AvrRps4 effector family processing and recognition in lettuce

Abstract

During pathogenesis, effector proteins are secreted from the pathogen to the host plant to provide virulence activity for invasion of the host. However, once the host plant recognizes one of the delivered effectors, effector-triggered immunity activates a robust immune and hypersensitive response (HR). In planta, the effector AvrRps4 is processed into the N-terminus (AvrRps4^N ) and the C-terminus (AvrRps4^C ). AvrRps4^C is sufficient to trigger HR in turnip and activate AtRRS1/AtRPS4-mediated immunity in Arabidopsis; on the other hand, AvrRps4^N induces HR in lettuce. Furthermore, AvrRps4^N -mediated HR requires a conserved arginine at position 112 (R112), which is also important for full-length AvrRps4 (AvrRps4^F ) processing. Here, we show that effector processing and effector recognition in lettuce are uncoupled for the AvrRps4 family. In addition, we compared effector recognition by lettuce of AvrRps4 and its homologues, HopK1 and XopO. Interestingly, unlike for AvrRps4 and HopK1, mutation of the conserved R111 in XopO by itself was insufficient to abolish recognition. The combination of amino acid substitutions arginine 111 to leucine with glutamate 114 to lysine abolished the XopO-mediated HR, suggesting that AvrRps4 family members have distinct structural requirements for perception by lettuce. Together, our results provide an insight into the processing and recognition of AvrRps4 and its homologues.

Keywords: AvrRps4; XopO; effector-triggered immunity; hypersensitive response; processing; recognition.

FIGURE 1

Like AvrRps4^N and HopK1^N, XopO^N can trigger a hypersensitive response in Lactuca sativa ‘Kordaat’. (a) N‐terminally HA‐tagged proteins and empty vector pTA7002 (EV) were transiently expressed in L. sativa ‘Kordaat’ using Agrobacterium at an optical density of 0.4. Two days postinfiltration, infiltrated leaves were sprayed with dexamethasone (Dex) solution (50 μM). The photographs were taken under white light and UV light 1 day after Dex treatment. This experiment was repeated twice with identical results. (b) Cell death level was quantified by conductivity as a measure of electrolyte release by cells. Three hours after Dex treatment, lettuce leaf discs were harvested and placed in double‐distilled water containing 0.005% Silwet and 50 μM Dex to initiate measurements. Values represent averages from four replicates and error bars denote SD. Two‐way analysis of variance was performed for the statistical tests. Letter codes indicate groups that are significantly different to others according to Tukey's tests (p < 0.0001). This experiment was repeated twice with identical results. (c) Protein expression of tested constructs in L. sativa ‘Koordat’ was confirmed by western blots. Samples were collected 3 h after Dex treatment. Ponceau S staining confirmed equal loading.

FIGURE 2

R111L blocks XopO processing but fails to abolish the XopO‐mediated hypersensitive response in Lactuca sativa ‘Kordaat’. (a) N‐terminally HA‐tagged proteins and empty vector pTA7002 (EV) were transiently expressed in L. sativa ‘Kordaat’, as described in Figure 1. This experiment was repeated twice with identical results. (b) Cell death level was quantified by conductivity as a measure of electrolyte release by cells. Three hours after dexamethasone (Dex) treatment, lettuce leaf discs were harvested and placed in double‐distilled water containing 0.005% Silwet and 50 μM Dex to initiate measurements. Values represent averages from four replicates and error bars denote SD. Two‐way analysis of variance was performed for the statistical tests. Letter codes indicate groups that are significantly different to others according to Tukey's tests (p < 0.05). This experiment was repeated twice with identical results. (c) Protein expression of tested constructs in L. sativa ‘Kordaat’ was confirmed by western blots. Samples were collected 3 h after Dex treatment. Ponceau S staining confirmed equal loading.

FIGURE 3

R88L blocks AvrRps4^F processing but still triggers a hypersensitive response in Lactuca sativa ‘Kordaat’. (a) N‐terminally HA‐tagged proteins and empty vector pTA7002 (EV) were transiently expressed in L. sativa ‘Kordaat’, as described in Figure 1. This experiment was repeated twice with identical results. (b) Cell death level was quantified by conductivity as a measure of electrolyte release by cells. Three hours after dexamethasone (Dex) treatment, lettuce leaf discs were harvested and placed in double‐distilled water containing 0.005% Silwet and 50 μM Dex to initiate measurements. Values represent averages from four replicates and error bars denote SD. Two‐way analysis of variance was performed for the statistical tests. Letter codes indicate groups that are significantly different to others according to Tukey's tests (p < 0.01). This experiment was repeated twice with identical results. (c) Protein expression of tested constructs in L. sativa ‘Kordaat’ was confirmed by western blots. Samples were collected 3 h after Dex treatment. Ponceau S staining confirmed equal loading.

FIGURE 4

Double mutant R111L, E114K is sufficient to abolish the XopO‐mediated hypersensitive response in Lactuca sativa ‘Kordaat’. (a) N‐terminally HA‐tagged proteins and empty vector pTA7002 (EV) were transiently expressed in L. sativa ‘Kordaat’, as described in Figure 1. This experiment was repeated twice with identical results. (b) Cell death level was quantified by conductivity as a measure of electrolyte release by cells. Three hours after dexamethasone (Dex) treatment, lettuce leaf discs were harvested and placed in double‐distilled water containing 0.005% Silwet and 50 μM Dex to initiate measurements. Values represent averages from four replicates and error bars denote SD. Two‐way analysis of variance was performed for the statistical tests. Letter codes indicate groups that are significantly different to others according to Tukey's tests (p < 0.05). This experiment was repeated twice with identical results. (c) Protein expression of tested constructs in L. sativa ‘Kordaat’ was confirmed by western blots. Samples were collected 3 h after Dex treatment. Ponceau S staining confirmed equal loading. (d) Proposed models for AvrRps4 and XopO processing and recognition in lettuce. In lettuce, effector processing is not required for effector recognition. The putative resistance protein can recognize both full‐length and N‐terminal effectors. Blue arrows indicate the process of effector processing. Black arrows indicate the process of effector recognition. In AvrRps4, R88 or R112 is important for AvrRps4^F processing. However, only R112 is necessary for AvrRps4^N/F recognition (top). In XopO, the conserved R111, not the conserved R87, is important for XopO^F processing. However, R111 and E114, together, are necessary for XopO^N/F recognition. Compared to AvrRps4, XopO is more easily recognized by lettuce due to the two key residues (bottom).