A phosphoinositide 5-phosphatase from Solanum tuberosum is activated by PAMP-treatment and may antagonize phosphatidylinositol 4,5-bisphosphate at Phytophthora infestans infection sites.

Potato (Solanum tuberosum) plants susceptible to late blight disease caused by the oomycete Phytophthora infestans display enhanced resistance upon infiltration with the pathogen-associated molecular pattern (PAMP), Pep-13. Here, we characterize a potato gene similar to Arabidopsis 5-phosphatases which was identified in transcript arrays performed to identify Pep-13 regulated genes, and termed StIPP. Recombinant StIPP protein specifically dephosphorylated the D5-position of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ) in vitro. Other phosphoinositides or soluble inositolpolyphosphates were not converted. When transiently expressed in tobacco (Nicotiana tabacum) pollen tubes, a StIPP-YFP fusion localized to the subapical plasma membrane and antagonized PtdIns(4,5)P2 -dependent effects on cell morphology, indicating in vivo functionality. P. infestans -infection of N. benthamiana leaf epidermis cells resulted in relocalization of StIPP-GFP from the plasma membrane to the extra-haustorial membrane. Colocalizion with the effector protein RFP-AvrBlb2 at infection sites is consistent with a role of StIPP in the plant-oomycete interaction. Correlation analysis of fluorescence distributions of StIPP-GFP and biosensors for PtdIns(4,5)P2 or phosphatidylinositol 4-phosphate (PtdIns4P) indicate StIPP activity predominantly at the extrahaustorial membrane. In Arabidopsis protoplasts, expression of StIPP resulted in the stabilization of the PAMP receptor, FLAGELLIN-SENSITIVE 2, indicating that StIPP may act as a PAMP-induced and localized antagonist of PtdIns(4,5)P2 -dependent processes during plant immunity.


Introduction
The hemibiotrophic oomycete Phytophthora infestans is the causal agent of late blight, the most devastating potato (Solanum tuberosum) disease worldwide. Attempts to generate resistant potato plants have previously concentrated on introducing resistance genes from wild species into cultivated potato (Fry, 2008). In addition, enhanced resistance can be induced in susceptible plants by treatment with chemicals such as b-amino butyric acid (BABA) (Cohen, 2002) or with the pathogen-associated molecular pattern (PAMP), Pep-13 (Brunner et al., 2002;Halim et al., 2004). The establishment of both types of resistance is dependent on salicylic acid (SA), as transgenic potato plants unable to accumulate SA fail to mount the induced resistance response (Halim et al., 2009;Eschen-Lippold et al., 2010). Pep-13-activated defense responses, moreover, require jasmonic acid (Halim et al., 2009).
The oligopeptide Pep-13 originates from an extracellular transglutaminase from Phytophthora species, and as a PAMP activates a multicomponent immune response (Brunner et al., 2002). Infiltration of potato leaves with Pep-13 leads to the accumulation of SA and jasmonic acid, the activation of defense genes and to hypersensitive cell death (Halim et al., 2009). An inactive analog of Pep-13, the peptide W2A, does not induce these responses (Brunner et al., 2002). To elucidate the downstream mechanisms of Pep-13-mediated resistance, we have previously identified Pep-13-activated genes by microarray analyses (Landgraf et al., 2014). Functional characterization of selected candidate genes revealed a contribution of vesicle trafficking processes to the defense against P. infestans (Eschen-Lippold et al., 2012), consistent with earlier work from the Arabidopsis model (Collins et al., 2003). Based on data from Arabidopsis, the interplay of secretory and endosomal pathways contributes to controlling the abundance of immune receptors at the plasma membrane (Ben Khaled et al., 2015), as receptors are actively internalized upon activation and degraded (Robatzek et al., 2006). At the same time, activation of the immune response induces the secretory pathway and the delivery of receptors to the plasma membrane (Saeed et al., 2019;Wang et al., 2020). Vesicle trafficking is also important for the secretion of antimicrobial compounds and of callose (Schulze-Lefert, 2004;Lipka et al., 2007), and transgenic potato plants with reduced expression of SYNTAXIN-RELATED 1 (SYR1) display altered membrane trafficking and altered defense responses in response to penetration by P. infestans (Eschen-Lippold et al., 2012).
Several previous studies link phosphoinositides to plant responses to biotic stress. Suppressed accumulation of the soluble second messenger inositol 1,4,5-trisphosphate in transgenic Arabidopsis thaliana plants results in a changed Ca 2+ signature and in altered susceptibility against the pathogenic bacterium Pseudomonas syringae DC3000 (Hung et al., 2014). A role for phosphoinositides in plant pathogen defense was recently proposed based on the observation that a fluorescent reporter for PtdIns(4,5)P 2 as well as a PI4P 5-kinase mediating the biosynthesis of PtdIns(4,5)P 2 accumulated in extra-invasive hyphal membranes upon infection of Arabidopsis thaliana with the pathogenic fungus Colletotrichum higginsianum (Shimada et al., 2019). Moreover, Qin et al. (2020) recently identified PtdIns(4,5)P 2 as a susceptibility factor associating with the EHM after powdery mildew infection of Arabidopsis.
A pathogen-induced localized change in membrane phosphoinositide composition may influence the lifetime and abundance of plasma membrane proteins, which may benefit either plant or microbe. Regulation of membrane trafficking by phosphoinositides will also pertain to defense-related membrane proteins and receptors for PAMPs, such as FLAGELLIN-SENSITIVE 2 (FLS2), which are inserted into their target membranes by secretion and are recycled by endocytosis, ending their plasma membrane lifetime (Robatzek et al., 2006;Wang et al., 2020). A number of plasma membrane proteins, such as the NADPH-oxidase RbohD, are activated upon PAMP perception (Aibara & Miwa, 2014;Kadota et al., 2014), and it might weaken an acute defense response to recycle these activated proteins at a constant rate. We hypothesized that modulation of phosphoinositides and membrane trafficking during responses to pathogen attack would contribute to the transient stabilization of activated defense proteins at the cell surface. In support of this notion, we recently demonstrated that the inhibition of the PI4P 5-kinase PIP5K6 upon perception of the bacterial PAMP flg22 results in reduced endocytosis of the NADPH-oxidase RbohD and other cargoes, correlating with increased production of reactive oxygen species in Arabidopsis (Menzel et al., 2019).
In our efforts to elucidate the role of vesicle trafficking in pathogen defense in potato, we identified a Pep-13-activated gene, which we predicted to encode an inositolpolyphosphate phosphatase (IPP) and termed StIPP. In Arabidopsis thaliana, IPPs are represented as several large gene families, including 5phosphatases (5-PTases) (Gillaspy, 2013;Gerth et al., 2017b) and SUPPRESSOR OF ACTIN (SAC) phosphatases (Gillaspy, 2013;Gerth et al., 2017b). Previously characterized IPPs display phosphatase activities against inositol-containing compounds, such as phosphoinositides or inositol polyphosphates, and are often promiscuous with regard to their accepted substrates (Gillaspy, 2013;Gerth et al., 2017b). While an influence of IPPs on Arabidopsis development has previously been reported (Berdy et al., 2001;Ercetin & Gillaspy, 2004;Gunesekera et al., 2007;Ercetin et al., 2008;Golani et al., 2013), it has remained largely unclear which relevant inositol-containing metabolites were the primary reason for these effects.
Here, we provide a detailed characterization of StIPP function. Our biochemical in vitro studies reveal an unusually specific preference of recombinant StIPP for dephosphorylating the D5-position of PtdIns(4,5)P 2 , a phosphoinositide with well-characterized roles in plant membrane trafficking. The subsequent cell biological analyses support in vivo functionality of StIPP in plant cells and specifically at P. infestans infection sites, likely serving as an antagonist of PtdIns(4,5)P 2dependent processes that are part of the interaction of the host plant with the oomycete.
A StIPP-RNAi fragment was cloned from cDNA using the primers 5 0 -CACCAACTTCGTCTCATCTTTGCATC-3 0 and 5 0 -CTTCTGATTAACCAACCCAATC-3 0 . The resulting 312 bp fragment was cloned into the pENTR TM /D-TOPO ® vector and subsequently into the binary vector pHellsgate12 (Wesley et al., 2001) by LR recombination. This was used for Agrobacterium tumefaciens AGL0-mediated transformation into S. tuberosum cv D esir ee plants.

Expression of StIPP in Escherichia coli and preparation of protein samples
pDEST-N112-StIPP or the empty vector coding for maltose binding protein (MBP) only were transferred into Escherichia coli Rosetta gami cells. Protein expression was induced with 1 mM IPTG. After resuspension in the presence of lysozyme and Halt TM protease inhibitor cocktail (Thermo Fisher Scientific), cells were lysed by sonication and centrifuged. The cleared lysate was used for enzyme assays.

Cultivation and treatment of plants
Growth and treatment of potato plants (S. tuberosum cv D esir ee) was performed as described (Dobritzsch et al., 2016).

Transformation of tobacco pollen by particle bombardment
Mature pollen was collected from four to six flowers of 8-wk-old tobacco (N. tabacum L.) plants. Transient expression in pollen tubes was performed as previously described (Stenzel et al., 2012).

Preparation and transient expression in Arabidopsis protoplasts
Arabidopsis thaliana L. mesophyll protoplasts were isolated and transformed with 10 µg pUGW14-StIPP_oS/100 µl protoplasts or 1 µg pUGW15-CFP/100 µl protoplasts according to (Yoo et al., 2007). Transfected protoplasts were harvested 16 h later by centrifugation, the supernantant was removed and the cell pellets frozen in liquid nitrogen.

Western blot
For standard western blot, protein extraction from protoplasts was done by direct application of Laemmli sodium dodecyl sulfate (SDS) sample buffer. Protein separation by SDS-PAGE (polyacrylamide gel electrophoresis) and immunoblotting on nitrocellulose membrane or polyvinylidene difluoride (PVDF) membrane were performed with standard protocols. Proteins were detected with anti-His, anti-FLS2, anti-mouse and anti-rabbit antibodies. For FLS2 signal quantification, a fluorescent western blot was performed combining the SPL Kit (NH DyeAGNOSTICS GmbH, Halle (Saale), Germany) and an IRcoupled antibody (Li-Cor Biosciences GmbH, Bad Homburg, Germany). Polyclonal rabbit antibodies against FLS2 were produced and affinity purified against the C-terminal peptide KANSFREDRNEDREV (Immunoglobe) as previously described . For protein extraction, sample buffer (100 mM NaCl, 20 mM DTT, 0.1% Triton X-100, 0.1% SDS, 0.1% NP-40, 50 mM Tris pH 9.6, 1 mM PMSF, 19 Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific)) was added to protoplasts. SPL sample buffer was added according to the manufacturer's instructions. The samples were incubated at 65°C for 15 min and centrifuged for 15 min at 21 130 g. Protein separation was done with 8%-SDS-PAGE. Afterwards, the gel was separated at the size of 63 kDa and the detection of total protein and the provided standard was performed with a laser scanner (Typhoon FLA 9500; GE Healthcare, Freiburg, Germany) according to the SPL kit manufacturer's instructions.

Results
StIPP was identified in microarray analyses as a transcript accumulating in response to treatment by Pep-13 in wild type potato plants as well as in transgenic potato plants impaired in biosynthesis (StAOC-RNAi and StOPR3-RNAi) or perception (StCOI1-RNAi) of jasmonic acid (Halim et al., 2009). The 60mer on the potato chips (Kloosterman et al., 2008) corresponds to gene locus PGSC0003DMG400016891, annotated to encode an inositol polyphosphate phosphatase (http://sola naceae.plantbiology.msu.edu/). In this study, the StIPP protein is characterized in vitro and in vivo to elucidate its roles in the defense of potato against P. infestans.
StIPP is a Pep-13-activated gene with similarity to sequences for inositol polyphosphate phosphatases The coding region of the StIPP gene covers eight exons and is 1017 bp in length (Fig. 1a). The deduced gene product of 338 amino acids (Sotub04g033080.  (Majerus & York, 2009). Phylogenetic analysis confirms association of StIPP with a well-funded clade, which also includes the sequence for At5PTase11 (Fig. 1c).
The up to 10-fold accumulation of StIPP transcript determined by microarray analyses for potato leaves treated with Pep-13 was independent of oxo-phytodienoic acid or jasmonic acid (Fig. 2a). Pep-13-dependent activation of StIPP was verified by independent qRT-PCR experiments, and Pep-13 treatment resulted in significantly enhanced StIPP transcript levels as early as 1 h after Pep-13 infiltration, compared to the transcript levels of leaves infiltrated with the inactive W2A-peptide (Fig. 2b), and StIPP transcript levels continued to increase after 24 h. Pep-13induced accumulation of StIPP transcripts was also observed in potato leaves infected with P. infestans (Fig. 2c) and after wounding ( Fig. 2d), with significantly enhanced StIPP transcript levels detectable after 1 d and 6 h, respectively. Together, the data indicate that the StIPP gene is induced in potato leaves upon PAMP treatment, pathogen infection and wounding.
StIPP is an inositol polyphosphate 5-phosphatase specific for PtdIns(4,5)P 2 For biochemical characterization of the StIPP gene product, the StIPP coding region was cloned into the bacterial expression vector pDEST-N112-MBP (Dyson et al., 2004) encoding a translational fusion of StIPP to an N-terminal MBP and a His-tag, as was previously used for the expression of phosphoinositide-modifying enzymes Stenzel et al., 2008Stenzel et al., , 2012Hempel et al., 2017). Transformation of Rosetta gami cells with the StIPP expression construct resulted in bacterial lysates that contained soluble MBP-StIPP fusion protein of calculated c. 83 kDa, as determined by western blot analyses using anti-His antibodies (Fig. 3a). Catalytic activity and substrate preference were determined by incubation of lysates containing MBP or MBP-StIPP with different phosphoinositide substrates, subsequent lipid extraction and the analysis of the hydrophobic reaction products by TLC and CuSO 4 -staining (Fig. 3b). In these The deduced amino acid sequences of StIPP, its closest homologs and the corresponding Arabidopsis genes are shown. The evolutionary history was inferred using the neighbor-joining method (Saitou & Nei, 1987). The optimal tree with the sum of branch length = 5.94410666 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl & Pauling, 1965) and are in the units of the number of amino acid substitutions per site. The analysis involved 31 amino acid sequences. All positions containing gaps and missing data were eliminated. There was a total of 200 informative positions in the final dataset. Evolutionary analyses were conducted using MEGA6 (Tamura et al., 2011).
Dephosphorylation of PtdIns(4,5)P 2 by StIPP can occur either at the D4 or the D5 phosphate, respectively yielding either PtdIns5P or PtdIns4P. To determine the regiospecificity of the StIPP-mediated dephosphorylation, PtdIns(4,5)P 2 was incubated with recombinant MBP-StIPP protein, and the reaction product was subsequently isolated and used as a substrate for re-phosphorylation assays. These assays were performed in the presence of c [ 33 P]ATP and either commercial human PI4P 5-kinase or human PI5P 4-kinase, which specifically act on PtdIns4P or PtdIns5P substrates, respectively. The specificity of these helper enzymes was first confirmed in vitro by converting the respective substrates, PtdIns4P and PtdIns5P (Fig. 3c). While both human enzymes were active as expected, re-phosphorylation of the StIPP

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New Phytologist reaction product was only observed with the PI4P 5-kinase (Fig. 3d), identifying the reaction product of MBP-StIPP-mediated conversion of PtdIns(4,5)P 2 as PtdIns4P. A quantification of the re-phosphorylation results is shown in Fig. 3(e). The in vitro data indicate that the StIPP protein acts as a PtdIns(4,5)P 2 -specific 5-phosphatase producing PtdIns4P.

StIPP is functional in vivo
As recombinant StIPP used PtdIns(4,5)P 2 in vitro as a substrate, we next addressed whether the expression of StIPP would influence PtdIns(4,5)P 2 also in vivo. For this purpose, we first used tobacco pollen tubes, which represent a well-characterized model to assess phosphoinositide-dependent membrane trafficking defects (Ischebeck et al., , 2010Sousa et al., 2008;Stenzel et al., 2012;Hempel et al., 2017). A fluorescent biosensor for PtdIns(4,5)P 2 , RedStar-PLC PH , decorated a well-defined subapical plasma membrane region of pollen tubes when coexpressed with YFP as a control protein (Fig. 4a, left panels). By contrast, coexpression of RedStar-PLC PH with StIPP-YFP under identical conditions resulted in a substantially reduced dimension of the plasma membrane region decorated by The reaction products were re-extracted, separated by thin-layer chromatography, visualized by staining with copper sulfate (CuSO 4 ) and identified according to comigration with authentic lipid standards, as indicated. The result shown is representative for three independent experiments. The open arrowhead indicates the migration of the reaction product of PtdIns(4,5)P 2 hydrolysis, a PtdIns-monophosphate. (c-e) To determine the regiospecificity of the dephosphorylation of PtdIns (4,5)P 2 , the PtdIns-monophosphate reaction product was re-extracted and subjected to a rephosphorylation assay. RedStar-PLC PH (Fig. 4a, right panels). This pattern is consistent with an in vivo function of the StIPP protein as a PtdIns(4,5)P 2specific phosphatase as determined in vitro (Fig. 3). The StIPP-YFP fusion localized to the subapical plasma membrane of the pollen tube cells (Fig. 4a, right panels) in a pattern similar to that shown by intrinsic tobacco enzymes hydrolyzing PtdIns(4,5)P 2 , such as NtPLC3 (Helling et al., 2006;Stenzel et al., 2020). Quantification of the dimensions of the plasma membrane region occupied by RedStar-PLC PH indicates a significant reduction upon coexpression of StIPP-YFP (n = 30 cells for YFP; n = 20  (Fig. 4b, top). The proportion of plasma membrane-associated and cytosolic RedStar-PLC PH fluorescence did not significantly change upon expression of StIPP-GFP compared to values upon expression of YFP (Fig. 4b,  bottom). Overall, the data are consistent with an in vivo effect of expressed StIPP-YFP on PtdIns(4,5)P 2 accumulation in tobacco pollen tubes.
To further test whether the expression of StIPP would interfere with PtdIns(4,5)P 2 -dependent aspects of pollen tube growth in vivo, we assessed the impact of StIPP-YFP expression on pollen tube cell morphologies arising from the modulation of PtdIns (4,5)P 2 . Pollen tubes display characteristic cell morphologies upon overproduction of PtdIns(4,5)P 2 , which can easily be scored, including pollen tubes with branched or stunted tips (Ischebeck et al., , 2010Stenzel et al., 2012;Hempel et al., 2017) or displaying tip swelling (Ischebeck et al., 2011;Stenzel et al., 2012Stenzel et al., , 2020. The range of pollen tube morphologies observed is represented in Fig 4(c), as indicated. The expression of StIPP-YFP alone in pollen tubes resulted in a significantly increased proportion of cells in which pollen tube growth was aborted shortly after tubes emerged from the pollen grains, whereas pollen tube growth was not affected by expression of the YFP control (n = 76 cells for YFP; n = 88 cells for StIPP-YFP/ mCherry; P ≤ 0.0001) (Fig. 4d). To test whether StIPP-YFP expression interfered with PtdIns(4,5)P 2 -dependent processes, the morphological defects caused by the overexpression of the PI4P 5-kinase, PIP5K5 , were assessed during coexpression of PIP5K5-YFP with an mCherry control or during coexpression of PIP5K5-CFP with StIPP-YFP (Fig. 4e).

StIPP-GFP localizes to the plasma membrane of uninfected pavement cells of potato and N. benthamiana
Based on the results so far, we next investigated the subcellular localization of a StIPP-GFP fusion in vegetative plant tissues. StIPP-GFP was transiently expressed, in an initial experiment, in potato leaves under the control of the cauliflower mosaic virus (CaMV) 35S promotor (Fig. 5a). Coexpression with an mCherry control indicates plasma membrane localization of StIPP-GFP (Fig. 5a). To enable the coexpression with additional fluorescence markers, further characterizations were performed upon expression of StIPP-GFP in N. benthamiana leaves (Fig. 5b-h). Plasma membrane localization of StIPP-GFP was assessed for uninfected N. benthamiana pavement cells relative to the coexpressed plasma membrane aquaporin, PLASMA MEMBRANE INTRINSIC PROTEIN 2A (PIP2A) (Johanson et al., 2001) fused to mCherry (Fig. 5b). Quantification of fluorescence intensities of the two markers along the dashed line in Fig. 5(b) indicates close Fig. 4 StIPP-YFP impacts on PtdIns(4,5)P 2 in vivo and antagonizes PtdIns(4,5)P 2 -dependent effects on cell morphologies in tobacco pollen tubes. The in vivo function of StIPP was assessed upon transient expression in tobacco (Nicotiana tabacum) pollen tubes. (a) Effects of StIPP on PtdIns(4,5)P 2 were assessed in vivo by monitoring the distribution of RedStar-PLC PH , a fluorescent biosensor for PtdIns(4,5)P 2 . The RedStar-PLC PH reporter decorated a subapical plasma membrane region when coexpressed with a YFP control (left panels). The dimension of the plasma membrane region occupied by RedStar-PLC PH was reduced upon coexpression with StIPP-YFP (right panels). (b) Quantification of the dimension of the plasma membrane region (distance of the lower edge from the tip) decorated by RedStar-PLC PH upon coexpression with YFP or with StIPP-YFP, as indicated (upper panel). Ratio of plasma membrane vs cytosolic fluorescence of RedStar-PLC PH upon coexpression with YFP or with StIPP-YFP, as indicated. Data are the mean AE SD from 30 and 20 experiments, respectively. (c) Polar tip growth of pollen tubes is sensitive to perturbation of PtdIns(4,5)P 2 production, giving rise to aberrant cell shapes. Pollen tube morphologies that have previously been reported include normal growth, here upon expression of a YFP control; branched growth, here upon expression of the PI4P 5-kinase PIP5K5 (the image is a three-dimensional projection of a confocal image stack); stunted growth, here upon strong expression of PIP5K5 (with apical membrane infolding); tip swelling, here upon expression of PIP5K11; and aborted growth, here upon expression of StIPP-YFP, where expansion of the pollen tube apex (arrowhead) ceases immediately upon emergence from the pollen grain. These cell morphologies were scored to assess the impact of StIPP on PI4P 5-kinase-mediated defects. (d) Control experiments illustrating cell morphologies observed upon expression of YFP or of coexpressed StIPP-YFP and mCherry, as indicated. The expression of StIPP-YFP resulted in an increased incidence of aborted pollen tubes. Data represent 76 cells for YFP and 88 cells for StIPP-YFP/mCherry. (e) An antagonistic function of StIPP-YFP towards PtdIns(4,5)P 2 -dependent effects was tested in vivo by coexpressing the Arabidopsis PI4P 5-kinase AtPIP5K5 with either a fluorescent protein alone (black bars) or with StIPP-YFP (gray bars) and scoring the resulting cell morphologies. Data represent 86 cells for PIP5K5-YFP/mCherry and 92 cells for PIP5K5-CFP/StIPP-YFP. Cell morphologies were categorized as normal, branched, stunted, swollen or aborted, as indicated. (f, g) Control experiments were performed in which the effects of bona fide PtdIns(4,5)P 2 -specific lipid phosphatases were tested, such as AtPTase11 (f) or Sac9 (g). Data represent 86 cells for PIP5K5-YFP/ mCherry and 78 cells for PIP5K5-CFP/AtPTase11-YFP (d) or 86 cells for PIP5K5-YFP/mCherry and 96 cells for PIP5K5-CFP/Sac9-YFP (e). Data in panels (d-g) represent means AE SD. Statistical analyses were performed using unpaired two-tailed Student's t-tests. Asterisks indicate statistical differences, as indicated (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001). ns, not significant. Bars, 10 µm.
Ó 2020 The Authors New Phytologist Ó 2020 New Phytologist Foundation New Phytologist (2021) 229: 469-487 www.newphytologist.com colocalization of the markers (Fig. 5c) with a Pearson coefficient (R) for the colocalization of 0.89 (Fig. 5d). The localization of StIPP-GFP was also analyzed relative to that of the fluorescencetagged pathogen effector, RFP-AvrBlb2 (Fig. 5e,f). This effector was previously shown to localize at the plasma membrane upon expression in epidermal N. benthamiana cells, and to re-localize to the extrahaustorial membrane (EHM) upon P. infestans infection (Bozkurt et al., 2011(Bozkurt et al., , 2015. In uninfected cells, StIPP-GFP showed a distinct plasma membrane signal at the cell periphery, where it co-localized with RFP-AvrBlb2 (Fig. 5e,f), with a high

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New Phytologist degree of colocalization indicated by the R = 0.92. Pearson coefficients for the colocalization of StIPP-GFP with PIP2A-mCherry or with RFP-AvrBlb2 were determined for 10 experiments, each, and indicate a consistently high degree of colocalization of StIPP-GFP at the plasma membrane of uninfected pavement cells with either marker (Fig. 5h).

StIPP-GFP relocalizes to pathogen entry sites upon infection
The localization of coexpressed StIPP-GFP and RFP-AvrBlb2 was further analyzed in epidermal cells of N. benthamiana upon infection with P. infestans 88069. After 3-4 d post infection (dpi), StIPP-GFP displayed clear association with the haustorium (Fig. 6a-c). In cells coexpressing StIPP-GFP and RFP-AvrBlb2, infection with P. infestans resulted in a re-localization of both StIPP-GFP and RFP-AvrBlb2 from the plasma membrane to the infection sites and developing EHM, with some retained plasma membrane association observed for either marker (Fig. 6d,g). In the majority of cases (52 out of 79 cells analyzed), StIPP-GFP but not RFP-AvrBlb2 retained a pronounced plasma membrane localization. Colocalization was consistently observed at the EHM, as is illustrated by the intensity plots (Fig. 6e) and the corresponding colocalization analysis (Fig. 6f). In a smaller number of cases (16 out of 79 cells analyzed), both StIPP-GFP and RFP-AvrBlb2 retained a similar degree of plasma membrane association in addition to localizing to the EHM (Fig. 6g), as illustrated by the intensity plots (Fig. 6h) and corresponding colocalization coefficient (Fig. 6i). Pearson coefficients for the colocalization of StIPP-GFP with RFP-AvrBlb2 were determined for 10 experiments and indicate a consistently high degree of colocalization of StIPP-GFP with RFP-AvrBlb2, regardless of whether the colocalization was analyzed globally or with a focus on the EHM (Fig. 6j). The patterns suggest that StIPP-GFP and RFP-AvrBlb2 are re-localizing from the plasma membrane to the EHM. As plasma membrane association of StIPP-GFP and RFP-AvrBlb2 appears to be retained independently, it appears possible that the markers may follow independent modes of relocalization to the EHM.
StIPP-GFP may act on PtdIns(4,5)P 2 specifically at infection sites As StIPP specifically hydrolyzed PtdIns(4,5)P 2 to PtdIns4P in vitro and associated with P. infestans infection sites in vivo, we next tested the subcellular localization of StIPP-GFP in relation to fluorescent reporters for PtdIns(4,5)P 2 or PtdIns4P in uninfected and infected tobacco leaf epidermis cells (Fig. 7). PtdIns (4,5)P 2 and PtdIns4P can be visualized in vivo by monitoring the subcellular distribution of the fluorescent reporters mCherry PLC-PH (van Leeuwen et al., 2007;Simon et al., 2014) or mCherry FAPP1-PH (Mishkind et al., 2009;Simon et al., 2014), respectively. In uninfected cells, the PtdIns(4,5)P 2 -biosensor mCherry PLC-PH localized to the cytosol, the nucleus (n) and the plasma membrane, where it colocalized with StIPP-GFP (Fig. 7a), as illustrated by the representative intensities (Fig. 7b) recorded along the dashed line in Fig. 7(a), with a colocalization coefficient for StIPP-GFP and mCherry PLC-PH in uninfected cells of around 0.88 (Fig. 7c). In uninfected cells, StIPP-GFP colocalized at the plasma membrane also with the PtdIns4P-biosensor mCherry FAPP1-PH , which also showed additional fluorescence in the nucleus (n) (Fig. 7d). Fluorescence intensities for StIPP-GFP and mCherry FAPP1-PH (Fig. 7e) recorded along the dashed line in Fig. 7(d), indicate a high degree of colocalization with a colocalization coefficient around 0.95 (Fig. 7f). The patterns for uninfected cells indicate that StIPP-GFP and biosensors for PtdIns (4,5)P 2 or PtdIns4P all colocalized at the plasma membrane.
Upon infection, both StIPP-GFP and mCherry PLC-PH decorated additional areas around the infection sites and the EHM (Fig. 7g) in a pattern consistent with recent reports on the accumulation of PtdIns(4,5)P 2 at pathogen infection sites in Arabidopsis (Shimada et al., 2019;Qin et al., 2020). StIPP-GFP and mCherry PLC-PH also associated with punctate structures in the vicinity of infection sites (arrows in Fig. 7g; Fig. S2). When the fluorescence intensities of StIPP-GFP and mCherry PLC-PH was quantified across the neck regions of penetrating P. infestans hyphae, a strong signal for StIPP-GFP was observed at the EHM, whereas the fluorescence intensity of mCherry PLC-PH was decreased in this area (Fig. 7g,h). The analysis of relative fluorescence intensities (Fig. 7h) across the infection structure (dashed line A in Fig. 7g) resulted in a negative Pearson coefficient for StIPP-GFP and mCherry PLC-PH (Fig. 7i), indicating a loss of the PtdIns(4,5)P 2 -biosensor fluorescence where StIPP-GFP intensity was high, which is consistent with StIPP-mediated hydrolysis of PtdIns(4,5)P 2 . By contrast, when the intensities for StIPP-GFP and mCherry PLC-PH were recorded across the plasma membrane of an infected cell (dashed line B in Fig. 7g), no inverted correlation was found (Fig. 7j,k) and the resulting pattern at the plasma membrane was similar to that observed in uninfected cells www.newphytologist.com (Fig. 7a-c). The data suggest that conversion of PtdIns(4,5)P 2 by StIPP might occur predominantly at the EHM. When the subcellular distribution of mCherry FAPP1-PH was analyzed relative to that of StIPP-GFP in infected cells, both StIPP-GFP and mCherry FAPP1-PH displayed fluorescence at the periphery of the neck region of penetrating hyphae (Fig. 7l), and the quantification of fluorescence intensities indicates close colocalization of StIPP-GFP and mCherry FAPP1-PH in this area (Fig. 7m) with a high colocalization coefficient around 0.9 (Fig. 7n). StIPP-GFP and mCherry FAPP1-PH also associated with

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New Phytologist punctate structures in the vicinity of infection sites (Fig. S2). Pearson coefficients for the colocalization of StIPP-GFP with the two lipid biosensors were determined for 10 experiments and indicate a high degree of colocalization of StIPP-GFP with the biosensors for PtdIns(4,5)P 2 and for PtdIns4P at the plasma membrane of uninfected and of infected cells (Fig. 7o). A notable exception is the inverse correlation (mean R = À0.62) between high StIPP-GFP fluorescence and low intensity of the PtdIns (4,5)P 2 reporter mCherry PLC-PH , which was observed only at the EHM of infected cells (Fig. 7o), a pattern consistent with StIPPmediated hydrolysis of PtdIns(4,5)P 2 predominantly at the EHM. Together, the data suggest StIPP-GFP action at P. infestans infection sites, which is accompanied by reduced intensity of a PtdIns(4,5)P 2 -specific biosensor.

StIPP overexpression in Arabidopsis protoplasts leads to accumulation of FLS2
Experiments so far identified StIPP as a Pep-13-activated gene encoding a PtdIns(4,5)P 2 -specific 5-phosphatase, which may cause the dephosphorylation of PtdIns(4,5)P 2 at infection sites. As local interference with PtdIns(4,5)P 2 or enhanced formation of PtdIns4P may influence membrane trafficking, the abundance of membrane proteins with roles in defense might be altered upon StIPP action. To test this hypothesis, we analyzed the effects of StIPP-HA expression on the abundance of the receptor kinase FLS2 in Arabidopsis cells. Arabidopsis mesophyll protoplasts were transfected with StIPP-HA or HA-CFP expressed under the control of the CaMV 35S promotor. The abundance of intrinsic FLS2 protein was then analyzed 16 h post-transfection (hpt) by immunodetection using an FLS2-specific antibody (Fig. 8). The presence of the expressed proteins was verified by immunodetection using anti-HA antibodies (Fig. 8). FLS2 was detected at low levels in protoplasts expressing HA-CFP (Fig. 8a). By contrast, expression of StIPP-HA resulted in much stronger signals for FLS2 (Fig. 8a), indicating an increased abundance of the FLS2 protein. Increased abundance of FLS2 in cells expressing StIPP was also observed when FLS2 signals were quantified with a fluorescent western blot (Fig. 8b). In these experiments, mesophyll protoplasts were transfected as described earlier, FLS2 abundance was detected using a FLS2-specific antibody, and then a secondary antibody conjugated with a fluorescent dye was applied, and the fluorescence intensity of the dye was quantified. The data were normalized to the total loaded protein prelabeled with the fluorescent dye (Fig. 8b). Analogous immunodetection experiments performed for PIN1, which does not accumulate at infection sites, did not indicate a significant change in PIN1 abundance (Fig. 8c). The combined observations indicate that modulation of PtdIns(4,5)P 2 /PtdIns4P levels by StIPP at infection sites (Fig. 7) might impact on the trafficking of defense-related membrane proteins, such as FLS2.
PtdIns(4,5)P 2 accumulates in Arabidopsis plants at sites of pathogen infection (Shimada et al., 2019;Qin et al., 2020), and PtdIns(4,5)P 2 was identified as a susceptibility factor in Arabidopsis (Qin et al., 2020). Therefore, the induction of StIPP, an enzyme hydrolyzing PtdIns(4,5)P 2 at the EHM, might be part of a defensive strategy of the host plant that involves limiting the availability of PtdIns(4,5)P 2 at infection sites. Our experiments provide evidence that in tobacco leaves PtdIns(4,5)P 2 correlates with P. infestans at infection sites (Fig. 7). While the association of PtdIns(4,5)P 2 with infection structures suggests a role in infection or defense processes, the precise molecular function of the lipid in this context is currently unclear. In our in vitro tests, StIPP activity was very specific and only converted PtdIns(4,5)P 2 to PtdIns4P, enabling a focused study of StIPP effects on cellular functions of these lipids. PtdIns(4,5)P 2 , and possibly PtdIns4P, influence membrane trafficking by controlling both secretion and endocytosis. In pollen tubes the intricate balance of secretion and vesicle recycling is very sensitive to perturbation of cellular PtdIns(4,5)P 2 contents, which results in aberrant deposition of pectin and morphological defects of the cells, such as tip branching and other characteristic shapes (Ischebeck et al., , 2011Sousa et al., 2008;Zhao et al., 2010;Hempel et al., 2017). When StIPP-YFP was

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New Phytologist expressed in pollen, pollen tube germination from the grains was often aborted (Fig. 4d), consistent with defects observed in Arabidopsis pip5k4 pip5k5 double mutants deficient in PtdIns(4,5) P 2 formation Sousa et al., 2008). Furthermore, StIPP-YFP expression antagonized the enhanced pollen tube tip branching and stunted tube growth caused by PIP5K5mediated overproduction of PtdIns(4,5)P 2 ( Fig. 4e-g), consistent with PtdIns(4,5)P 2 dephosphorylation and a functional effect on membrane trafficking much like that of other PtdIns(4,5)P 2specific 5-phosphatases, such as AtPTase11 (Ercetin et al., 2008) or Sac9 (Williams et al., 2005). While the results from the pollen tube model may at first appear unrelated to a role of StIPP in defense, it has recently been shown that PtdIns(4,5)P 2 production at the apical plasma membrane of pollen tubes is controlled by phosphorylation of PI4P 5-kinases by the MAP-kinase MPK6 (Hempel et al., 2017). Importantly, MPK6 also has a demonstrated role in PAMP-responses and pathogen defense (Meng & Zhang, 2013), and a PAMP-triggered MAPK-cascade involving MPK6 has recently been shown to inhibit the formation of PtdIns(4,5)P 2 in Arabidopsis (Menzel et al., 2019). Evidently, the limitation of cellular PtdIns(4,5)P 2 by means of inhibiting its biosynthesis might be part of the plant defensive strategy against pathogen attack. Our data from the potato/P. infestans model provide further evidence for this concept, with the noted difference that here PtdIns(4,5)P 2 is reduced by enhancing its breakdown through the activation of StIPP.
In infected tobacco epidermal cells, StIPP relocalized from general plasma membrane association (as also observed in pollen tubes) to sites of P. infestans penetration (Fig. 6). The specific recruitment to infection sites suggests a change in protein-protein or protein-lipid interactions of StIPP that is mediated by the infection. Importantly, the imaging data also provide evidence for StIPP-mediated localized hydrolysis of PtdIns(4,5)P 2 (Fig. 7g-k) and for the concomitant formation of PtdIns4P ( Fig. 7l-n), suggesting that StIPP locally inhibits PtdIns(4,5)P 2dependent processes. The quantitative in vivo imaging of processes at dynamic infection structures represents a substantial experimental challenge. Therefore, the results from our imaging approach should not be overinterpreted. Negative Pearson coefficients for the relative localizations of StIPP-GFP and mCherry PLC-PH were only observed at the EHM of infected cells, whereas in other subcellular locations the respective Pearson coefficients were positive, regardless of whether cells were infected or not. Taking these data at face-value, this pattern suggests that conversion of PtdIns(4,5)P 2 by StIPP occurred predominantly at the EHM. A possible conclusion is that StIPP might require a post-translational activation step at the EHM in addition to the induction of its transcript and its relocalization from the plasma membrane to the EHM. Our observation of a significant accumulation of the immune receptor FLS2 upon StIPP expression in Arabidopsis protoplasts (Fig. 8) supports the hypothesis that reduced PtdIns(4,5)P 2 results in attenuated rathes of clathrin-mediated endocytosis (CME). As PtdIns(4,5)P 2 is a key mediator of CME (K€ onig et al., 2008b;Zhao et al., 2010;Ischebeck et al., 2013;Tejos et al., 2014;Menzel et al., 2019), the observed accumulation of FLS2 (Fig. 8) may reflect modulated endocytosis, which results in the stabilization of the receptor at the plasma membrane. Future studies will show whether StIPP regulates vesicular trafficking of specific membrane proteins during the immune response.
It is possible that the activation of StIPP and its recruitment to sites of infection is part of a mechanism to locally and transiently stabilize plasma membrane proteins with roles in immunity only where an infection actually occurs. As plant defense responses are highly complex, it is clear that StIPP will only be one element in the multi-layered series of events. In line with this notion, we did not observe altered susceptibility or resistance against P. infestans in transgenic potato plants expressing RNAi constructs against StIPP, despite substantial downregulation of StIPP transcript abundance in these plants (Fig. S3). Nonetheless, our data suggest that the StIPP protein contributes to defense, likely through its localized effects on the phosphoinositide system and vesicle trafficking at infection sites. As illustrated in the model shown in Fig. 9, StIPP may, thus, be part of transient reprogramming of membrane trafficking processes at the plasma membrane, which concentrates at the perceived sites of P. infestans infection. , the expression of StIPP is activated, resulting in the association of the enzyme with the plasma membrane, concentrated around the sites of penetration. As a PtdIns(4,5)P 2 -specific 5-phosphatase, StIPP may attenuate PtdIns(4,5)P 2 -dependent recycling of plasma membrane-resident proteins, possibly resulting in reduced recycling, indicated by the gray oval, and their transient stabilization at the plasma membrane. Other explanations are possible. Thicker arrows indicate an enhanced effect. cw, cell wall; cyt, cytosol; EHM, extrahaustorial membrane; n, nucleus; pm, plasma membrane; PRR, pattern recognition receptor; red circle, StIPP; vac, vacuole.

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