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A phytobacterial TIR domain effector manipulates NAD+ to promote virulence

Summary The Pseudomonas syringae DC3000 type III effector HopAM1 suppresses plant immunity and contains a Toll/interleukin‐1 receptor (TIR) domain homologous to immunity‐related TIR domains of plant nucleotide‐binding leucine‐rich repeat receptors that hydrolyze nicotinamide adenine dinucleotide (NAD+) and activate immunity. In vitro and in vivo assays were conducted to determine if HopAM1 hydrolyzes NAD+ and if the activity is essential for HopAM1’s suppression of plant immunity and contribution to virulence. HPLC and LC‐MS were utilized to analyze metabolites produced from NAD+ by HopAM1 in vitro and in both yeast and plants. Agrobacterium‐mediated transient expression and in planta inoculation assays were performed to determine HopAM1’s intrinsic enzymatic activity and virulence contribution. HopAM1 is catalytically active and hydrolyzes NAD+ to produce nicotinamide and a novel cADPR variant (v2‐cADPR). Expression of HopAM1 triggers cell death in yeast and plants dependent on the putative catalytic residue glutamic acid 191 (E191) within the TIR domain. Furthermore, HopAM1’s E191 residue is required to suppress both pattern‐triggered immunity and effector‐triggered immunity and promote P. syringae virulence. HopAM1 manipulates endogenous NAD+ to produce v2‐cADPR and promote pathogenesis. This work suggests that HopAM1’s TIR domain possesses different catalytic specificity than other TIR domain‐containing NAD+ hydrolases and that pathogens exploit this activity to sabotage NAD+ metabolism for immune suppression and virulence.


DNA manipulation
Desired DNA regions were amplified from Pto DC3000 genomic DNA using DreamTaq polymerase (Thermo Fisher Scientific). Standard protocols were followed for conventional ligation or gateway cloning. For conventional cloning, PCR products were digested with restriction enzymes and ligated to vectors with T4 DNA Ligase (New England Biolabs). For gateway cloning, amplicons were cloned into a pENTR vector (Invitrogen) and the resulting pENTR plasmids with insertion were recombined into gateway destination vectors with LR clonase to create recombinant constructs. Site-directed mutagenesis of the hopAM1 gene was achieved by amplifying two overlapping fragments of hopAM1 by PCR using primers introduced with specific mutation, then fusing the fragments in a second round of PCR with hopAM1 specific forward and reverse primers. The resulting PCR products were cloned into pENTR and the constructs containing desired mutation were confirmed by sequencing. All constructs generated for this study are listed in supplementary Table S1.

Purification of recombinant protein
Overnight E. coli BL21 cultures carrying pET28a(+) plasmids that express hopAM1, hopAM1 E191A, as well as empty vector (mock) were diluted at a ratio of 1:50 into 100 ml LM and grown with shaking at 37°C till an optical density (OD600nm) of 0.6 (~4 hours). Cultures were induced with 1 mM IPTG (Invitrogen) for 2.5 hours at 37°C. The induced cultures were pelleted by centrifugation at 16,000 g and resuspended with 10 ml CelLytic B (Sigma-Aldrich) buffer amended with Benzonase (50 U ml -1 ) (Sigma-Aldrich), Lysozyme (200 µg ml -1 ) (Sigma-Aldrich), and Protease Inhibitor Cocktail for His-tagged protein purification (10 µl ml -1 ) (Sigma-Aldrich). Cells were agitated at room temperature (25°C) for 15 minutes, then centrifuged at 16,000 g for 10 minutes. The supernatant cell lysates were mixed with 200 µl HIS-select Nickle Affinity resin (Sigma-Aldrich) for 15 minutes at room temperature (25°C) with agitation. The resin was washed three times with 2 ml 50 mM sodium phosphate, pH 8.0, 0.3 M sodium chloride and then eluted in 4 ml 50 mM sodium phosphate, pH 8.0, 0.3 M sodium chloride, and 250 mM imidazole. Eluate was concentrated for 45 minutes at 4,000 g using an Amicon Ultracel 3K Centrifugal Filter (Millipore) and then mixed with 2 ml of 0.64x PBS and re-concentrated two more times to a final volume of 100 µL.

Proteins and immunoblot analysis
For plant protein extraction, 48 hours after infiltration leaves were sampled with a 16 mm diameter cork borer. Samples were frozen in liquid nitrogen and ground with a plastic pestle, then resuspended in 200 µL x1.5 sample buffer, vortexed and boiled for 10 minutes before storage at -20°C until SDS-PAGE.
For yeast protein extraction, 1 ml of culture was spun down and resuspended in 0.1 M NaOH for five minutes as described by (Kushnirov, 2000), then spun down for 30 seconds at 15,000 g and resuspended in 100 µL x1.5 sample buffer, vortexed and boiled for 10 minutes before storage at -20°C until SDS-PAGE.
Extracted proteins were separated by SDS-PAGE and transferred to PVDF membrane using a TransBlot Turbo transfer system (Bio-RAD) with the standard setting. Immunoblots were performed with appropriate antibodies following standard protocols. The following primary antibodies were used: Rabbit anti-Lex (Millipore), Rat anti-HA (Roche), and Mouse anti-HIS (Sigma-Aldrich). All secondary antibodies were conjugated with alkaline phosphatase (Sigma-Aldrich). Immunoblots were visualized using CDP-Star (Roche).

Unmarked mutagenesis of P. syringae DC3000
Upstream and downstream DNA regions of avrPto, avrPtoB, hopAM1-1, or hopAM1-2 were PCR-amplified using DreamTaq or Phire II DNA polymerase (Thermo Fisher Scientific) and cloned into a gateway pENTR vector (Invitrogen). The resulting pENTR constructs were recombined with pLN5841 by LR clonase creating sacB-based constructs for unmarked mutagenesis. The resulting mutagenesis constructs were conjugated using tri-parental or biparental mating and integrated into the chromosome of Pto DC3000 by homologous recombination. Desired deletions were screened on KB medium containing sucrose (5%) to counter-select for the survived colonies that lack of antibiotic resistance. The final mutants were further verified by PCR.

Yeast cell death assay
Yeast strain EGY48 carrying pGilda derivatives were grown overnight at 30 °C in synthetic dropout glucose media lacking histidine (glucose-His). The cells were washed and resuspended in ddH2O to an OD600nm of 0.1. A 10-fold dilution series was plated on glucose-His agar or galactose-His and grown at 30 °C. Colonies were assessed for survival after three days.

Plant materials
All Arabidopsis plants were grown at 24 °C with a 10 hour light/14 hour dark cycle in micro-climate controlled growth chambers. N. benthamiana and N. tabacum cv. Xanthi plants were grown in standard greenhouses.

Agrobacterium-mediated transient assays
For assays with tobacco, Agrobacterium tumefaciens C58C1 strains carrying binary vectors were grown overnight, centrifuged at 4,696 g for 10 min and resuspended in induction medium (0.5 g L -1 sodium citrate, 10 mM MES, 1 g L -1 ammonium sulfate, 250 µM magnesium sulfate, 0.1% glycerol, 0.2% glucose, 10.5 g L -1 potassium phosphate dibasic, 4.5 g L -1 potassium phosphate monobasic) containing 150 µM acetosyringone (Fisher Scientific) and incubated with shaking at 30 °C for 6 hours. The induced Agrobacterium cultures were pelleted by centrifugation at 4,696 g for 10 min and resuspended in infiltration medium (2.15 g L -1 MS basal salts, 10 mM MES, pH5.6) containing 300 µM acetosyringone adjusted to an OD600nm of 0.8. Leaves of N. tabacum cv. Xanthi or N. benthamiana were infiltrated with needless syringe and plants were kept at room temperature. For inducible-expression constructs, twenty-four hours after infiltration infiltrated leaves were sprayed with 20 µM estradiol (Sigma-Aldrich) containing 0.02% Silwet-L77 (Lehle Seeds) using a spray bottle. Samples were harvested at indicated time points for metabolite analysis and immunoblot analyses.

Confocal microscopy
GFP fusion proteins were transiently expressed in N. benthamiana via Agrobacterium tumefaciens infiltration. After 48 hours infiltrated leave discs were detached to monitor green fluorescence and imaged at 40x magnification with a Nikon A1-NiE confocal microscope. Images were visualized using NIS-Elements software with internal ruler.

Plant hypersensitivity response assays
For assays in tobacco, the development of a hypersensitive response was assessed and photographed 72 hours after infiltration with Agrobacterium cultures. For assays with Arabidopsis, overnight cultures of Pto DC3000 D28E strains were resuspended in 10 mM MgCl2 and adjusted to an OD600nm of 0.1 (equivalent to a cell density of 1x10 8 CFU ml -1 ). Leaves of four-week-old Arabidopsis accessions Xan-2, Xan-5, or Col-0 plants were infiltrated with a needleless syringe. Plants were covered with a plastic lid and maintained for moisture at room temperature (25°C). The development of a hypersensitive response or symptoms was assessed and photographed after 2-4 days.

In vitro NAD hydrolase and enzyme kinetics assays
Ten microliters of concentrated protein extract were mixed with 3 µL of 250 µM NAD + (Selleck Chemicals) in 0.64x PBS and incubated at room temperature (25°C). Samples at 0 and 60 minutes were mixed with 250 µL 50% methanol at -40°C and vortexed, then mixed with 250 µl chloroform at -40°C. Samples were then centrifuged at 15,000 g for 5 minutes at -10°C. The aqueous/methanol layer was removed, lyophilized, and stored at -80°C for HPLC analysis.
To calculate HopAM1 enzyme kinetics, in vitro reactions consisting of 10 microliters of protein laden bead suspension and 40 µl of 1, 10, 50, 100, 400, or 600 µM NAD + in 25 mM HEPES buffer pH=7.5 at room temperature with constant agitation. At 1 hour, reactions were quenched by pulling the beads to the side and transfer 40 microliters of the reaction mixture to a new tube containing 160 microliters of ice cold 0.5 M HClO4. Ten microliters of 2x Laemmli buffer (BioRad 1610737) supplemented with β-mercaptoethanol were added to the beads and boiled for ten minutes and subjected to gel electrophoresis on a 4-12% gradient Bis-TRIS polyacrylamide gel (Invitrogen NW04125). Protein concentration was approximated by staining the SDS-page gel with Coomassie blue stain (instant blue, AbCam AB119211) and application of densitometry using ImageJ with comparison to a set of BSA standards. NAD + consumption rates for HopAM1 were calculated by finding NAD + peak area reduction compared to empty vector control and using NAD + calibration curve to convert peak area reduction to change in number of moles of NAD + . Average change in NAD moles per minute of reaction was then calculated. The Curve Fitting Tool on MATLAB (Version R2020a) was used to fit NAD + consumption kinetics data to the Michaelis-Menten equation, V = Vmax * (NAD + Concentration) / (Km + NAD + Concentration) where parameters Vmax and Km are constants. Optimal values were output for the parameters Vmax and Km as well as an R 2 value for the data's fit to the model.

ROS assay
ROS production was determined following a previously described protocol (Asai et al., 2008). Briefly, Pseudomonas fluorescens 55 (Pf) (pLN1965) strains were grown overnight, suspended in 10 mM MgCl2 and infiltrated with a needleless syringe into leaves of five-week-old Arabidopsis plants at a density of 1x10 7 CFU ml -1 . After 24 hours leaf discs were excised using a 4 mm diameter cork borer and incubated in H2O in white 96 well plate overnight. H2O is then replaced with 0.5 mM L-012 and 1 µM flg22 in 10 mM MOPS-KOH at pH 7.4. The production of ROS was determined by counting photons using with a Synergy 5 luminometer (BioTek, Winooski, VT, USA).

Callose deposition assay
Pf(pLN1965) strains were grown overnight, suspended in 10 mM MgCl2 and infiltrated with a needleless syringe into leaves of five-week-old Arabidopsis Col-0 plants at a density of 1x10 7 CFU ml -1 . After 48 hours, callose deposits in infiltrated leaves were stained with aniline blue and visualized with a Zeiss Axioplan 2 microscope. The numbers of callose deposits were quantified using ImageJ (http://imagej.nih.gov/ij/) following a procedure previously reported (Guo et al., 2016).
In planta bacterial growth assay P. syringae strains were grown overnight, resuspended in 10 mM MgCl2 and infiltrated with a needleless syringe into leaves of five-week-old Arabidopsis at 1x10 5 cells ml -1 or sprayinoculated onto tomato cultivar 'Moneymaker' plants with 0.02% Silwet-L77 at a density of 1x10 8 cells ml -1 . Plants were maintained with moisture in covered trays at room temperature. A 6 mm diameter cork borer was used to sample inoculated leaves at the indicated time points. The samples were ground in 250 µl sterile ddH2O using a plastic pestle and 20 µl each of a 10-fold serial dilution series was plated on KB agar and incubated at 30°C. Bacterial colonies were numerated after 2-3 days.