The Pseudomonas syringae type III effector HopF2 suppresses Arabidopsis stomatal immunity

Abstract

Pseudomonas syringae subverts plant immune signalling through injection of type III secreted effectors (T3SE) into host cells. The T3SE HopF2 can disable Arabidopsis immunity through Its ADP-ribosyltransferase activity. Proteomic analysis of HopF2 interacting proteins identified a protein complex containing ATPases required for regulating stomatal aperture, suggesting HopF2 may manipulate stomatal immunity. Here we report HopF2 can inhibit stomatal immunity independent of its ADP-ribosyltransferase activity. Transgenic expression of HopF2 in Arabidopsis inhibits stomatal closing in response to P. syringae and increases the virulence of surface inoculated P. syringae. Further, transgenic expression of HopF2 inhibits flg22 induced reactive oxygen species production. Intriguingly, ADP-ribosyltransferase activity is dispensable for inhibiting stomatal immunity and flg22 induced reactive oxygen species. Together, this implies HopF2 may be a bifunctional T3SE with ADP-ribosyltransferase activity required for inhibiting apoplastic immunity and an independent function required to inhibit stomatal immunity.

Figure 1. Identification of HopF2/RIN4 complexes by gel filtration chromatography and immunoblotting.

Clarified extracts from HopF2:HA overexpressing plants were subjected to gel filtration chromatography on a Sephacryl S-300 HR 16/60 column. Every second fraction from the void volume was resolved by SDS-PAGE and immunoblotted with anti RIN4 immunosera and anti-HA IgG. Bands were visualized with HRP-conjugated secondary anti-bodies and Amersham ECL Advance detection kit. (a) Elution profile of dexamethasone treated Arabidopsis HopF2:HA clarified extract fractionated by gel filtration chromatography. Solid Arrow indicates elution of HA and RIN4 immunoreactive bands. Dashed arrow indicates elution of HA immunoreactive bands alone. Elution volumes of six molecular weight standards are shown as triangles. (b) Immunoblots (IB) showing co-elution of RIN4 and HA immunoreactive bands at high molecular weight. Results are representative of 3 independent replicates. Arrows indicate expected band size for RIN4 (25 kDa) and HopF2 (25 kDa), respectively.

Figure 2. HopF2 and RIN4 co-purifty during immunoaffinity purification from high-molecular weight FPLC fractions.

High Mr fractions containing HA immunoreactive (V_e 36–46 ml) bands were pooled (input, pool) and concentrated for one hour in a 10,000 Da Mr cutoff concentrator (input, [pool]). Concentrated pooled fractions were incubated with anti-HA magnetic resin (µMACS), immobilized, flow through (FT) collected and resin washed with low salt (LSW), no salt (NSW) buffer then eluted over four fractions with 0.1 M NH₄OH. Samples were resolved by SDS-PAGE and immunoblotted (IB) with α (RIN4) immunosera and α (HA) IgG. All bands are from the same blot exposure. Blots were cropped to remove molecular weight standards between lanes containing NSW and eluate fractions. Blots of purifications from -DEX tissue were overexposed relative to blots from +DEX purifications. Results are representative of 3 independent replicates.

Figure 3. Transgenic expression of HopF2 results in altered stomatal immunity.

Four week old Col-0 or transgenic Arabdiopsis expressing HopF2 or HopF2^D175A under the control of a dexamethasone inducible promoter were sprayed with 30 µM dexamethasone. Leaf discs were prepared 24 hours after dexamethasone treatment and incubated in water or 1×10⁸ CFU suspensions of wild type Pto DC3000. Epidermal peels were performed after one (a) or three (b) hours incubation and stomatal aperture was measured. Values are means ± S.E.M of n = 60 stomata from 3–5 leaves. Results are representative of 3 independent replicates. Asterisks denote significant differences between means (t-test, p<0.05). (c) ABA treatment induces stomatal closure in transgenic Arabidopsis expressing HopF2. Two week old Col-0 or transgenic Arabdiopsis expressing HopF2 or HopF2^D175A under the control of a dexamethasone inducible promoter were sprayed with 30µM dexamethasone. First and second true leaves were detached one hour after dexamethasone treatment and incubated in buffer overnight then buffer or 10µM ABA for two hours. Epidermal peels were performed and stomatal aperture was measured. Values are means ± S.E.M of n = 60 stomata from 3–5 leaves.

Figure 4. Transgenic expression of HopF2 increases virulence of surface inoculated, coronatine deficient Pto DC3118.

Four week old transgenic Arabidopsis expressing HopF2:HA under the control of a dexamethasone inducible promoter were sprayed with dexamethasone (+DEX) or water (–DEX). Plants were dipped into 1×10⁸ CFU suspensions of wild type Pto DC3000 or coronatine deficient Pto DC3118. (a) Wild-type HopF2:HA in Col-0 plants. (b) HopF2^D175A::HA in Col-0 plants. (c) Wild-type HopF2:HA in rin4/rps2 plants. Bacterial growth curve analyses were performed 3 days post inoculation. Values are means ± S.E.M from n = 12 plants. Asterisks denote significant differences between means (t-test, p<0.05). Results are representative of 3 independent replicates.

Figure 5. HopF2 inhibits PTI through distinct molecular mechanisms.

(a) Transgenic expression of HopF2 subverts MAP kinase activation in Arabidopsis. Eleven-day-old seedlings of Col-0, transgenic Arabidopsis expressing HopF2 and mutant Arabidopsis lacking mpk3 or mpk6 were incubated with 3 µM DEX (+DEX) or water (–DEX) 24 h prior to treatment with 1 µM flg22 (+flg22) or water. Samples were collected 20 min after treatment with flg22 (see Methods). Activated MAPKs were detected by immunoblotting using anti-p44/42 MAPK antibody. Mutant Arabidopsis lacking mpk3 or mpk6 represent negative controls for the respective MAPK bands. Ponceau staining is presented as a loading control. Results are representative of 5 independent replicates. (b) Transgenic expression of HopF2 inhibits callose deposition triggered by infiltration of Pto DC3000ΔhrcC. Four week old Col-0 or transgenic Arabidopsis expressing HopF2 were sprayed with 30 µM DEX (+DEX) or water (–DEX) 24 h prior to inoculation. Leaves were stained with aniline blue and visualized 24 h after inoculation of 1×10⁸ cfu/mL of Pto DC3000ΔhrcC. Callose deposition was quantified using Image J by counting number of papillae in 12 field of views per treatment. Values are means ± SEM from n = 12 leaves. Asterisk denotes statistically significant differences between DEX treatment samples as determined by a pairwise Student’s t-test (P<0.05). Results are representative of 3 independent replicates. (c) Transgenic expression of HopF2 increases growth of syringe inoculated Pto DC3000ΔhrcC. Four week old Col-0 or transgenic Arabidopsis expressing HopF2 were treated with 30 µM DEX (+DEX) or water (–DEX) immediately after bacterial inoculation (1×10⁵ cfu/ml). Bacterial counts were performed 1 h after inoculation (day 0; filled bars) or 3 days postinoculation (day 3; open bars). Values are means ± SEM from n = 12 leaves. Asterisk denotes statistically significant differences between day 3 samples as determined by a pairwise Student’s t-test (p<0.05). Results are representative of 3 independent replicates. (d) Transgenic expression of HopF2 inhibits flg22 triggered ROS production. Four week old transgenic Arabidopsis expressing HopF2 were sprayed with 30 µM DEX (+DEX) or water (–DEX) 24 h prior to inoculation and leaf discs floated in 2 µM flg22. ROS production was quantified using luminol and horseradish peroxidase with luminescence recorded every two minutes for 60 min. Values are mean sums of total luminescence ± SEM from from n = 6 wells. Asterisk denotes statistically significant differences between DEX treatment samples as determined by a pairwise Student’s t-test (P<0.05).