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. 2012 Oct 2;109(40):16371-6.
doi: 10.1073/pnas.1212332109. Epub 2012 Sep 17.

Distinct regions of the Pseudomonas syringae coiled-coil effector AvrRps4 are required for activation of immunity

Kee Hoon Sohn  1 , Richard K Hughes, Sophie J Piquerez, Jonathan D G Jones, Mark J Banfield
Affiliations

Distinct regions of the Pseudomonas syringae coiled-coil effector AvrRps4 are required for activation of immunity

Kee Hoon Sohn et al. Proc Natl Acad Sci U S A. .

Abstract

Gram-negative phytopathogenic bacteria translocate effector proteins into plant cells to subvert host defenses. These effectors can be recognized by plant nucleotide-binding-leucine-rich repeat immune receptors, triggering defense responses that restrict pathogen growth. AvrRps4, an effector protein from Pseudomonas syringae pv. pisi, triggers RPS4-dependent immunity in resistant accessions of Arabidopsis. To better understand the molecular basis of AvrRps4-triggered immunity, we determined the crystal structure of processed AvrRps4 (AvrRps4(C), residues 134-221), revealing that it forms an antiparallel α-helical coiled coil. Structure-informed mutagenesis reveals an electronegative surface patch in AvrRps4(C) required for recognition by RPS4; mutations in this region can also uncouple triggering of the hypersensitive response from disease resistance. This uncoupling may result from a lower level of defense activation, sufficient for avirulence but not for triggering a hypersensitive response. Natural variation in AvrRps4 reveals distinct recognition specificities that involve a surface-exposed residue. Recently, a direct interaction between AvrRps4 and Enhanced Disease Susceptibility 1 has been implicated in activation of immunity. However, we were unable to detect direct interaction between AvrRps4 and Enhanced Disease Susceptibility 1 after coexpression in Nicotiana benthamiana or in yeast cells. How intracellular plant immune receptors activate defense upon effector perception remains an unsolved problem. The structure of AvrRps4(C), and identification of functionally important residues for its activation of plant immunity, advances our understanding of these processes in a well-defined model pathosystem.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
AvrRps4C is an antiparallel α-helical coiled coil. (Left) Cartoon representation of the α-helices with residues in sticks (from the N terminus) Ala157, Arg159, Glu163, Leu167, Glu175, Gln177, Ile180, Glu182, Pro185, Ala186, Glu187, Arg196, Arg199, and Arg213 shown with carbon atoms in yellow (structure-based mutants) or carbon atoms in cyan (natural variation mutants). In gray are the residues forming the “knobs” of the coiled coil. (Right) Electrostatic surface representation with the prominent negative patch highlighted by the arrow.
Fig. 2.
Fig. 2.
Glu175Ala and Glu187Ala variants interfere with AvrRps4-triggered HR and/or immunity. (A) HR assays in different Arabidopsis lines using PF0-1(T3S) strains carrying avrRps4:pBBR1MCS-5 constructs [Ws-0 and RLD are wild-type Arabidopsis lines, rps4-21 is a loss-of-function mutant in rps4, and RLD(RPS4Ler) is a transgenic RLD line carrying functional RPS4 from Landsberg]. One or two red asterisks indicate weak or strong HR, respectively. The photograph was taken at 20 hpi. This experiment was repeated four times with similar results. (B and C) In planta bacterial growth assays of Pto DC3000, carrying the constructs used in A, in Ws-0, and in mutant lines (rrs1-1 is a loss-of-function mutant in rrs1). Bacterial growth in the eds1-1 line (eds1 loss of function) is shown in Fig. S5. Means labeled with the same symbols are not statistically different at the 5% confidence level based on Tukey’s test. These experiments were performed three times with similar results.
Fig. 3.
Fig. 3.
Natural variation in AvrRps4 alleles confers distinct RPS4/RRS1-dependent and RPS4/RRS1-independent recognition specificities. HR assay in Arabidopsis using PF0-1(T3S) strains carrying avrRps4 natural variants. The photograph was taken at 20 hpi. This experiment was performed three times with similar results.
Fig. 4.
Fig. 4.
Leu167 is specifically required for RPS4/RRS1-independent HR. Arabidopsis leaves infiltrated with PF0-1(T3S) carrying wild-type or variant avrRps4Ppi151:pBBR1MCS-5 constructs show RPS4/RRS1-dependent and RPS4/RRS1-independent HR except Leu167Thr, which loses RPS4/RRS1-independent recognition. The photograph was taken at 20 hpi.
Fig. 5.
Fig. 5.
AvrRps4C and EDS1 do not interact in yeast cells or in co-IP from plants. (A) AvrRps4C does not interact with EDS1 in N. benthamiana total protein extracts following transient expression. Full-length, epitope-tagged AvrRps4, PAD4 (both C-terminal tags), and EDS1 (N- or C-terminal tag as indicated) were expressed in N. benthamiana leaves. For AvrRps4-HA and AvrRps4-GFP, only the processed AvrRps4C was detectable. IP was performed using anti-HA (Sigma) or anti-GFP (Chromotek) agarose beads. Ponceau staining shows equal loading of proteins. IB and IP indicate immunoblot and immunoprecipitation, respectively. This experiment was repeated four times with similar results. (B) AvrRps4 does not interact with EDS1 in yeast cells. Yeast strains carrying AD-EDS1 and the indicated binding domain constructs were tested for reporter gene activation on SD-Ura/Trp/His/Leu agar medium containing X-gal [−UTHL(X-Gal)]. The image was taken 3 d after transferring colonies to fresh medium. This experiment was repeated twice with similar results.
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