The bacterial type III-secreted protein AvrRps4 is a bipartite effector

<div><p>Bacterial effector proteins secreted into host plant cells manipulate those cells to the benefit of the pathogen, but effector-triggered immunity (ETI) occurs when effectors are recognized by host resistance proteins. The RPS4/RRS1 pair recognizes the <i>Pseudomonas syringae</i> pv. pisi effector AvrRps4. AvrRps4 is processed <i>in planta</i> into AvrRps4^N (133 amino acids), homologous to the N-termini of other effectors including the native <i>P</i>. <i>syringae</i> pv. tomato strain DC3000 effector HopK1, and AvrRps4^C (88 amino acids). Previous data suggested that AvrRps4^C alone is necessary and sufficient for resistance when overexpressed in heterologous systems. We show that delivering AvrRps4^C from DC3000, but not from a DC3000 <i>hopK1</i>^<i>-</i> strain, triggers resistance in the Arabidopsis accession Col-0. Delivering AvrRps4^C in tandem with AvrRps4^N, or as a chimera with HopK1^N, fully complements AvrRps4-triggered immunity. AvrRps4^N in the absence of AvrRps4^C enhances virulence in Col-0. In addition, AvrRps4^N triggers a hypersensitive response in lettuce that is attenuated by coexpression of AvrRps4^C, further supporting the role of AvrRps4^N as a bona fide effector domain. Based on these results we propose that evolutionarily, fusion of AvrRps4^C to AvrRps4^N may have counteracted recognition of AvrRps4^N, and that the plant <i>RPS4/RRS1</i> resistance gene pair was selected as a countermeasure. We conclude that AvrRps4 represents an unusual chimeric effector, with recognition in Arabidopsis by RPS4/RRS1 requiring the presence of both processed effector moieties.</p></div

AvrRps4^N overexpression enhances bacterial virulence.

<p><i>In planta</i> bacterial growth was measured in two independent Col-0 AvrRps4^N lines (N2 and N8) and untransformed Col-0 after inoculation with DC3000 (top) and DC3000 <i>hopK1</i>^<i>-</i> (bottom) at 5x10⁴ cfu/mL in the presence of dexamethasone (Dex) or ethanol (mock). Values are averages from two independent experiments with quadruplicate samples, and error bars denote standard deviation. Asterisks denote statistically significant differences compared to bacterial growth in mock- and Dex-treated Col-0 based on two-tailed Student's t-tests (ns: non-significant, *<i>P</i><0.05, **<i>P</i><0.01, ****<i>P</i><0.0001).</p

Bacteria secreting AvrRps4^N and AvrRps4^C in tandem trigger resistance comparable to wild-type AvrRps4.

<p>Col-0 plants were inoculated with 5x10⁴ cfu/mL suspensions of the indicated DC3000 <i>hopK1</i>^<i>-</i> strains. AvrRps4 variants were delivered from separate broad host range plasmids (top: pML123 constructs; bottom: pVSP61 constructs). Error bars denote standard deviation. Values are averages from four independent experiments with triplicate samples, and error bars denote standard deviation, with letters indicating statistically significant differences (<i>P</i><0.05).</p

ETI is attenuated when AvrRpm1SP-AvrRps4 is delivered from DC3000 <i>hopK1</i>^- compared to DC3000.

<p>(A) Col-0 plants were inoculated with 5x10⁴ cfu/mL suspensions of either wild-type DC3000 or the <i>hopK1</i> deletion strain DC3000 <i>hopK1</i>^<i>-</i> containing empty vector (EV), or expressing AvrRpm1SP-AvrRps4 (A1SP-AvrRps4) or AvrRps4. Values are averages from three (DC3000) or four (DC3000 <i>hopK1</i>^<i>-</i>) independent experiments with triplicate samples, and error bars denote standard deviation, with letters indicating statistically significant differences (<i>P</i><0.0001). (B) Model of HopK1^N contribution to AvrRpm1SP-AvrRps4-triggered immunity.</p

AvrRps4^N triggers a hypersensitive response in lettuce which is attenuated by expression of AvrRps4^C.

<p>(A) N-terminally HA-tagged AvrRps4 full-length (4^FL), AvrRps4^N (4^N), AvrRps4^C (4^C), combined AvrRps4^N and AvrRps4^C (4^N+4^C), or empty-vector HA-pBA (EV) were expressed in <i>L</i>. <i>sativa</i> cv. Kordaat at an O.D. of 0.3. Cell death was imaged three days after infiltration. This experiment was repeated four times with identical results. (B) Conductivity as a measure of electrolyte release by cells undergoing HR. Measurements were taken at the indicated time points after vacuum infiltration of lettuce leaf discs with ddH₂O, 36 hours after infiltration with agrobacterium strains. Values represent averages from two independent experiments with quintuplicate samples normalized to maximal conductivity observed with AvrRps4^N at 48 h, and error bars denote SD. (C) Western blots confirming expression of proteins in <i>L</i>. <i>sativa</i>.</p

AvrRps4^N and HopK1^N are sufficient to trigger a hypersensitive response in lettuce.

<p>(A) Agrobacterium C58C1 containing the constructs for transient expression of N-terminally HA-tagged AvrRps4 full-length (4^FL), AvrRps4^N (4^N), AvrRps4^C (4^C), and HA-pBA empty vector (EV) was infiltrated into <i>Lactuca sativa</i> cv. Kordaat at an O.D. of 0.3. (B) Equivalent experiment with HopK1 full-length (K1^FL), HopK1^N (K1^N), HopK1^C (K1^C), and HA-pBA empty vector (EV). Cell death phenotypes in (A) and (B) were visualized three days post-infiltration. These experiments were repeated four times with identical results.</p

HopK1^N/AvrRps4^C chimeras are functional in ETI.

<p>(A) The indicated AvrRps4 and HopK1 constructs were delivered from the <i>P</i>. <i>fluorescens</i> strain EtHAn into Arabidopsis Ws-0. HR response was observed after 24 hours. This experiment was repeated at least twice with similar results. (B) <i>In planta</i> bacterial growth analysis of DC3000 <i>hopK1</i>^<i>-</i> secreting indicated wild-type or chimeric effectors. Ws-0 plants were inoculated with 5x10⁴ cfu/mL suspensions of bacteria. Values are averages from two independent experiments with triplicate samples, and error bars denote standard deviation, with letters indicating statistically significant differences (<i>P</i><0.0001).</p

AvrRps4^N and AvrRps4^C directly interact with EDS1.

<p>(A) Co-IP of microsomal EDS1 with AvrRps4^N and AvrRps4^C expressed in <i>N</i>. <i>benthamiana</i>. This experiment was repeated once with similar results. (B) <i>In vitro</i> interaction of AvrRps4^N and AvrRps4^C with EDS1 in <i>E</i>. <i>coli</i>. Proteins were pulled down and subjected to immunoblot analysis with either His or T7 antibodies. This experiment was repeated once with similar results.</p

Localization of GFP-tagged AvrRps4 variants.

<p>AvrRps4 full-length, AvrRps4^N, and AvrRps4^C with or without the AvrRpm1 signal peptide were C-terminally tagged with GFP and expressed in <i>N</i>. <i>benthamiana</i>. Cells were visualized after two days. For each construct, quantification of twenty random cells showed no variation in subcellular localization of GFP signal. Arrows indicate nuclei. This experiment was repeated twice with similar results.</p