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. 2010 Feb 2;107(5):2349-54.
doi: 10.1073/pnas.0904739107. Epub 2010 Jan 19.

The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence

Mike Wilton  1 , Rajagopal Subramaniam, James Elmore, Corinna Felsensteiner, Gitta Coaker, Darrell Desveaux
Affiliations

The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence

Mike Wilton et al. Proc Natl Acad Sci U S A. .

Abstract

Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes that serve as "non-self" features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as "non-self" features or induce a "modified-self" state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2(Pto) also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2(Pto) were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2(Pto) interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2(Pto). In support of this hypothesis, HopF2 (Pto) interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2(Pto) did not induce ETI and instead promoted P. syringae growth in Arabidopsis. This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2(Pto) and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Transgenic expression of HopF2Pto suppresses AvrRpt2-mediated ETI. (A) Half-leaves of untreated HopF2Pto transgenic plants (−DEX) or HopF2Pto transgenic plants treated with DEX for 24 h (+DEX) were infiltrated with Pto DC3000 (5 × 107 cfu/mL) expressing the indicated avirulence gene or empty vector (EV). Photographs of AvrB, AvrRpm1, or corresponding EV control were taken 6 h after inoculation, whereas HopZ1aPsyA2, AvrRpt2, or corresponding EV were taken ≈20 h after inoculation. Asterisks indicate leaves with visible HR collapse. (B) Trypan blue staining of untreated (−DEX) or 24 h DEX-treated (+DEX) HopF2Pto transgenic leaves 14 h postinoculation. (C) Electrolyte leakage of untreated (−DEX) or 24 h DEX-treated (+DEX) HopF2Pto transgenic leaf disks after infiltration with Pto DC3000 expressing the indicated constructs. (D) Growth analysis of Pto DC3000 expressing the indicated avirulence gene or the empty vector (EV) infiltrated into HopF2Pto or HopF2Pto D175A transgenic plants. Plants were treated with 30 μM DEX (+DEX) or water (−DEX) immediately after bacterial inoculation. Bacterial counts were performed 1 h postinoculation (day 0; filled bars) and 2 days postinoculation (day 2; open bars). Although Pto DC3000 empty vector did not grow significantly better in DEX-treated HopF2Pto:HA plants after 2 days of growth, a significant growth enhancement was observed after 3 days of growth (Fig. S7). Results are representative of three independent replicates. Error bars represent the SD from eight samples. “a” or “b” above the bar denotes statistically significant [Fisher’s protected least significant difference post hoc (FLSD) test, P < 0.05] differences between samples. Similar results were obtained with an independent transgenic HopF2Pto line, as well with a lower DEX concentration of 3 μM (Fig. S8).
Fig. 2.
Fig. 2.
HopF2Pto inhibits AvrRpt2-mediated degradation of RIN4. (A) Immunoblot analysis of RIN4 levels in Arabidopsis HopF2Pto and HopF2Pto D175A transgenic leaves infiltrated with Pto DC3000 empty vector (EV) or Pto DC3000 expressing AvrRpt2. Expression of HopF2Pto:HA or HopF2Pto D175A:HA was induced with 30 μM DEX 24 h before infection. Pto DC3000 was infiltrated at 5 × 107 cfu/mL, and samples were collected at the indicated time after infiltration. (B) HopF2Pto does not inhibit AvrRpt2 activation and self-processing in vitro. In vitro cleavage reactions (see Methods) were probed with HA antisera. Cleavage reactions were performed five times with similar results. ○, full-length AvrRpt2; *, processed AvrRpt2. (C) HopF2Pto interferes with AvrRpt2-mediated RIN4 proteolysis in vitro. Reactions were conducted as in B and probed with RIN4 antisera. Arrows indicate full-length RIN4 (I) and proteolytically cleaved RIN4 (II).
Fig. 3.
Fig. 3.
HopF2Pto interacts with RIN4 both in vitro and in vivo. (A) GST:RIN4- or GST-bound glutathione resin was incubated with recombinant 6xHIS:HopF2Pto, washed and probed for the presence of bound 6xHIS:HopF2Pto (see Methods). Equal volumes of 6xHIS:HopF2Pto added to GST:RIN4- or GST-bound glutathione resin were immunoblotted and probed with HIS antisera before washes (Top) or following washes (Middle). (Bottom) Ponceau stain before washes as a loading control. The GST and full-length RIN4 bands are indicated by an arrow. Bands below the full-length RIN4 band are RIN4 degradation products as determined by an anti-RIN4 blot (Fig. S9). (B and C) HopF2Pto associates with RIN4 in vivo. (B) Uninduced HopF2Pto transgenic leaves were harvested 24 h after mock treatment. Ten micrograms of input protein (Input) or entire elution (50 μL) of the immunoprecipitation (IP) by α-HA or α-RIN4 was loaded onto the SDS/PAGE gel and probed with the indicated antisera (see Methods). (C) HopF2Pto transgenic leaves were harvested 24 h after 30 μM DEX application and treated as described in B. α-HA immunoblot detects the presence of HopF2Pto:HA.
Fig. 4.
Fig. 4.
HopF2Pto virulence function requires the RIN4 Arabidopsis protein. (A and B) Growth of Pto DC3000 hopF2 − mutant expressing the indicated HopF2Pto ATG construct or the empty vector (EV) in Arabidopsis Col-0 leaves. Bacterial counts were performed 1 h postinoculation (day 0; filled bars) and 3 days postinoculation (day 3; open bars). Error bars represent the SD of eight samples. Experiments are representative of three independent trials. “a” or “b” above a bar denotes statistically significant [Fisher’s protected least significant difference post hoc (FLSD) test, P < 0.05] differences between samples. (C) Immunoblot analysis of indicated HopF2Pto ATG:HA protein expression in the Pto hopF2 − mutant. Bacteria were grown in minimal media to induce the type III secretion system, and equal amounts of protein were immunoblotted with HA antisera (33). Ponceau red staining is shown as a loading control. (D and E) Growth of Pto DC3000 hopF2 − mutant expressing HopF2Pto ATG:HA (HopF2Pto) or the empty vector (EV) in Arabidopsis Col-0 wild-type, rin4/rps2, rin4/rps2/rpm1, rps2, or rpm1 leaves. Bacterial counts were performed as in A and B. This experiment is representative of three independent trials.
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Comment in

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