembrane segments, suggesting a potential role in membrane integration or subcellular localisation (Figure 1A). To explore the evolutionary relationship of ORPs among different biological groups, a phylogenetic analysis was conducted on ORPs from multiple species, including fungi, animals, plants, and oomycetes (Table S1). The ORP sequences of oomycetes had low similarity with the homologous protein sequences of other eukaryotes and formed an independent branch on the phylogenetic tree, indicating a distinct evolutionary branch (Figure 1B).

FIGURE 1.

FIGURE 1

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Structural classification and phylogenetic analysis of ORP family proteins. (A) Schematic diagram illustrating the domain architectures of representative ORP subfamilies, with key structural domains labelled. Roman numerals denote distinct ORP subfamilies with high sequence homology. (B) Maximum‐likelihood phylogenetic tree of ORP homologues from oomycetes, Arabidopsis thaliana , Saccharomyces cerevisiae , Homo sapiens and other organisms. Bootstrap values are based on 1000 replicates labelled on each branch, and different background colours correspond to distinct taxonomic groups. The phylogenetic tree was constructed using MEGA‐X software, and iTOL was applied only for tree visualisation and annotation. Detailed information of all ORP protein sequences used in this study is summarised in Table S1.

We attempted to generate a homozygous knockout mutant of PsORP1 using conventional CRISPR/Cas9‐mediated gene editing; however, no homozygous transformants were obtained (data not shown). However, we generated three PsORP1‐inducible transformants in P. sojae using a Tet‐on/CRISPR‐Cas9 inducible expression system (Li et al. 2025) in which the native promoter of PsORP1 was successfully replaced with an inducible promoter (Figure S1A,B). In the absence of inducer doxycycline (Dox), the silencing efficiency of PsORP1 exceeded 90% in three inducible expression transformants (Figure S1C). Their hyphal growth was severely suppressed (inhibition rate > 90%; Figure 2A), and they failed to produce sporangia, zoospores, and oospores (Figure 2B–D). Upon Dox addition, PsORP1 expression significantly increased to approximately 40% of normal levels (Figure S1C). Following the restoration of PsORP1 expression, the transformants exhibited partial recovery in hyphal growth and the production of sporangia, zoospores, and oospores. Zoospore production in all three transformants and sporangium production in the siORP1‐T3 transformant were restored to wild‐type levels (Figure 2B,C). The above results indicate that PsORP1 is a crucial functional protein involved in the growth and asexual/sexual development of P. sojae.

FIGURE 2.

FIGURE 2

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Phenotypic characterisation of PsORP1‐inducible expression transformants. (A) Mycelial colony morphology (scale bar, 2 cm) and growth rate of the indicated strains cultured on V8 medium at 25°C for 8 days. (B–D) Micrographs and number of sporangia (B), zoospores (C), and oospores (D) in PsORP1‐induced mutants compared to the wild‐type (Phytophthora sojae P6497) and control (CK) strains, with or without doxycycline (Dox) induction (scale bars, 100 μm). For all bar charts, data points are grouped by colour to represent three biological replicates, with total sample sizes of n = 18 (A, C, D) or n = 27 (B). Data are presented as mean ± SD. Ordinary one‐way ANOVA and Dunnett's multiple comparisons test were used, with p ≤ 0.05 indicating significance.

2.2. PsORP1 Specifically Localises to ER–Golgi MCSs

ORPs typically function at MCSs between distinct organelles (Mesmin et al. 2013; Olkkonen and Li 2013; Chung et al. 2015; Pietrangelo and Ridgway 2018; Kawasaki et al. 2022). To investigate the localisation of PsORP1, we constructed GFP‐tagged PsORP1 and co‐expressed it with the early endosome marker mCherry‐fused Rab5, late endosome marker Rab7, mitochondrial marker ATPase, Golgi marker MAN, and ER marker Sec61β in P. sojae (Li et al. 2024; Ah‐Fong and Judelson 2011; Zhu et al. 2018). Confocal fluorescence microscopy revealed partial co‐localisation of PsORP1 with the ER and Golgi, suggesting potential MCS involvement (Figure S2; Figure 3A,B). To specifically test targeting to ER–Golgi MCSs, we engineered an MCS reporter (PH‐FFAT‐mCherry) (De Matteis and Rega 2015) for co‐expression with PsORP1 variants (Figure S3). Co‐localisation analysis demonstrated significant enrichment of full‐length PsORP1 at ER–Golgi MCSs (Figure 3C). Strikingly, deletion of the ORD had no effect on the MCS localisation of PsORP1 (Figure 3D,E).

FIGURE 3.

FIGURE 3

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Subcellular localisation of PsORP1 and its OSBP‐related domain (ORD)‐truncated mutant PsORP1∆ORD in Phytophthora sojae. (A and B) Co‐localisation of PsORP1‐GFP with Golgi marker MAN‐mCherry (A) and ER marker Sec61β‐mCherry (B) in wild‐type strain P6497 (scale bar, 10 μm). (C and D) Co‐localisation of PsORP1‐GFP (C) and PsORP1∆ORD‐GFP (D) with ER–Golgi membrane contact site marker PH‐FFAT‐mCherry in wild‐type strain P6497 (scale bar, 10 μm). The outlined regions in the images are magnified on the right and the white arrow indicates the region used for the fluorescence intensity profile indicated in the graph to the right of the images. (E) Pearson's correlation coefficient for PsORP1‐GFP and its splice variants with PH‐FFAT‐mCherry (ER–Golgi MCSs marker protein). Data represent mean ± SD, n = 65 (PsORP1‐GFP), n = 81 (PsORP1∆ORD‐GFP) regions of interest (ROI) were selected from 3 trials. t‐test was used, and p > 0.05 represent non‐significant.

2.3. ORD of PsORP1 Mediates PS/PI4P Counter‐Transport at ER–Golgi MCSs

PsORP1 possesses the hallmark lipid transfer ORD, but its specific lipid cargo remains to be firmly established. To identify which lipids serve as cargo for PsORP1, we successfully purified the recombinant ORD of PsORP1 and characterised its lipid‐binding attributes by microscale thermophoresis (MST) and lipid dot‐blot screen assays. Results showed that the ORD can bind phosphatidylserine (PS), phosphatidylinositol 4‐phosphate (PI4P), PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 (Figure S4; Figure 4A–D). Further comparative analysis of ORP homologues capable of binding PS and PI4P revealed that the residue clusters mediating this specific PI4P and PS binding in other ORDs (Chung et al. 2015; Moser von Filseck et al. 2015) were also conserved in the ORD of PsORP1 (Figure 4E). To determine the functional significance of these conserved sites, we purified the ORD containing targeted mutations and quantified lipid‐binding affinities. Mutations in PS‐binding residues (ORDM‐PS, L656D, K713/N716A) impaired interaction with PS, increasing the dissociation constant more than 100‐fold (Figure 4A,C). Similarly, PI4P‐binding mutations (ORDM‐PI4P, H740/741/K936A) reduced affinity, with the dissociation constant increasing from 4.7 to 38.2 μM (Figure 4B,D). To further validate the functional importance of the PS/PI4P‐binding and transfer activities of the ORD, we overexpressed PsORP1 variants carrying point mutations in the PS‐ or PI4P‐binding sites (designated PsORP1M‐PS and PsORP1M‐PI4P, respectively) in the PsORP1‐inducible expression transformants. Notably, neither of these mutant variants was able to rescue the phenotypic defects, demonstrating that the ORD‐mediated lipid‐binding is indispensable for the biological function of PsORP1 (Figure 4F–I; Figure S5).

FIGURE 4.

FIGURE 4

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OSBP‐related domain (ORD) binds phosphatidylinositol 4‐phosphate (PI4P) and phosphatidylserine (PS) through a conserved binding mechanism. (A, B) Binding affinity of the ORD for PS (A) and PI4P (B) determined by microscale thermophoresis. ORD/OX denotes the binding measured in the presence of fungicide oxathiapiprolin. Data represent mean ± SEM from three independent assays. (C, D) Lipid dot‐blot assays show the binding of the recombinant ORD to PS (C) and PI4P (D), with phosphatidylcholine (PC) as a control. The ORDM‐PS (L656D, K713A, N716A) and ORDM‐PI4P (H740/741A, K936A) mutants exhibited impaired binding to PS and PI4P, respectively. (E) Amino acid sequences involved in recognition of the head group of PI4P and PS in PsORP1 are aligned with corresponding sequences HmORP5, HmORP8 ( Homo sapiens ), ScOSH6, and ScOSh7 ( Saccharomyces cerevisiae ). Sequence conservation figures were generated from multiple sequence alignment using Jalview software. Residues that directly contact PS (red) and PI4P (green) are indicated by triangles. (F–I) Mycelial colony growth rate (F), sporangia number (G), zoospore number (H), and oospore number (I) from complemented strains expressing PsORP1‐GFP, PsORP1M‐PS‐GFP, and PsORP1M‐PI4P‐GFP on the PsORP1‐inducible expression background compared with wild‐type (P6497) and the PsORP1‐inducible expression transformant. For all bar charts, data points are grouped by colour to represent three biological replicates, with total sample sizes of n = 18 (F, H, I) or n = 27 (G). Data are presented as mean ± SD. Ordinary one‐way ANOVA and Dunnett's multiple comparisons test were used, with p ≤ 0.05 indicating significance.

These findings prompted us to investigate the transport activity of PsORP1 towards its ligands PI4P and PS. To assess the transport of lipids, we performed an in vitro lipid transport assay as described previously (Chung et al. 2015; Tan and Finkel 2022). For the ORD‐mediated PS transport assays, the donor liposomes contained 93% DOPC, 5% 18:1–12:0 NBD‐PS, and 2% Rhod‐PE, the acceptor liposomes contained 96% DOPC and 4% PI4P, and the neutral acceptor liposomes contained 100% DOPC. The fluorescence of NBD‐PS was quenched due to FRET with Rhod‐PE. Adding ORD would result in dequenching if the proteins can transport PS to LB liposomes (Figure 5A,B).

FIGURE 5.

FIGURE 5

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OSBP‐related domain (ORD) mediates PI4P/PS transport between liposomes. Schematic of the assay employed to examine phosphatidylserine (PS) (A, B) and phosphatidylinositol 4‐phosphate (PI4P) (C, D) transport by the ORD. PS transport was measured by injecting the protein into a suspension of LA liposomes containing 5% NBD‐PS and 2% Rhod‐PE, mixed with LB liposomes (with 0 or 4% PI4P). PI4P transport was measured by adding the protein to LA liposomes containing 4% PI4P and 2% Rhod‐PE, mixed with NBD‐PHFAPP1 and LB liposomes (with or without 5% PS). Changes in the NBD fluorescence signals serve as a reporter for lipid transport activity. Data represent mean ± SEM from three independent assays.

For PI4P transport assays, NBD‐PHFAPP1 was mixed with two liposome populations: the donor liposome containing 94% DOPC, 4% PI4P, and 2% Rhod‐PE; and the acceptor liposome containing either 100% DOPC alone or 95% DOPC and 5% PS. The fluorescence of NBD‐PHFAPP1 was quenched when bound to donor liposomes through PI4P due to FRET with Rhod‐PE. Adding the ORD would result in dequenching if the proteins are capable of transporting PI4P to acceptor liposomes (Figure 5C,D).

In the case of PsORP1, PS transport by its ORD to acceptor liposomes containing only PC was minimal, whereas transport to PI4P‐containing acceptor liposomes was significantly enhanced (Figure 5B). Similarly, PI4P transport increased substantially when acceptor liposomes contained the reciprocal ligand PS (Figure 5D). Mutations in either the PS‐ or PI4P‐binding sites markedly impaired transport of their cognate lipids (Figure 5B,D). These results clearly demonstrate that ORD is able to transport PI4P and PS between liposomes. Furthermore, the enhanced transport of a lipid due to the presence of another lipid in acceptor liposomes, which is indicative of counter‐transport activity by the ORD, suggests a possible role of PsORP1 as a lipid exchanger (Figure 5B,D).

2.4. Oxathiapiprolin Disrupts ORD‐Dependent PS and PI4P Transfer by PsORP1

Previous studies have shown that point mutations in the ORD domain of ORP1 in Phytophthora spp. can confer high‐level resistance to oxathiapiprolin (Miao et al. 2020). Therefore, we speculated that oxathiapiprolin could exert its fungicidal activity by interfering with the function of the ORD. Our MST assays demonstrated that oxathiapiprolin indeed impaired the binding of the ORD to both PS and PI4P (Figure 4A,B). We hypothesised that the compound competitively binds to the ORD, thereby inhibiting ORD‐mediated transport of PS and PI4P. Subsequent in vitro lipid transport assays confirmed that oxathiapiprolin treatment significantly reduced the ORD‐dependent transport efficiency for both PS and PI4P (Figure 5B,D).

To validate these findings in vivo, we co‐expressed a GFP‐tagged PS biosensor with an mCherry‐labelled ER marker (Figure 6A) alongside a GFP‐tagged PI4P biosensor with a Golgi marker (Figure 6B). This approach enabled quantitative assessment of the accumulation of PS on the ER and accumulation of PI4P on the Golgi apparatus following oxathiapiprolin treatment. The results revealed elevated PS levels associated with the ER and increased PI4P accumulation on the Golgi upon exposure to oxathiapiprolin, indicating impaired trafficking of these lipids between their respective organelles (Figure 6C,D). Collectively, these data demonstrate that oxathiapiprolin disrupts the transport of both PS and PI4P by competitively inhibiting the function of the ORD, underlying its exceptional efficacy against oomycete pathogens.

FIGURE 6.

FIGURE 6

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Oxathiapiprolin blocks phosphatidylinositol 4‐phosphate (PI4P)/phosphatidylserine (PS) counter‐transport. (A, B) Subcellular localisation of phosphatidylserine (PS) sensor Lact‐C2‐GFP with endoplasmic reticulum (ER) marker Sec61β‐mCherry or PI4P sensor 2 × PHFAPP1‐GFP with Golgi marker MAN‐mCherry in wild‐type strain P6497, with or without oxathiapiprolin (scale bar, 10 μm). The outlined regions are magnified on the right and the white arrow indicates the region used for the fluorescence intensity profile indicated in the graph to the right of the images. (C) Pearson's correlation coefficient for Lact‐C2‐GFP with Sec61β‐mCherry in wild‐type strain P6497, with or without oxathiapiprolin. Data represent mean ± SD; n = 178 (dimethyl sulphoxide [DMSO]) and n = 187 (oxathiapiprolin) regions of interest selected from three trials. t‐test was used with p ≤ 0.05 indicating significance. (D) Pearson's correlation coefficient for 2 × PHFAPP1‐GFP with MAN‐mCherry in wild‐type strain P6497, with or without oxathiapiprolin. Data represent mean ± SD; n = 108 (DMSO) and n = 126 (oxathiapiprolin) regions of interest selected from three trials. t‐test was used with p ≤ 0.05 indicating significance.

3. Discussion

In this study, we comprehensively characterised PsORP1 derived from P. sojae, covering its structural architecture, evolutionary trajectory, biological function, subcellular localisation and lipid transport activity. Furthermore, we mechanistically deciphered the inhibitory mechanism of oxathiapiprolin, an oomycete‐specific fungicide, which selectively targets PsORP1 to constrain pathogen proliferation. Our results identify PsORP1 as an indispensable lipid transporter localised at ER–Golgi MCSs, which mediates counter‐transport of PS and PI4P. Critically, we validated that oxathiapiprolin directly interferes with PS/PI4P exchange mediated by the PsORP1 ORD, thereby disrupting intracellular lipid homeostasis of P. sojae. Collectively, these findings establish a refined mechanistic framework for the rational design and structural optimisation of next‐generation oomycete‐selective fungicides.

ORP family proteins are conserved lipid transfer proteins widely distributed in eukaryotes, and canonical long‐chain ORPs from animals, plants, and fungi share unified domain composition and functional motifs (Raychaudhuri and Prinz 2010; Mesmin et al. 2013; Olkkonen and Li 2013; Chung et al. 2015; Pietrangelo and Ridgway 2018; Kawasaki et al. 2022). Our structural and phylogenetic analysis verified that oomycete ORP1 is an evolutionarily unique clade distinct from ORPs of other eukaryotic kingdoms. Different from reported eukaryotic long‐chain ORPs, Phytophthora ORP1 lacks the classic FFAT motif responsible for binding ER‐anchored VAP proteins, which is a typical structural variation of oomycete ORP1. Notably, the START domain exclusive to oomycete ORP1 is not annotated in any known ORP homologues across eukaryotes, and this domain carries two predicted transmembrane segments. Given that deletion of the ORD did not alter PsORP1 localisation, we reasoned that its subcellular targeting likely relies on the PH domain, START domain, and the transmembrane motif. This differs from the MCS targeting pattern of conventional eukaryotic ORPs that rely on FFAT‐PVAP interaction, revealing a novel MCS localisation mechanism mediated by PH‐START dual domains in oomycete ORP1, which is a key evolutionary adaptation of pathogenic oomycetes to adapt to intracellular lipid transport modes.

In most eukaryotes, the ORP family comprises numerous members with functional redundancy and overlap, which are critical for cell growth and survival. Accordingly, deletion of all four ORP genes in Caenorhabditis elegans is embryonic lethal upon cholesterol restriction (Kobuna et al. 2010). Similarly, yeast cells lacking the seven Osh proteins are not viable (Beh et al. 2001; Beh and Rine 2004; Beh et al. 2012). Notably, deletion of individual ORPs maintains viability; loss of any single ORP among the five homologues in Aspergillus nidulans merely reduces conidiation (Takeshita et al. 2008), and in S. cerevisiae , single deletions affect endocytosis, polarised secretion, PI4P metabolism, and sterol organisation in the plasma membrane (PM) (Beh et al. 2001; Beh and Rine 2004; Kobuna et al. 2010; Beh et al. 2012). However, Phytophthora has undergone significant contraction of the ORP repertoire, retaining only ORP1 and ORP2. Genetic evidence, including the inability to generate homozygous PsORP1 knockout mutants and near‐lethal phenotypes from its inducible silencing, establishes ORP1 as indispensable for oomycete viability. In contrast, the deletion of PsORP2 has no discernible impact, though its potential role in lipid metabolism remains to be elucidated. Notably, PsORP1 is not transcriptionally up‐regulated in ΔPsORP2 mutants, arguing against obvious transcriptional‐level compensatory effects (Miao et al. 2018). Nevertheless, we cannot fully exclude weak or condition‐dependent functional redundancy between PsORP1 and PsORP2 under untested environmental stresses. These observations suggest that ORP1 may have integrated or supplanted core functions performed by multiple ORPs in other eukaryotes during evolution. Consequently, ORP1 has become a central hub and vulnerable node within the oomycete lipid homeostasis network whose functional indispensability makes it an ideal target for highly selective fungicides.

ORPs primarily mediate the counter‐transport of two distinct lipid species across organelle membranes. ORP3 targets the PM via the binding of its PH domain to PI(4,5)P2, catalysing calcium‐dependent PI4P/PC exchange (Weber‐Boyvat et al. 2015). ORP5 and ORP8 mainly mediate lipid exchange between the ER and PM, transporting PS synthesised in the ER to the PM while retrieving PI4P or PI(4,5)P2 in the opposite direction (Chung et al. 2015; Ghai et al. 2017). ORP9, ORP10, and ORP11 participate in the reciprocal transport of PS and PI4P between the ER and Golgi apparatus (He et al. 2023; Cabukusta et al. 2024). In this study, we demonstrated that the ORD of PsORP1 possesses intrinsic PS/PI4P transport activity, as evidenced by in vitro reconstitution assays. Together with its specific localisation at ER–Golgi MCSs, we propose that PsORP1 regulates organelle function by mediating PS delivery to the Golgi and PI4P retrieval to the ER in vivo. This hypothesis is further supported by liposome‐based transport assays, which show that oxathiapiprolin specifically inhibits ORD‐mediated lipid transfer. Critically, direct tracing of in vivo lipid dynamics confirmed that oxathiapiprolin blocks PS efflux from the ER and PI4P recycling from the Golgi, thereby providing a molecular basis for its antifungal activity. Although our data confirm the transport capacity of the ORD in vitro, direct visualisation and quantitative analysis of ORP1‐mediated lipid fluxes in living cells remain technically challenging. Currently, no method exists for real‐time quantitative monitoring of PS/PI4P transport kinetics between the ER and Golgi, a limitation that hampers precise evaluation of ORP1's transport efficiency and its contribution to cellular lipid homeostasis. Furthermore, although the inhibitory effect of oxathiapiprolin on lipid transport has been validated, the downstream physiological cascade it triggers remains to be further investigated. Perturbed PS/PI4P homeostasis at ER–Golgi membrane contact sites may alter membrane lipid composition and identity, destabilise MCS architecture, and impair vesicle trafficking and membrane remodelling (Thanh et al. 2020; Kim and Burd 2023). Such lipid‐ and membrane‐associated abnormalities likely underpin the defects in hyphal growth, sporangium formation, zoospore release and oospore development observed in PsORP1‐silenced strains. Nevertheless, the complete molecular cascade by which lipid network perturbation leads to developmental failure in P. sojae remains to be fully elucidated. Beyond its canonical PS/PI4P counter‐transport activity, PsORP1‐ORD displays affinity for PI(3,4)P2 and PI(4,5)P2. Such binding interactions may confer lipid‐sensing and allosteric regulatory capabilities on PsORP1, as observed for human ORP1L (Dong et al. 2019); however, the physiological relevance of these properties in oomycetes remains uncharacterised.

In summary, this study constructs a complete functional model of PsORP1: the evolutionarily unique PH‐START module anchors PsORP1 to ER–Golgi MCSs, and the conserved ORD mediates bidirectional PS/PI4P counter‐transport to maintain intracellular lipid homeostasis, which is essential for the growth, asexual, and sexual reproduction of P. sojae. Oxathiapiprolin competitively inhibits ORD lipid transport activity to break organellar lipid balance and exert antifungal activity. Although our study has precisely defined the functions of oomycete ORP1, we still lack high‐resolution three‐dimensional structures of full‐length ORP1 protein and its multidomain complexes. Without such structural data, our understanding of key details, including the precise spatial arrangement of domains, presence of allosteric regulation, and specific insertion mode of transmembrane helices into the ER membrane, remains largely inferential. Furthermore, directly observing and quantifying the ORP1‐mediated lipid transport rate and flux in live oomycete hyphae remains extremely challenging. Although PS and PI4P accumulation following drug inhibition provides strong indirect evidence, precise quantification of transport kinetics continues to pose a technical hurdle in the field. Notably, it remains unclear whether the lipid‐binding specificity and transport mode of the START domain allosterically regulate the lipid exchange activity of the ORD. Future research could focus on resolving the crystal structure of the oxathiapiprolin–ORD complex and employing structure‐based strategies to modify lead compounds for the development of novel fungicides with enhanced affinity and resistance‐breaking properties. Meanwhile, targeting the oomycete‐specific START domain for fungicide discovery, in combination with ORD‐directed inhibitors, would form a synergistic system that broadens the arsenal of fungicidal mechanisms and mitigates the emergence of field resistance in oomycete pathogens.

4. Experimental Procedures

4.1. Strains, Culture, and Special Reagents

The P. sojae P6497 strain was used as the wild‐type strain in this study. All strains were cultured on 10% V8 vegetable medium in the dark at 25°C. Oxathiapiprolin (Corteva Agriscience) was dissolved in dimethyl sulphoxide (DMSO) and stored in aliquots at 4°C. We used 4% paraformaldehyde (PFA) solution (Thermo Fisher Scientific, J19943.K2) for tissue fixation. We obtained 18:1‐12:0 NBD‐PS (catalogue no. 810195P), 18:1 Liss Rhod PE (catalogue no. 810133P), DOPC (catalogue no. 850375P), DOPE (catalogue no. 850725P), DOPS (catalogue no. 840035P), 18:1‐12:0 NBD‐PC (catalogue no. 810195P), 18:1/18:1 PI3P, brain PI4P (catalogue no. 840045P), brain PI(4,5)P2 (catalogue no. 840046P), 18:1 PI(3,4,5)P3 (catalogue no. 850156P), 18:1 PI(3,4)P2 (catalogue no. 850153P), and 18:1 PI5P (catalogue no. 850151P) from Avanti Polar Lipids.

4.2. Plasmid Construction

The 1000 bp upstream and downstream regions of the PsORP1 promoter were amplified as homologous arms, which were then directionally assembled together with the synthesised PHam34‐rtTA‐THam34‐TetO‐Pmin cassette into the pBS SKII+ vector, constructing a replacement donor vector for the native PsORP1 promoter (Li et al. 2025). cDNAs encoding fragments of PsORP1‐GFP and its variants (PsORP1ΔORD‐GFP, PsORP1M‐PS‐GFP, PsORP1M‐PI4P‐GFP), MAN‐mCherry, Sec61‐mCherry, ATPase‐mCherry, Lact‐C2‐GFP, 2 × PHFAPP1‐GFP, VAMP‐mCherry, PH‐FFAT‐GFP, and PH‐FFAT‐BFP were subcloned into the pYF3 vector (resistant to G418). The coding sequences of PsORP1, Rab5, Rab7, MAN, Sec61, VAMP, and ATPase were amplified from P. sojae cDNA. Sequences for Lact‐C2 (the C2 domain of lactadherin), 2 × PHFAPP1 (two cDNAs encoding the PH domain of phosphatidylinositol‐4‐phosphate adapter protein‐1), and PH‐FFAT (residues 76–332 of human OSBP) (Chung et al. 2015; De Matteis and Rega 2015) were optimised based on the codon usage bias of P. sojae and custom‐synthesised by Beijing Tsingke Biotechnology Co. Ltd. For bacterial expression, cDNAs encoding ORDPsORP1 (amino acids 591–962) were cloned into pCold‐TF vector with an N‐terminal 6×His tag. PHFAPP1 mutant (amino acids 1–100) (C37S/C94S/T13C) was cloned into the pET22b vector with a C‐terminal 6×His tag. All primers and corresponding sequences used in vector construction are listed in Tables [Link], [Link].

4.3. Phytophthora sojae Transformation and Phenotype Analysis

Transformants were generated through polyethylene glycol (PEG)‐CaCl2‐mediated protoplast transformation and selected on V8 medium supplemented with 50 μg/mL G418. Genomic DNA was extracted from P. sojae mycelia using the CTAB method. For mycelial growth assessment, a 5 mm mycelial plug was inoculated onto 10% V8 agar and incubated at 25°C in the dark for 8 days. Oospore production was quantified under a microscope at 200× magnification after culturing the transformants on V8 agar plates for 10 days under the same conditions. Sporangia were counted after the plates had been rinsed 10 times with sterile water. Zoospore production was evaluated by adding 5 mL of sterile water to each plate following sporangium formation, with the number of zoospores per 1 μL counted microscopically. All experiments were performed with at least three biological replicates.

4.4. Total RNA Extraction and qPCR Analysis

Total RNA was isolated from freshly transformed hyphae using the SV Total RNA Isolation System (Promega) following the manufacturer's protocol. RNA integrity was verified by agarose gel electrophoresis, whereas concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Scientific). First‐strand cDNA was reverse‐transcribed from 1 μg of total RNA using PrimeScript Reverse Transcriptase (Takara). Quantitative PCR was carried out on a CFX Real‐Time PCR System (Bio‐Rad) with TB Green Fast qPCR Mix (Takara). Gene expression levels were normalised to the actin reference gene and calculated via the 2−ΔΔCt method, consistent with established protocols. All qPCR analyses were performed with three technical replicates from three biologically independent samples. Primer sequences and amplification efficiencies are provided in Table S2.

4.5. Confocal Imaging and Analysis

Transformants expressing fluorescently tagged target proteins were cultured in V8 liquid medium for 48 h with 50 μg/mL G418 and visualised using an LSM 900 laser scanning confocal microscope (Zeiss) at specific excitation and emission wavelengths (excitation wavelengths: GFP, 488 nm; mCherry, 561 nm; and BFP, 405 nm). Colocalisation analysis was quantified using the Coloc2 plug‐in in ImageJ. Pearson's coefficient (measuring correlation in variations between two channels) was used to assess the degree of colocalisation.

4.6. Protein Extraction and Immunoblot Analysis

Transformants were cultured in V8 liquid medium for 3 days and then total proteins extracted using the BestBio Thick‐Wall microbial protein extraction kit. Proteins were fractionated using 12% SDS‐PAGE and transferred to a PVDF membrane. Incubation with primary antibody (anti‐His, 1:5000, AB0005, Abways) was performed at 4°C overnight. The membrane was then washed and incubated with a secondary antibody. The signals were amplified by horseradish peroxidase (HRP)‐streptavidin and detected by chemiluminescent assays.

4.7. Recombinant Protein Expression and Purification

Plasmids encoding ORDPsORP1 (amino acids 591–962), M‐PSPsORP1 (amino acids 591–962) (L656D, K713/N716A), M‐PI4PPsORP1 (amino acids 591–962) (H740/741/K936A), and PHFAPP1 (amino acids 1–100) (C37S/C94S/T13C) were transformed into Escherichia coli BL21 (DE3) cells. Cells were grown in 5–10 L Luria Bertani broth at 37°C until OD600 reached 0.6–0.8. The temperature of the culture was brought down to 15°C and protein expression initiated by induction with 0.5 mM IPTG and allowed to grow at 15°C overnight. The cells were washed with phosphate‐buffered saline (PBS), pelleted, and resuspended in lysis buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10% glycerol, and protease inhibitor cocktail. Cells were sonicated and centrifuged at 17,000 g for 60 min. His‐tagged proteins from the supernatant were purified using Ni‐NTA agarose and eluted into elution buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10% glycerol, and 250 mM imidazole. Protein purity was verified by Coomassie‐blue‐stained SDS‐PAGE (Figure S6) and qualified peak fractions were aliquoted and stored at −80°C.

4.8. Protein Labelling

For NBD labelling of PHFAPP1 (amino acids 1–100) (C37S/C94S/T13C), the protein (2 mg/mL) was incubated with a 10‐fold molar excess of N,N′‐dimethyl‐N‐(iodoacetyl)‐N′‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl) ethylenediamine (Thermo Fisher Scientific, D2004) in labelling buffer (20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 5% glycerol, 1 mM Tris(2‐carboxyethyl)phosphine [TCEP]) for 16 h at 4°C in the dark. The free dye was removed using Amicon Ultra Centrifugal Filter 3 kDa MWCO (UFC9003, Millipore) and the labelled protein analysed by SDS‐PAGE and absorption spectroscopy.

4.9. MST

The phospholipid‐binding specificity and affinity of ORDORP1 and various mutants were investigated by MST. Lipids were dissolved in chloroform and dried under nitrogen gas without heating before being kept dry in a desiccator overnight at room temperature. The dried lipids were hydrated in PBST buffer 1 (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.005% Tween 20, pH 7.5) by vortexing for at least 3 h at room temperature and then centrifuged at 15,000 g for 10 min at room temperature to remove any possible remaining lipid pellet. The lipid solution was serially diluted 15 times in a 1:1 ratio with PBST buffer 1. All recombinant proteins were labelled with His‐Tag Labeling Kit RED‐Tris‐NTA 2nd Generation (Nano Temper, MO‐L018). The dye was diluted to 5 μM with PBST buffer 1, aliquoted, and stored at −20°C. A working solution of 100 nM was prepared before use. The target protein was diluted to 200 nM and mixed with an equal volume of the dye working solution. After incubation at room temperature for 30 min, the mixture was centrifuged, and the supernatant containing the labelled protein was collected. For MST assays, the diluted lipid solutions were mixed with the same volume of the labelled protein solution, and then the mixtures were loaded into capillaries. The assays were carried out using a Monolith NT.115 instrument. The thermophoresis signals and dissociation constants were analysed using MO.Affinity Analysis software.

4.10. Lipid Dot‐Blot Assay

Lipid dissolved in chloroform was spotted onto nitrocellulose filters and dried for 30 min at room temperature. The filters were preblotted in PBST buffer 2 (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 0.05% Tween 20, pH 8.0) containing 5% (w/v) fatty‐acid‐free bovine serum albumen (BSA) for 1 h, and then incubated with 2 μg/mL recombinant proteins for 2 h at room temperature. After washing thoroughly with PBST buffer 2, the filters were incubated with anti‐His antibody at 4°C overnight. The membrane was then washed and incubated with a secondary antibody. The signals were amplified by HRP‐streptavidin and detected using chemiluminescent assays.

4.11. Liposome Preparation

Lipids dissolved in chloroform were mixed, dried to thin films under nitrogen flow, and desiccated overnight. After further drying in a vacuum for 1 h, the lipid films were rehydrated in HKM buffer (50 mM HEPES, pH 7.2, 120 mM potassium acetate, 1 mM MgCl2) to obtain a suspension of multilamellar liposomes. The multilamellar liposome suspension was subjected to 10 freeze–thaw cycles using alternating liquid nitrogen and a 37°C water bath, and then extruded through a 100 nm filter 13 times. Liposomes were stored at 4°C in the dark and were used within 2 days.

4.12. Lipid‐Transfer Assays

Lipid transport was measured in vitro in 100 μL reaction buffer (50 mM HEPES, pH 7.2, 120 mM potassium acetate, 1 mM MgCl2) in a 96‐well plate using a FRET‐based assay. The donor and acceptor liposomes (200 μM total lipids for each) were mixed and the NBD fluorescence monitored (excitation at 460 nm, emission at 535 nm) upon the addition of 10 μM purified proteins. For ORD‐mediated PS transfer, donor liposomes (93% DOPC, 5% 18:1–12:0 NBD‐PS, 2% Rhod‐PE) were incubated with acceptor liposomes (96% DOPC, 4% PI4P) for 10 min, and the initial NBD signal (F 0) was recorded at 528 nm (excitation: 460 nm). Protein (10 μM) was then injected, and the NBD signal (F) was tracked for 60 min. Similarly, for PI4P transfer, NBD‐PHFAPP1 was mixed with donor liposomes (94% DOPC, 4% PI4P, 2% Rhod‐PE) and acceptor liposomes (95% DOPC, 5% PS) for 10 min before recording F 0. After the addition of protein (10 μM), NBD fluorescence was monitored for 60 min. To evaluate the effect of oxathiapiprolin on PS and PI4P transport, the protein was pre‐incubated with 10 μM oxathiapiprolin before being added to the liposome mixture. The NBD signal was then recorded as described. In all assays, the time‐dependent change in NBD emission (F/F 0) served as a quantitative measure of lipid transport.

4.13. Oxathiapiprolin Effects on PS/PI4P Transport In Vivo

The effects of oxathiapiprolin on PS and PI4P transport in vivo were determined by co‐expression of PS probe C2‐lact‐GFP with ER marker Sec61β‐mCherry and co‐expression of PI4P probe 2 × PHFAPP1‐GFP with Golgi marker MAN‐mCherry in P. sojae, respectively. The obtained transformants were cultured in V8 liquid medium supplemented with 50 μg/mL G418 for 48 h in the dark at 25°C, followed by a 2‐h treatment with oxathiapiprolin at the same temperature. Transformants were then immediately fixed in 4% PFA for 30 min. Fixed mycelium was washed three times with PBS and visualised using an LSM 900 laser scanning confocal microscope. Colocalisation between C2‐lact‐GFP and Sec61β‐mCherry, and between 2 × PHFAPP1‐GFP and MAN‐mCherry, was quantified using Pearson's correlation coefficient.

4.14. Statistical Analysis

Statistical analyses were performed in Prism 9 (GraphPad). One‐way ANOVA was performed with Dunnett's multiple comparisons test. Pairwise comparisons were performed using Student's t‐test.

Author Contributions

Qin Peng: investigation. Xiaofei Liu: writing – original draft, writing – review and editing, visualization, validation, investigation, formal analysis, data curation, resources, methodology, conceptualization, software. Haixia Wang: investigation. Guangda Shao: investigation. Chengcheng Li: conceptualization, methodology, investigation, formal analysis, data curation, writing – original draft. Xili Liu: conceptualization, methodology, writing – original draft, resources, supervision, funding acquisition. Jianqiang Miao: writing – review and editing, supervision, resources, methodology, funding acquisition, conceptualization.

Funding

This work was supported by the National Key R&D Program of China (Grant 2022YFD1401300), National Natural Science Foundation of China (32372602) and Natural Science Foundation of Shaanxi Province (Grant 2024JC‐YBQN‐0165).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Verification of PsORP1‐induced expression transformants. (A) Schematic of the PsORP1 promoter substitution strategy. The native PsORP1 promoter was replaced with a tetracycline‐inducible minimal promoter using a CRISPR/Cas9‐mediated gene editing system. (B) Validation of the transformants by gel electrophoresis of PCR products. The primer pairs for PCR were used at the positions labelled in (A). (C) Relative expression levels of PsORP1 in the wild‐type, CK, and PsORP1‐induced transformants as determined by RT‐qPCR.

MPP-27-e70331-s008.docx (655.8KB, docx)

Figure S2: PsORP1 subcellular localisation. (A–C) Co‐localisation of PsORP1‐GFP with early endosome marker Rab5‐mCherry (A), late endosome marker Rab7‐mCherry (B), and mitochondrial marker ATPase‐mCherry (C) in wild‐type strain P6497 (scale bar, 10 μm). The outlined regions are magnified on the right and the white arrow indicates the region used for the fluorescence intensity profile indicated in the graph to the right of the images.

MPP-27-e70331-s003.docx (858.2KB, docx)

Figure S3: Construction of an ER–Golgi membrane contact site (MCS) marker. (A) Schematic of the construction strategy. An artificial protein based on human OSBP was engineered to visualise ER–Golgi MCSs. Its dual‐targeting strategy relies on the PI4P‐binding PH domain for Golgi attachment and the VAP‐binding FFAT motif for ER association, confining it to the interface between these organelles. (B) Co‐localisation of ER–Golgi MCS marker PH‐FFAT‐GFP with ER membrane protein VAMP‐mCherry (scale bar, 10 μm). The outlined regions are magnified on the right and the white arrow indicates the region used for the fluorescence intensity profile indicated in the graph to the right of the images.

MPP-27-e70331-s010.docx (398.6KB, docx)

Figure S4: Identification of lipid ligands for the ORD of PsORP1. The binding affinity of the ORD for PI3P (A), PI5P (B), PI(3,4)P2 (C), PI(3,5)P2 (D), PI(4,5)P2 (E), and PI(3,4,5)P3 (F) was determined by microscale thermophoresis. Data represent mean ± SEM from three independent assays.

MPP-27-e70331-s006.docx (684.1KB, docx)

Figure S5: Phenotypic characterisation of the complemented mutants. (A–D) Mycelial colony morphology (A) (scale bar, 2 cm) and micrographs of sporangia (B) (scale bar, 100 μm), zoospores (C) (scale bar, 50 μm), and oospores (D) (scale bar, 100 μm) from complemented strains expressing PsORP1‐GFP, PsORP1M‐PS‐GFP (L656D, K713A, N716A), or PsORP1M‐PI4P‐GFP (H740/741A, K936A) on the PsORP1‐inducible expression background compared with wild‐type (P6497) and the PsORP1‐inducible expression transformant (siORP1‐T3).

MPP-27-e70331-s001.docx (2.8MB, docx)

Figure S6: Size‐exclusion chromatography profiles and Coomassie‐stained SDS‐PAGE analysis of recombinant proteins used for in vitro biochemical assays. (A–C) Gel‐filtration chromatograms of wild‐type ORD, ORDMPS and ORDMPI4P mutant proteins of PsORP1. (D, E) Size‐exclusion profiles and SDS‐PAGE of the PI4P biosensor PHFAPP1and PS biosensor C2Lact. Peak fractions were collected for microscale thermophoresis, lipid dot‐blot and liposome transfer assays.

MPP-27-e70331-s009.docx (621.6KB, docx)

Table S1: Accession numbers of proteins used in this study.

MPP-27-e70331-s004.docx (21.9KB, docx)

Table S2: Primers used in the Tet‐on/CRISPR‐Cas9‐induced expression system to obtain PsORP1‐induced expression transformants.

MPP-27-e70331-s002.docx (16.1KB, docx)

Table S3: Primers used to generate fluorescence constructs.

MPP-27-e70331-s007.docx (18KB, docx)

Table S4: Primers used for the construction of prokaryotic expression vectors.

MPP-27-e70331-s011.docx (14.6KB, docx)

Table S5: Sequences of Lact‐C2, PH‐FFAT and 2 × PHFAPP1.

MPP-27-e70331-s005.docx (15.9KB, docx)

Acknowledgements

We appreciate Prof. Xin Bian's (Nankai University) valuable suggestions and discussion.

Contributor Information

Jianqiang Miao, Email: mjq2018@nwafu.edu.cn.

Xili Liu, Email: seedling@nwafu.edu.cn.

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.

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Associated Data

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Supplementary Materials

Figure S1: Verification of PsORP1‐induced expression transformants. (A) Schematic of the PsORP1 promoter substitution strategy. The native PsORP1 promoter was replaced with a tetracycline‐inducible minimal promoter using a CRISPR/Cas9‐mediated gene editing system. (B) Validation of the transformants by gel electrophoresis of PCR products. The primer pairs for PCR were used at the positions labelled in (A). (C) Relative expression levels of PsORP1 in the wild‐type, CK, and PsORP1‐induced transformants as determined by RT‐qPCR.

MPP-27-e70331-s008.docx (655.8KB, docx)

Figure S2: PsORP1 subcellular localisation. (A–C) Co‐localisation of PsORP1‐GFP with early endosome marker Rab5‐mCherry (A), late endosome marker Rab7‐mCherry (B), and mitochondrial marker ATPase‐mCherry (C) in wild‐type strain P6497 (scale bar, 10 μm). The outlined regions are magnified on the right and the white arrow indicates the region used for the fluorescence intensity profile indicated in the graph to the right of the images.

MPP-27-e70331-s003.docx (858.2KB, docx)

Figure S3: Construction of an ER–Golgi membrane contact site (MCS) marker. (A) Schematic of the construction strategy. An artificial protein based on human OSBP was engineered to visualise ER–Golgi MCSs. Its dual‐targeting strategy relies on the PI4P‐binding PH domain for Golgi attachment and the VAP‐binding FFAT motif for ER association, confining it to the interface between these organelles. (B) Co‐localisation of ER–Golgi MCS marker PH‐FFAT‐GFP with ER membrane protein VAMP‐mCherry (scale bar, 10 μm). The outlined regions are magnified on the right and the white arrow indicates the region used for the fluorescence intensity profile indicated in the graph to the right of the images.

MPP-27-e70331-s010.docx (398.6KB, docx)

Figure S4: Identification of lipid ligands for the ORD of PsORP1. The binding affinity of the ORD for PI3P (A), PI5P (B), PI(3,4)P2 (C), PI(3,5)P2 (D), PI(4,5)P2 (E), and PI(3,4,5)P3 (F) was determined by microscale thermophoresis. Data represent mean ± SEM from three independent assays.

MPP-27-e70331-s006.docx (684.1KB, docx)

Figure S5: Phenotypic characterisation of the complemented mutants. (A–D) Mycelial colony morphology (A) (scale bar, 2 cm) and micrographs of sporangia (B) (scale bar, 100 μm), zoospores (C) (scale bar, 50 μm), and oospores (D) (scale bar, 100 μm) from complemented strains expressing PsORP1‐GFP, PsORP1M‐PS‐GFP (L656D, K713A, N716A), or PsORP1M‐PI4P‐GFP (H740/741A, K936A) on the PsORP1‐inducible expression background compared with wild‐type (P6497) and the PsORP1‐inducible expression transformant (siORP1‐T3).

MPP-27-e70331-s001.docx (2.8MB, docx)

Figure S6: Size‐exclusion chromatography profiles and Coomassie‐stained SDS‐PAGE analysis of recombinant proteins used for in vitro biochemical assays. (A–C) Gel‐filtration chromatograms of wild‐type ORD, ORDMPS and ORDMPI4P mutant proteins of PsORP1. (D, E) Size‐exclusion profiles and SDS‐PAGE of the PI4P biosensor PHFAPP1and PS biosensor C2Lact. Peak fractions were collected for microscale thermophoresis, lipid dot‐blot and liposome transfer assays.

MPP-27-e70331-s009.docx (621.6KB, docx)

Table S1: Accession numbers of proteins used in this study.

MPP-27-e70331-s004.docx (21.9KB, docx)

Table S2: Primers used in the Tet‐on/CRISPR‐Cas9‐induced expression system to obtain PsORP1‐induced expression transformants.

MPP-27-e70331-s002.docx (16.1KB, docx)

Table S3: Primers used to generate fluorescence constructs.

MPP-27-e70331-s007.docx (18KB, docx)

Table S4: Primers used for the construction of prokaryotic expression vectors.

MPP-27-e70331-s011.docx (14.6KB, docx)

Table S5: Sequences of Lact‐C2, PH‐FFAT and 2 × PHFAPP1.

MPP-27-e70331-s005.docx (15.9KB, docx)

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.

Articles from Molecular Plant Pathology are provided here courtesy of Wiley

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