Bacterial pathogens deliver water- and solute-permeable channels to plant cells

Many animal- and plant-pathogenic bacteria use a type III secretion system to deliver effector proteins into host cells1,2. Elucidation of how these effector proteins function in host cells is critical for understanding infectious diseases in animals and plants3–5. The widely conserved AvrE-family effectors, including DspE in Erwinia amylovora and AvrE in Pseudomonas syringae, have a central role in the pathogenesis of diverse phytopathogenic bacteria6. These conserved effectors are involved in the induction of ‘water soaking’ and host cell death that are conducive to bacterial multiplication in infected tissues. However, the exact biochemical functions of AvrE-family effectors have been recalcitrant to mechanistic understanding for three decades. Here we show that AvrE-family effectors fold into a β-barrel structure that resembles bacterial porins. Expression of AvrE and DspE in Xenopus oocytes results in inward and outward currents, permeability to water and osmolarity-dependent oocyte swelling and bursting. Liposome reconstitution confirmed that the DspE channel alone is sufficient to allow the passage of small molecules such as fluorescein dye. Targeted screening of chemical blockers based on the predicted pore size (15–20 Å) of the DspE channel identified polyamidoamine dendrimers as inhibitors of the DspE/AvrE channels. Notably, polyamidoamines broadly inhibit AvrE and DspE virulence activities in Xenopus oocytes and during E. amylovora and P. syringae infections. Thus, we have unravelled the biochemical function of a centrally important family of bacterial effectors with broad conceptual and practical implications in the study of bacterial pathogenesis.

Many animal-and plant-pathogenic bacteria use a type III secretion system to deliver effector proteins into host cells 1,2 .Elucidation of how these effector proteins function in host cells is critical for understanding infectious diseases in animals and plants [3][4][5] .The widely conserved AvrE-family effectors, including DspE in Erwinia amylovora and AvrE in Pseudomonas syringae, have a central role in the pathogenesis of diverse phytopathogenic bacteria 6 .These conserved effectors are involved in the induction of 'water soaking' and host cell death that are conducive to bacterial multiplication in infected tissues.However, the exact biochemical functions of AvrE-family effectors have been recalcitrant to mechanistic understanding for three decades.Here we show that AvrE-family effectors fold into a β-barrel structure that resembles bacterial porins.Expression of AvrE and DspE in Xenopus oocytes results in inward and outward currents, permeability to water and osmolarity-dependent oocyte swelling and bursting.Liposome reconstitution confirmed that the DspE channel alone is sufficient to allow the passage of small molecules such as fluorescein dye.Targeted screening of chemical blockers based on the predicted pore size (15-20 Å) of the DspE channel identified polyamidoamine dendrimers as inhibitors of the DspE/AvrE channels.Notably, polyamidoamines broadly inhibit AvrE and DspE virulence activities in Xenopus oocytes and during E. amylovora and P. syringae infections.Thus, we have unravelled the biochemical function of a centrally important family of bacterial effectors with broad conceptual and practical implications in the study of bacterial pathogenesis.
In this study, we carried out AlphaFold2 analysis of the threedimensional models of AvrE-family proteins.Unexpectedly, AlphaFold2 predicts that this family of proteins fold into a porin-like β-barrel structure.This prediction prompted us to conduct a series of cryogenic electron microscopy (cryo-EM) imaging, Xenopus oocyte, liposome and in planta experiments.Our results show that AvrE-family effectors are water-and solute-permeable channels that can be blocked by polyamidoamine (PAMAM) dendrimers.The discovery of the waterand solute-permeable channel function of AvrE/DspE-family effectors solves a decades-long puzzle regarding one of the most important families of phytobacterial type III effectors and marks a major advance in understanding bacterial pathogenesis.prominent central β-barrel forming the stem, which is surrounded by a globular amino-terminal domain (E.amylovora DspE: K298-H672), a WD40 repeat domain (H673-P912) and two perpendicularly arranged helix bundles (E998-T1222 and A1567-H1647) on the top.The predicted domain arrangement is supported by our cryo-EM imaging of E. amylovora DspE, for which the two-dimensional class averages clearly reveal an overall similar top view to that of the AlphaFold model, with circularly arranged globular domains surrounding a central pore (Fig. 1a,b).
Further examination of the predicted E. amylovora DspE threedimensional model reveals that surface β-barrel residues facing outside are enriched with hydrophobic amino acids (Fig. 1c), whereas inward-facing pore residues are predominantly hydrophilic (Fig. 1d).The length of the lower portion of the β-barrel stem covered by hydrophobic residues is estimated to be about 40 Å, roughly the thickness of a cellular membrane.As AvrE has previously been reported to be membrane anchored 20 , the β-barrel stem of AvrE-family effectors probably inserts into the membrane and functions as a channel, similar to that of bacterial porins 28 .Such a mode of insertion is distinct from that of pore-forming bacterial toxins, such as staphylococcal α-haemolysin and Clostridium perfringens β-toxin, for which the β-barrel is assembled through oligomerization of two long β-strands 29,30 .

DspE and AvrE currents in Xenopus oocytes
The prediction that AvrE-family effectors are channel-forming proteins prompted us to conduct experiments to test the hypothesis that AvrE and DspE may allow ion conductance when expressed in Xenopus oocytes.As shown in the current-voltage relationship (Fig. 2b,c), inward currents and outward currents at negative and positive test potentials, respectively, were detected from oocytes injected with dspE or avrE complementary RNA (cRNA).The reversal potentials for DspE and AvrE channels are approximately −25 mV.The currents were not affected by niflumic acid, which blocks an endogenous Ca 2+ -activated Cl − channels 31 , or fipronil, which inhibits GABA-gated Cl − channels and glutamate-gated Cl − channels 32 (Extended Data Fig. 3a,b).Surface biotinylation experiment with oocytes expressing DspE confirmed that this protein is anchored across the oocyte membrane (Extended Data Fig. 4a).
We further characterized whole-cell currents from DspE-expressing oocytes by conducting ion permeability experiments.Replacing extracellular sodium in ND96 recording solution (Methods) with potassium or other cations caused only minor variations in the magnitude of DspE currents (Extended Data Fig. 3c).Similarly, only minor variations in the magnitude of DspE currents were observed when extracellular Cl − was replaced with various anions, except for 4-morpholineethanesulfonic acid, sodium salt (Na-MES; Extended Data Fig. 3d,e).When 50% or 100% of the NaCl was replaced by Na-MES, progressively smaller outward currents were observed, and the reversal potential was shifted to a less negative value (Extended Data Fig. 3e).However, the negative reversal potential was not affected by replacement of other ions.These results suggest that Cl − may have a major role in carrying the outward current and that the DspE channel seems to have some selectivity towards anions including Cl − .Future research is needed to comprehensively survey possible ion selectivity of these channels.

DspE and AvrE induce cell swelling
During voltage-clamp current recording experiments, we noticed that many oocytes injected with dspE or avrE cRNA showed baseline swelling (Extended Data Fig. 5a), reminiscent of oocytes expressing plant aquaporins 33 .This raised the possibility that AvrE and DspE proteins may function like aquaporin channels to allow water to pass through cell membranes along an osmotic gradient, assuming that the osmolarity of the oocyte bathing medium (about 200 mOsm) may be lower than that of the oocyte cytoplasm.To more directly test this possibility, we adopted an oocyte swelling assay used for aquaporins and transferred oocytes from 200 mOsm to 40 mOsm bathing medium to create a larger osmotic difference.Notably, both DspE-and AvrE-expressing oocytes markedly swelled (Fig. 2d) and eventually burst (Supplementary Videos 1 and 2).We conducted further experiments to determine whether plant cells expressing AvrE would swell using our previously produced transgenic DEX::avrE plants 20 .As shown in Extended Data Fig. 5b,c, Arabidopsis leaf protoplasts expressing AvrE swelled to a greater extent compared to control Arabidopsis leaf protoplasts that have endogenous aquaporins.This provides further evidence that the AvrE channel has an ability to increase water permeability in planta [7][8][9][10][11][12][13][14][15][16][17][18][19] .

DspE channel size selectivity
The predicted AvrE/DspE channels have a diameter of 15-20 Å, much larger than the size of a water molecule or a simple ion.We reasoned that, in addition to ions and water, the AvrE and DspE channels may allow larger molecules to pass through.We conducted fluorescent dye permeability assays to test whether molecules smaller than the predicted pore size could pass through the membrane, whereas molecules larger than the predicted pore size could not.Two fluorescent molecules were tested: fluorescein (molecular mass of 332 Da with an estimated maximum molecular diameter of 7 Å) and enhanced green fluorescent protein (eGFP; molecular mass of 27 kDa with an estimated minimum diameter of 30 Å).As shown in Fig. 2e,f  DspE(Δβ-barrel) mutant, in which most β-barrel-forming sequences are deleted, exhibited substantial activity (Fig. 2g,h), suggesting that no other protein is needed for baseline passage of small molecules through the DspE channel.As in the oocyte experiment, although DspE readily induced carboxyfluorescein dye release from liposomes (Fig. 2h), no time-dependent release of large molecules, such as fluorescein isothiocyanate-polysucrose 40 (molecular mass of 30-50 kDa with an estimated diameter of 80 Å), was observed (Extended Data Fig. 5d).

Mutational analysis of the DspE channel
We made several mutant derivatives of DspE to evaluate their functional consequences.As a negative control, DspE(Δβ-barrel) failed to induce water-soaking symptoms in Nicotiana benthamiana leaves, conduct ion currents, cause oocyte swelling or allow fluorescein release in the liposome assay (Fig. 2h and Extended Data Fig. 6).Similarly, triple substitution of three conserved hydrophobic, outward-facing residues (L1776, L1777 and L1778) of the predicted transmembrane region of DspE (location indicated by the orange asterisk in Fig. 1c) also abolished the DspE activities (Extended Data Fig. 6).Surface biotinylation of oocytes expressing DspE(Δβ-barrel) and DspE(L1776E/L1777E/L1778E) showed that they are no longer accessible by surface biotinylation and therefore probably cannot anchor across the membrane (Extended Data Fig. 4a).Finally, charge-reversal double substitution at K1399 and K1401, two inward-facing residues of the β-barrel (location indicated by the purple asterisk in Fig. 1d; not conserved among AvrE-family members), partially abolished the DspE activities (Extended Data Fig. 6).As a newly discovered family of channels, future research is needed to comprehensively define inward-facing residues that are critical for the function of DspE and other AvrE-family members.

Inhibitors of DspE and AvrE channels
Next, we attempted to identify compounds of molecular diameters that could fit the predicted pores of AvrE/DspE channels and might therefore block AvrE/DspE activities.We focused on a class of synthetic PAMAM dendrimers, which have programmable molecular diameters 34 .For example, PAMAM G0 has a diameter of 15 Å, whereas PAMAM G1 has a diameter of 22 Å (https://www.dendritech.com/pamam.html).Notably, the currents passing through the DspE and AvrE channels were reduced by G1 in a dose-dependent manner, reaching 71% inhibition on the DspE channel and 93% inhibition on the AvrE channel, respectively, at 10 mM G1 at 50 mV test pulse (Fig. 3a,b), while not affecting DspE expression (Extended Data Fig. 4c).Similar inhibition by G0 was observed on oocytes expressing DspE, reaching 68% inhibition at 10 mM G0 at 50 mV test pulse (Extended Data Fig. 7a).
When G1 was added to the ND96 incubation buffer, the baseline swelling over time was also inhibited in a dose-dependent manner, reaching a maximum of 76% inhibition for 5 mM G1 at 19 h after injection (Fig. 3c).Again, a similar effect was observed when this inhibitor was tested on AvrE-expressing oocytes, with 89% inhibition for 5 mM G1 (Fig. 3d).
We also tested the effect of G1 on fluorescein uptake by oocytes expressing DspE, and found that it inhibited fluorescent dye uptake, reaching 79% inhibition by the end of the assay (Fig. 3e).Finally, we tested G1 on purified DspE protein reconstituted in liposomes using the DspE-dependent liposome dye release assay.We found that G1 dose-dependently inhibited the release of fluorescein from the soybean liposomes after the addition of DspE (Fig. 3f).Fitting of the dye release yielded an IC 50 value of 31 µM for G1 (Fig. 3g).Together, these findings show that we have identified PAMAM G1 as an inhibitor of AvrE/DspE-family channels.

PAMAM G1 inhibits bacterial infection
The ability of PAMAM G1 to inhibit DspE and AvrE activities in oocytes and liposomes in vitro raised the possibility that we had identified a lead compound that could interfere with the AvrE and DspE virulence function in planta during bacterial infection.We first tested this possibility against the AvrE function during P. syringae infection.In many P. syringae strains, AvrE is functionally redundant to another effector, HopM1 (refs.11,13,19).Mutation of either avrE or hopM1 alone does not strongly affect Pst DC3000 virulence, but the avrE,hopM1 double mutant is severely impaired in virulence 11,19 .Notably, whereas avrE-family effector genes are conserved widely 35 , hopM1-family genes and/or their secretion chaperone genes are subjected to natural genetic mutations as in the case of the major pandemic bacterial pathogen P. syringae pv.actinidiae 36 .We found that G1 effectively inhibited Pst DC3000 infection of Arabidopsis in an AvrE-dependent manner (that is, inhibition occurs in the hopM1-deletion mutant, which simulates natural mutations in the hopM1 gene; Fig. 4a,b).Furthermore, inhibition of AvrE function by PAMAM G1 was not associated with induction of the PR1 protein, a marker for activation of salicylic acid-dependent immune responses in plants (Fig. 4c), or with negative effects on plant appearance (Extended Data Fig. 7b) or seed production (Extended Data Fig. 7c).Next we tested PAMAM G1 against E. amylovora infection.In E. amylovora, DspE plays an essential role in causing the devastating fire blight diseases, as the dspA/E mutant is largely nonpathogenic 7,8 .We found that G1 completely inhibited E. amylovora infection of highly susceptible pear fruits, phenocopying the dspE mutant of E. amylovora, an observation consistent with DspE being an indispensable virulence effector (Fig. 4d).
It is well known that type III secretion systems and their effectors are expressed and needed for bacterial growth only in host tissues, but not in vitro 1,2 .In accordance with this, G1 did not inhibit E. amylovora or Pst DC3000 growth in vitro (Extended Data Fig. 7d), providing further evidence that PAMAM G1 is truly a AvrE and DspE-specific virulence inhibitor, not just a nonspecific bactericidal antibiotic.

Discussion
Since the initial report of AvrE in P. syringae almost three decades ago 37 , the central importance of AvrE-family effectors in diverse pathogenic bacterial species has attracted the attention of researchers.Before this study, however, researchers largely pursued a working hypothesis that, like most other type III effectors, AvrE/DspE-family effectors would target host proteins, RNA or DNA to exert their virulence functions.Our finding that AvrE-family effectors could directly function as waterand solute-permeable channels in this study is therefore striking and unexpected.We propose a new integrated model for the function of AvrE-family effectors (Extended Data Fig. 8).The carboxy-terminal halves of AvrE-family effectors act primarily as a new class of waterand solute-permeable channels dedicated to creating osmotic/water potential perturbation and a water-and nutrient-rich apoplast in which bacteria multiply within the infected plant tissues.Future research is needed to determine how AvrE-family channel activity, discovered in this study, mediates water, solute and nutrient flows and apoplast osmolarity in planta to generate macroscopic water soaking, host cell death and defence suppression in the infected tissue, as shown previously 6,13,19,25,38,39 .We found that infiltrating the Arabidopsis leaf apoplast with water (to simulate water soaking) was sufficient to suppress flg22-induced callose deposition (Extended Data Fig. 9a,b), suggesting that water soaking and suppression of certain immune responses (for example, defence-associated callose deposition) are linked processes.As large proteins with potentially many protein-interacting interfaces, AvrE-family effectors can additionally engage host proteins, including plant protein phosphatase PP2A subunits, type one protein phosphatases and receptor-like kinases [22][23][24][25] , to affect aspects of AvrE/DspE functions.It is striking that the AvrE channel inhibitor PAMAM G1 can essentially phenocopy the avrE or dspE genetic mutations in abrogating all major virulence phenotypes associated with AvrE and DspE, including water soaking, tissue necrosis and bacterial multiplication in infected tissues (Fig. 4).Therefore, future research should examine the possibility that some of the identified AvrE, DspE-and WtsE-interacting host proteins may act through modulating AvrE-family channel properties and/or optimizing downstream pathogenic outcomes of AvrE-family channel activities.
In summary, this study has unravelled the long-sought-after function of AvrE-family effectors.In addition, a chemical inhibitor of AvrE-family channels has been identified that appears to be broadly effective in reducing AvrE-and DspE-mediated bacterial infections.As such, the discovery of the water-and solute-permeable channel function of AvrE-family effectors has broad implications in the study of bacterial pathogenesis and bacterial disease control in plants.

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Cloning, expression and purification of E. amylovora DspE and DspE(Δβ-barrel) in Escherichia coli
The dspE gene was PCR-amplified from the genomic DNA of E. amylovora strain Ea273 using the KOD hot start polymerase (Millipore Sigma) and the following primer set: DspE_Forward_primer: 5′-ATGGAATTAAAATCACTGGGAACTGAACACAAG -3′; DspE_Reverse_ primer: 5′-GCTCTTCATTTCCAGCCCTTCCTTC-3′.The PCR product was cloned into a modified pET28a vector (Millipore Sigma) as a C-terminal fusion to maltose-binding protein (MBP) containing a His 8 tag, a preScission protease cleavage site (PPX) and a Flag tag in the form of MBP-His 8 -PPX-Flag-DspE (hereinafter as MBP-DspE).Plasmid-transformed BL21(DE3) E. coli cells were grown in Luria-Bertani medium at 37 °C until the optical density at 600 nm (OD 600 nm ) reached 0.4-0.6, and were then induced with 0.1 mM IPTG and grown at 18 °C overnight.Collected cell pellets were resuspended in a lysis buffer containing 20 mM HEPES (pH 7.5), 300 mM NaCl and 2.5% glycerol, supplemented with cOmplete EDTA-free protease inhibitor tablet (Roche) and DNase I, and lysed by a French press.Following centrifugation at 20,000 r.p.m. for 30 min at 4 °C to remove cell debris, the fusion protein was purified using TALON Cobalt resin (Takara Bio).Following extensive washing in a buffer containing 20 mM HEPES (pH 7.5), 300 mM KCl, 2.5% glycerol, 1 mM ATP, 5 mM MgCl 2 and 10 mM imidazole, the fusion protein was eluted in a buffer containing 20 mM HEPES (pH 7.5), 300 mM NaCl, 2.5% glycerol and 250 mM imidazole, and was further purified on a Superose 6 Increase 10/300 GL column (Cytiva Life Science) pre-equilibrated with a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl and 1 mM dithiothreitol (DTT) at 4 °C.The protein fractions at the peak were aliquoted and flash-frozen at −80 °C for storage.
The construct of DspE(Δβ-barrel) (Δ1278-1566 + Δ1649-1813) was generated from the WT MBP-DspE construct described above through in-fusion cloning.The mutant protein was purified by following identical procedures as for the WT protein.
Representative size-exclusion chromatography profiles and SDS-polyacrylamide gel electrophoresis (PAGE) gels of the purified MBP-DspE and MBP-DspE(Δβ-barrel) proteins are shown in Extended Data Fig. 4g,h.

Cryo-EM sample preparation and data collection and processing
Cryo-EM grids were prepared using a Leica EM GP2 automatic plunge freezer in a humidity-controlled chamber operated at 10 °C and 85-90% relative humidity.Homemade gold Quantifoil R1.2/1.3 300-mesh grids were glow-discharged using the PELCO easiGlow glow discharge cleaning system (TED PELLA) before sample application.During sample freezing, a 3-µl sample of DspE (about 1.1 mg ml −1 ) was applied to freshly glow-discharged grids and incubated on grids for 60 s before blotting with Whatman #1 filter paper for 2.8 s.The grids were then immediately plunge-frozen in liquid ethane and stored in liquid nitrogen before data acquisition.
A total of 7,810 micrograph stacks were recorded on an FEI Titan Krios electron microscope (Thermo Fisher) operated at 300 kV equipped with a K3 direct electron detector (Gatan) operated in the counting mode.Micrograph stacks were collected at a nominal magnification of ×81,000 using a pixel size of 1.08 Å per pixel with a defocus range from −2.4 µm to −0.8 µm using the Latitude S (Version 3.51.3719.0,Gatan) automated image acquisition package.Each stack was exposed for 2.8 s with an exposure time of 0.047 s per frame, resulting in 60 frames per stack.The total dose was approximately 56.3 e − Å −2 for each stack.
Motion correction and contrast transfer function (CTF) estimation were carried out with the patch motion correction model and patch CTF estimation module in cryoSPARC 40 .A total of 7,141 micrographs were selected from a total of 7,810 images on the basis of the CTF fitting resolution using a cutoff value of 4.0 Å.A total of about 2.4 million particles were picked using pre-trained TOPAZ 41 models, of which about 167,000 particles corresponding to full-length protein with high-resolution features were selected to generate two-dimensional class averages.Cryo-EM samples of DspE showed a severe orientation bias of the particles, which prevented high-resolution reconstruction of the cryo-EM density maps.

Xenopus laevis oocyte preparation, injection and expression of DspE and AvrE
Oocytes were purchased as ovary from Xenopus1.The ovary was treated with 0.55 mg ml −1 collagenase B (0.191 U mg −1 ) in calcium-free ND96 saline 43 (96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 5 mM HEPES and 2.5 mM Na pyruvate, pH 7.5) for 20 min while on a nutating mixer at 21-22 °C.Immediately after treatment, the enzymatic solution was rinsed off the ovary with ND96 bath saline (96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES, 2.5 mM Na pyruvate and 0.5 mM theophylline, pH 7.5) several times, and cell clusters were spread on several 70-mm plastic culture dishes with ND96 bath saline for temporary storage in an 18 °C incubator.On the same day, the follicular cells and follicular membrane covering mature oocytes (stages IV and V) were manually peeled off with fine forceps, and oocytes were kept in ND96 bath saline at 18 °C until cRNA injection.cRNA was mixed with diethyl pyrocarbonate-treated water to defined concentrations necessary to deliver desired amounts (ranging from 0.01 ng to 20 ng) of cRNA per oocyte when injecting a volume of 27.6 nl.Injection was carried out with a nanoinjector (Nanoject II, Drummond Scientific) following the manufacturer's directions.Diethyl pyrocarbonate-treated water was injected in control oocytes.Oocytes were kept in a 6-well plastic culture plate at 18 °C to allow expression of proteins.Incubation solution was either control (ND96 bath saline), ND96 with inhibitor (PAMAM G0 or G1, niflumic acid or fipronil), ND96 with 0.0005% fluorescein or ND96 with 0.1% GFP protein.

Oocyte surface biotinylation assay
Oocytes were injected with 2 ng of WT or mutant dspE cRNA and incubated in bath ND96 saline for 15 h.Surface-exposed proteins were biotinylated and purified using the Pierce Cell Surface Protein Biotinylation and Isolation Kit (Thermo Fisher), following the manufacturer's protocol with some modifications, as described previously 44 .Five oocytes were used per treatment (with biotin, without biotin or total cell extract).In brief, cells were rinsed three times in OR2 buffer 44 before incubating in 2.5 ml of OR2 buffer for 10 min with or without sulfo-NHS-SS-biotin in 6-well culture plates in a benchtop orbital shaker, set at 85 r.p.m. at room temperature.Oocytes for the total cell extract treatment were immediately stored at −80 °C, whereas oocytes for biotinylation (with or without biotin) were rinsed three times in TRIS buffer 44 before being placed in a 1.5-ml tube with 500 µl of lysis buffer containing 10 µl of Halt Protease Inhibitor Cocktail (Thermo Fisher).Lysis mix containing oocytes was homogenized by passing through a 20-gauge (G) needle 10 times, before incubation at 4 °C on a nutating mixer.The remaining steps followed the kit manufacturer's protocol.Final samples were eluted with 200 µl elution buffer and mixed with 50 µl of 5× SDS sample buffer.In parallel, the five oocytes stored for total cell extract treatment were homogenized in 250 µl of 2× SDS sample buffer.For equal loading, 25 µl of total extract or avidin-pulldown biotinylated protein samples was added to each lane for SDS-PAGE.

TEVC clamp recordings
Oocytes were injected with 0.01 ng of WT or mutant dspE cRNA or with 0.1 ng of avrE cRNA.AvrE seems less functional in oocytes than DspE.After about 15 h of incubation in bath ND96 saline (96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl, 10 mM HEPES, pH 7.5), each oocyte was impaled by one voltage-sensing borosilicate microelectrode and one current-passing borosilicate microelectrode with a resistance of 0.5 ± 0.1 MOhm, while in 1 ml of an electrically grounded ND96 recording saline (96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl, 10 mM HEPES, pH 7.5).Of note, in initial preliminary experiments, when oocytes were injected with a high amount of dspE or avrE cRNA (for example, 1 ng of dspE or 20 ng avrE cRNA per oocyte), membrane potentials at 24 h dropped close to 0 mV (Extended Data Table 1) and current conductance was very large (>50 µA), a condition at which the TEVC equipment no longer works properly.Thus, we lowered the cRNA input to 0.01 ng dspE per oocyte and 0.1 ng avrE per oocyte and evaluation time to 15 h after injection, which yielded a resting potential similar to that of oocytes injected with water control (Extended Data Table 1) and modest currents that TEVC was able to record.
For ion replacement experiments, variations of ND96 were prepared by replacing the major salt (that is, 96 mM NaCl) with 96 mM LiCl, KCl, RbCl, CsCl, choline-Cl, NDMG-Cl (N-methyl-d-glucamine hydrochloride), NaBr, NaI, NaClO 3 , NaBrO 3 or Na-MES 45,46 .Currents were first recorded in ND96 recording buffer.To replace ND96 with a new cation or anion, 10 ml of a new ND96 solution was slowly added from one end of the recording chamber using a 10-ml plastic syringe with an 18-G needle, while the original ND96 was washed out using a vacuum outflow 20-G tube from the other end of the chamber.The glass microelectrodes were half-filled with 1.5% agar containing 3 M KCl.The electrodes were connected to an oocyte clamp amplifier (OC-725C, Warner Instrument) by chlorinated silver wires.The bath clamp headstage was connected to bath saline by two chlorinated silver wires inside a disposable polytetrafluoroethylene 18-G tubing filled with 1.5% agar containing 3 M KCl serving as agar bridges.The oocyte clamp amplifier was connected to a computer by an analog-digital interface (Digidata 1440A, Molecular Devices).The command voltage protocols and data acquisition were carried out in the pCLAMP v.10.7 software suite (Molecular Devices).Oocytes were clamped to a desired potential using the fast clamp mode with maximum clamp gain and current gain set to 0.1 V uA −1 .Signal for both voltage and currents was recorded.After impaling an oocyte and before clamping it, both electrodes are capable of measuring the resting potential of that oocyte.Oocytes were clamped at their resting potential and test pulses of 100 ms towards more positive or more negative potentials in 10 mV increments were applied.The resultant current was recorded and analysed.Current amplitude was determined 10 to 20 ms after the start of the test pulse, the time at which there was the smallest or no overlaps with membrane capacitance or Ca-dependent Cl − currents from endogenous oocyte channels 47 .As resting potential values across individual oocytes varied (probably owing to uncontrollable intrinsic differences in each oocyte, its size and in fine adjustment of electrode position and resistance), the voltage-current relationship data were fitted to a quadratic polynomial regression (SigmaPlot 12.5 Systat Software) providing intermediate values and 95% confidence intervals.This also allowed currents elicited by the test potentials on control oocytes to be subtracted from the currents in treatment oocytes, so the resultant values represent only DspE-or AvrE-mediated current flowing across the membrane.Comparison of the currents was carried out using a two-way ANOVA with Tukey's test, with significance set to a P value < 0.05.

Oocyte swelling assay
Oocytes injected with 1 or 2 ng dspE or 20 ng avrE cRNA per oocyte were imaged using Motic Images Plus 3.0 software connected to a Moticam X3 camera (Motic China) on an SHR Plan Apo 1×, working distance 60, magnification lens of a stereoscope (Nikon SMZ18).At 0.01 ng dspE or 0.1 ng avrE cRNA per oocyte that was used for TEVC recordings, no baseline oocyte swelling was observed.Baseline swelling began to be observed at >0.1 ng dspE or >10 ng avrE cRNA per oocyte.For baseline oocyte swelling, starting oocyte images were recorded immediately after each injection, and then every 2 h to 4 h for 24 h.Oocytes were kept in bath saline of 200 mOsm with or without PAMAM inhibitors in the stereoscope room at 18-19 °C for the entire period.Each picture depicting five oocytes (replicates) was analysed with Fiji v.2.3.0 software 48 .Data are presented as absolute volume at a given evaluation time or as change in volume in relation to the start point (immediately after cRNA injection).For hypoosmotic-induced swelling, oocytes expressing DspE or AvrE were transferred into a fivefold-diluted ND96 bath saline (40 mOsm) and were immediately imaged as described for baseline swelling once every 20 s for 10-20 min or until oocytes injected with dspE or avrE started to burst.Data are presented as change in volume in relation to the first picture in diluted saline.Pictures were also arranged in sequence to create time-lapse videos showing oocyte swelling and bursting.One-way ANOVA with Tukey's test was used for multiple comparisons within a dataset, with significance set to a P value < 0.05.For dataset with repeated measures over time, as in the hypoosmotic-induced swelling assay, a two-way repeated measures ANOVA with Dunnett's test was used instead, with significance also set to a P value < 0.05.

Oocyte dye uptake assay
Two hours after injection with 1 or 2 ng of dspE or 20 ng of avrE cRNA, oocytes were placed in ND96 bath saline with or without 5 µg ml −1 fluorescein, 1 mg ml −1 GFP and/or 5 mM PAMAM G1 inhibitor and incubated until evaluation time, as indicated in the figure legends.Oocytes were rinsed twice in ND96 bath saline and imaged as described above for oocyte swelling assay, with a few exceptions: they were imaged at a ×2 magnification with either a bright-field or GFP-B filter.In the Motic Images Plus software, the green channel gain was increased to improve green fluorescence detection.Although specific values of the green channel gain value varied across different independent assays, all configurations were kept the same across all treatments within the same experiment.Bright-field and fluorescence images of each oocyte were stacked using Fiji software and the integrated density of fluorescence was measured within oocyte boundaries and subtracted from the fluorescence values observed on the image background, so data are presented as corrected total cell fluorescence (for short: cell fluorescence).Two-way ANOVA, with Tukey's test, was used for multiple comparisons within a dataset, with significance set to a P value < 0.05.

eGFP purification
For eGFP purification, pET28-eGFP 49 was transformed into E. coli Rosetta(DE3).eGFP production was induced by adding 0.25 mM IPTG to bacterial culture for 4 h at 28 °C.eGFP was purified from total cell lysate using Ni-NTA agarose beads in the extraction buffer (50 mM Tris-Cl, pH 8.0, 250 mM NaCl, 5% glycerol, 0.1 mM phenylmethylsulfonyl fluoride).Before the oocyte uptake test, the buffer was exchanged to ND96 bath saline using Amicon Ultra-4 centrifugal filter units (MilliporeSigma).

Liposome preparation and liposome dye release assay
Soy extract lipids in chloroform were purchased from Avanti Polar Lipids and stored in glass vials 24 .These solutions were evaporated under a stream of nitrogen until a thin lipid film formed and then dried in a vacuum desiccator chamber overnight.On the second day, the lipid film was dissolved in a suspension buffer (HBS buffer: 20 mM HEPES, 300 mM NaCl, pH 8.0) containing 50 mM 5(6)-carboxyfluorescein (Novabiochem) or 50 mg ml −1 polysucrose 40-fluorescein isothiocyanate conjugate (FITC-polysucrose, molecular mass of 30-50 kDa with an estimated diameter of 80 Å, MilliporeSigma).To solubilize lipids, the solution in the glass vial was sonicated for 15 min and then incubated in a 37 °C water bath for at least 1 h.Then the lipid solution was subjected to eight freeze-thaw cycles, in which lipids were frozen in liquid nitrogen for 5 min and then thawed in a 37 °C water bath for 10 min, to reduce the formation of multilamellar liposomes.To control the liposome size, liposomes were extruded through a polycarbonate filter (200 nm, Whatman) 25 times using a mini extruder (Avanti Polar Lipids) with Hamilton glass syringes.Carboxyfluorescein-or FITC-polysucrose-loaded liposomes were purified by centrifugation at 41,000 r.p.m. for 20 min in a TLA 100.3 rotor incorporating three sequential wash steps.After the final wash, carboxyfluorescein-or FITC-polysucrose-loaded liposomes were resuspended in HBS buffer to give a final carboxyfluorescein-loaded liposome concentration of 1 mg ml −1 and FITC-polysucrose-loaded liposome concentration of 0.5 mg ml −1 (ref.51).
Release of the liposome contents was assessed using the selfquenching property and fluorescence of carboxyfluorescein-and FITC-polysucrose.The HBS buffer composition in and outside the liposome was the same (20 mM HEPES, 300 mM NaCl, pH 8.0).Permeability induced by DspE was evaluated by incubating 10 µl DspE protein solution with 90 µl carboxyfluorescein-or FITC-polysucrose-loaded liposomes (0.25 µg µl −1 ).The fluorescence intensity was measured every 30 s continuously for 2 h after addition of the purified DspE protein (WT or mutant) to the liposomes in a SpectraMax M3 (Molecular Devices).Then 5 µl of 20% Triton X-100 (Sigma-Aldrich) was added to the 100-µl solution to fully release the dye and its readings were measured for 20 min.The average reading of the last 3 min was used for normalization (100% dye release).In the compound inhibition assays, the buffer, DspE protein and PAMAM G1 inhibitor, at a total volume of 10 µl, were first mixed thoroughly with pipetting, and then 90 µl carboxyfluorescein-or FITC-polysucrose-loaded liposomes was added to a total volume 100 µl.The spectrofluorometric excitation and emission parameters were set at the wavelengths of 485 and 510 nm for carboxyfluorescein-and FITC-polysucrose molecules.
The DspE protein stock solutions (2.5−25 µM) contained 1 mM DTT.The liposome assays were carried out at 0.05-0.15µM DspE concentrations with the final DTT concentration less than 0.02 mM.The presence of DTT in the protein buffer did not affect the fluorescence of carboxyfluorescein (Extended Data Fig. 5e).Similarly, the presence of PAMAN G1 at concentrations in the range of 0.3-300 µM did not affect the intrinsic fluorescence of carboxyfluorescein (Extended In E. amylovora DspE, the β-barrel starts from a small β-hairpin from K932-E956, which is followed by a large insertion of multiple helices (including a vertical helix bundle) from E957-D1276.The main β-barrel follows from S1277-G1813, though it is disrupted by several insertion loops and helices, including T1403-E1430 (a helix-turn-helix motif), A1567-H1647 (a horizontal helix bundle), N1662-P1712 (an insertion loop followed by a helix-turn-helix motif), and K1723-N1753 (two antiparallel helices).The β-barrel is further appended with a C-terminal helix and an extended loop (G1814-S1838).Mutated outwardfacing residues (L1776, L1777, and L1778) and inward-facing residues (K1399 and K1401) of the β-barrel are labeled in pink and blue, respectively.

Fig. 1 |
Fig. 1 | Model and cryo-EM images of E. amylovora DspE.a, Threedimensional model of E. amylovora DspE generated by AlphaFold2 using MMseqs2 (ColabFold).DspE (residues 298-1838) is shown in the cartoon model in a rainbow colour gradient, with the N terminus in blue and the C terminus in red.b, Cryo-EM two-dimensional class averages of DspE, revealing a circular arrangement of domains around a pore.Scale bars, 5 nm.

Fig. 2 |
Fig. 2 | DspE and AvrE activities in Xenopus oocytes and liposome.a, Schematic of the three oocyte assays, showing details for the two-electrode voltage clamp (TEVC) to test ion conductance (bottom left) (Vc, voltage command; R e , electrode resistance; R m , membrane resistance; and C m , membrane capacitance), the baseline and induced swelling and burst assay to test water conductance (bottom middle) and dye uptake to test conductance to molecules larger than single ions (bottom right).b,c, DspE and AvrE induce ion currents in the two-electrode voltage clamp assay.Mean ± s.e.m. (n = 5 oocytes) current values at different test pulses from oocytes expressing DspE (0.01 ng cRNA per oocyte) or AvrE (0.1 ng cRNA per oocyte) were recorded.d, DspE (2 ng) and AvrE (20 ng) induced fast oocyte swelling and burst at 24 h after cRNA injection when placed in a low-osmolarity (40 mOsm) solution.The data are presented as mean ± s.e.m. (n = 5 oocytes) of increased oocyte

Fig. 3 |
Fig. 3 | Inhibition of DspE and AvrE channels by the PAMAM dendrimer G1.The assays were carried out according to Fig. 2a,g, except with inhibitors added to the bath saline.a,b, DspE-dependent (a) and AvrE-dependent (b) currents were inhibited by G1.Solid lines represent fit values for current (µA) across the entire voltage range, with dashed lines representing the lower and upper 95% confidence interval of a quadratic polynomial regression for each treatment after subtracting control values.See Extended Data Fig. 4c for DspE expression in G1-containing ND96.c,d, Baseline swelling of oocytes injected with 1 ng dspE (c) or 20 ng avrE (d) cRNA was reduced in the presence of G1.Data show mean ± s.e.m. (n = 5 oocytes) increase in volume from the start point (at the time of injection) after subtracting control values.e, Inhibition of fluorescein uptake.G1 reduced fluorescein entry into oocytes expressing DspE as evaluated 6 h after injection with 1 ng dspE per oocyte.Data are presented as in Fig. 2e.f, DspE-mediated dye release from liposomes in the presence of increasing concentrations of G1.The result is representative of three experimental replicates.g, Dose-dependent inhibition of liposome dye release.IC 50 , halfmaximal inhibitor concentration.Data show mean ± s.e.m. (n = 3 batches of liposome preparations for 0.3, 1, 3 and 300 µM G1; n = 4 batches of liposome preparations for 10, 30 and 100 µM G1).Experiments were independently carried out two times for a,b,e, four times for c,d, and seven times for f,g.P values were calculated using Two-way ANOVA (e) or two-way repeated measures ANOVA (c,d).

Extended Data Fig. 1 |
AlphaFold2 models of the AvrE-family effector proteins.The top-ranked models, predicted local distance difference test (pLDDT), and predicted aligned error (PAE) of E. amylovora DspE, P. carotovorum DspE, P. syringae pv.tomato AvrE, and P. stewartii WtsE are shown in panels a, b, c, and d, respectively.The models are predicted by AlphaFold2 using MMseqs2 (ColabFold).For each protein, the N-terminal disordered loop with low pLDDT scores is not shown, while the remaining residues are shown in Cα traces and colored in rainbow, with the N-terminus in blue and C-terminus in red.Extended Data Fig. 2 | Sequence alignment of representative AvrE-family effectors.Sequences of E. amylovora DspE, P. carotovorum DspE, Pst DC3000 AvrE, and P. stewartii WtsE are aligned using Clustal Omega 44 .Domain regions are indicated above the aligned sequences.Sequence identities between different protein pairs are labeled.The β-barrel is formed with multiple helices and long loops inserted within the primary sequence of the β-barrel.

Extended Data Fig. 3 |
Characterization of DspE-induced whole-cell currents.a,b, DspE-induced currents were not inhibited by niflumic acid or fipronil.Current values (mean ± s.e.m.; n = 5 oocytes) at different test pulses from oocytes expressing DspE proteins were recorded once in the ND96 recording buffer at 15 h after injection with 0.01 ng cRNA, and a second time after 10 min of incubation with 100 µM of each inhibitor.c, Cation replacement experiment.After 15 h of incubation, cells were recorded in the normal ND96 buffer, and then in a new recording solution where sodium in ND96 was replaced with another cation (see details in the Methods section).The data show mean ± s.e.m. (n = 5 oocytes) values at each test pulse for each cation after subtracting currents from control cells.d,e, Anion replacement experiment.Same as presented in c (mean ± s.e.m., n = 5 oocytes), but with chloride in ND96 being replaced by other elementary anions (d) the organic anion MES -(e).In e, either 100% or only 50% of the Cl -was replaced by MES -.Experiments were independently performed three (a,c,e) or two (b,d) times with similar results.Two-way ANOVA values and exact P-values for all comparisons are detailed in the Source Data files.Extended Data Fig. 4 | Immunoblot detection of AvrE and DspE proteins and elution profile of MBP-DspE and MBP-DspE Δβ-barrel proteins.a, Oocyte surface biotinylation assay of DspE.Two nanograms of wild-type or mutant dspE cRNA was injected into oocytes, which were incubated in ND96 media at 18 °C for 15 h before being subjected to surface protein biotinylation assay (see Methods).Anti-β-Actin antibody detect β-Actin, a cytoplasmic protein serving as a negative control.b,c,e,f, Detection of AvrE and DspE proteins expressed in oocytes.Oocytes injected with 1 ng (unless otherwise noted in the gel lane label) avrE, dspE or mutant dspE cRNA or H 2 O (control) were incubated in ND96 media at 18 °C for 19 h before being subjected to SDS-PAGE and immunoblot analysis.In c, oocytes were incubated in the absence or presence of 5 or 10 mM PAMAM G1. d, Detection of DspE proteins expressed in tobacco leaves. 1 × 10 8 CFU/mL of Agrobacterium tumefaciens GV3101 containing pER8 empty vector, pER-dspE or pER-dspE mutants were syringe-inoculated into leaves of Nicotiana benthamiana, kept at 22 °C for 24 h before leaves were painted with 90 µM estradiol to induce protein expression for 8 h and then subjected to SDS-PAGE and immunoblotting.Asterisks in the pER8 and DspE WT lanes indicate a faint nonspecific protein band.Because active DspE expressed much more poorly in plant cells than mutant DspE proteins, mutant DspE samples were diluted 20 times with 2×SDS sample buffer (See Supplementary Fig. 1d).Experiments were independently performed two times.See Supplementary Fig. 1 for image cropping.g,h, Purified WT MBP-DspE (g) or MBP-DspE Δβ-barrel (h) was eluted on a Suprose 6 increase 10/300 GL column and was analyzed on a SDS-PAGE gel.FPLC traces and gel images in g and h are representative of 3 experimental replicates.See Supplementary Information Fig. 1 for image cropping.Extended Data Fig. 5 | DspE activities in Xenopus oocytes, Arabidopsis and liposome.a, DspE and AvrE induced a baseline swelling of many oocytes in 200 mOsm bath saline.Oocytes were imaged and measured at 24 h after 2 ng dspE or 20 ng avrE cRNA injection.Plot shows mean ± s.e.m. (n = 10 oocytes) and individual replicate values for the increased oocyte volume in relation to its initial volume.See Extended Data Fig. 4e, f for immunoblotting of DspE and AvrE proteins expressing in oocytes.b, Five-week-old Arabidopsis wild type Col-0 and transgenic Col-0/DEX::his-avrE plants (basal expression without dexamethazone induction).c, Changes in protoplast volume (mean ± s.e.m.; n = 24 protoplasts) when isolated protoplasts were incubated in protoplast incubation buffer containing 320 mM (low osmolarity) mannitol for 1 h compared to protoplast incubation buffer containing normal 400 mM mannitol for 1 h before image capture and volume analysis with Image J software.d, Liposome dye release assay using fluorescein isothiocyanate conjugated polysucrose 40 (FITC-polysucrose 40) and carboxyfluorescein (CF).e, Normalized liposome dye release (mean ± s.e.m.; n = 3 batches of liposome preparations) after addition of triton.Raw fluorescence readings were normalized to the buffer control samples.Experiments presented in this figure were independently performed two times.One-way ANOVA on Ranks (a,e) or two-tailed student's t-test (c) values and exact P-values for all comparisons are detailed in the Source Data files.Extended Data Fig. 6 | Functional analysis of mutant DspE proteins in Xenopus oocytes and tobacco.a, Effect of mutations on DspE-induced currents in oocytes.Mean ± s.e.m. (n = 5 oocytes) current values at different test pulses from oocytes expressing wild-type or mutant DspE proteins at 15 h after injection with 0.01 or 0.1 ng cRNA.Note: At 0.01 cRNA injection, all mutant DspE proteins did not induce currents.Next, 10-fold increase in mutant dspE cRNA (i.e., 0.1 ng) was injected into oocytes, revealing current induction by DspE K1399E/K1401E , suggesting the K1399E/K1401E double mutations only partially affect ion conductance, consistent with results in panels b and c. b, Effect of mutations on DspE-induced baseline swelling in oocytes.Plots represent mean ± s.e.m. (n = 6 oocytes) and individual values of increased oocyte volume in relation to its initial volume 15 h after 1 ng cRNA injection.c, Water-soaking assay in tobacco leaves. 1 × 10 8 CFU/mL of Agrobacterium tumefaciens GV3101 containing pER8 empty vector, pER-dspE, pER-dspE mutants were syringe-inoculated into Nicotiana benthamiana leaves (infiltration areas circled) and kept at 22 °C for 24 h before leaves were painted with 90 µM estradiol to induce DspE expression.Water-soaking symptom (dark-coloured appearance) was assessed at 8 h and 24 h after estradiol induction, showing a complete loss of water-soaking induction by DspE β-barrel and DspE L1776E/L1777E/L1778E , but only delayed water-soaking by DspE K1399E/K1401E .Experiments were independently performed two times.Two-(a) or one-(b) way ANOVA values and exact P-values for all comparisons are detailed in the Source Data files.See Extended Data Figs.4b and d for immunoblotting of DspE and DspE mutant expression.Extended Data Fig. 7 | Effects of PAMAM inhibitors on DspE activities in oocytes and liposome and on plant phenotypes and bacterial growth.a, Inhibition of ion currents in oocytes.DspE-dependent currents were inhibited by G0 in a dose-dependent manner across all test pulses.Solid lines represent fit values for current (µA) across the entire voltage range, with dashed lines representing the lower and upper 95% confidence interval of a quadratic polynomial regression for each treatment after subtracting control values (n = 5 oocytes).b,c, PAMAM G1 does not affect plant growth or seed production of Arabidopsis Col-0 plant.Five-week-old Col-0 plants were sprayed with 10 µM PAMAM G1 every week until 12 weeks old.b, Picture of 8-week-old plants is shown here as an example.c, Seeds were collected at 15 weeks old.No obvious difference in plant appearance or seed production was observed.Data shown as mean ± s.e.m. (n = 3 pots of plants, each pot had 4 plants).d, PAMAM G1 does not impact bacteria multiplication in vitro (in LB agar medium).Ea273 or Pst DC3000 cells in the logarithmic growth phase were spotted, at a 10-fold serial dilution (from left to right), on low-salt LB agar plates with or without 50 µM PAMAM G1.Plates were kept at 28 °C for 2 days to visualize colonies.e, Normalized liposome dye release after addition of triton.Fluorescence readings of groups with increasing concentrations of G1 were normalized to the no compound controls and are shown as mean ± s.e.m. (n = 6 batches of liposome preparations for 0.3, 1, 3, and 300 µM G1; n = 8 batches of liposome preparations for 10, 30, and 100 µM G1).Experiments were performed two (a) or three (b,c,d,e) times with similar results.Two-tailed student's t-test (c), One-way ANOVA on Ranks (e), and regression (a) values are detailed in the Source Data files.Extended Data Fig. 8 | A working model for the molecular actions of AvrEfamily effectors in plants.AvrE-family effectors act primarily as a novel class of water/solute-permeable channels dedicated to creating osmotic/water potential perturbation and a water/nutrient-rich apoplast, in which bacteria multiply within the infected plant tissues.AvrE-family effectors can additionally engage host proteins, including plant protein phosphatase PP2A subunits, type one protein phosphatases (TOPPs) and receptor-like kinases (RLKs), possibly to modulate AvrE-family channel properties or to optimize the pathogenic outcomes of AvrE/DspE-family channel activities, including water soaking, host cell death and defence suppression.Extended Data Fig. 9 | Water infiltration is sufficient to dampen callose deposition in Arabidopsis leaves.Arabidopsis Col-0 leaves were syringeinfiltrated with 1 µM flg22 and immediately covered by a clear plastic dome to maintain infiltrated leaves water-soaked ("water-soaked"; spotty, darker appearance) or air-dried to let infiltrated leaves returned to pre-infiltration appearance ( ~ 1 h) and then covered by a clear plastic dome ("no water soaked"; uniform, lighter appearance).a, Leaf pictures and callose (bright dots) images were taken at 8 h post flg22 infiltration.White scale bar indicates 100 µm.b, Quantification of callose deposition (mean ± s.e.m.; n = 18 leaf images [1 mm 2 each] of callose deposit).Experiments were performed three times with similar results.Two-tailed student's t-test values are detailed in the Source Data files.

Extended Data Table 1 | Effects of DspE and AvrE expression on oocyte membrane resting potential
Experiments were independently performed three times with similar results.Exact P-values for all comparisons are detailed in the Supplementary Data.