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. 2012 Jul;14(7):1071-84.
doi: 10.1111/j.1462-5822.2012.01779.x. Epub 2012 Mar 27.

Activation of a plant nucleotide binding-leucine rich repeat disease resistance protein by a modified self protein

Brody J DeYoung  1 , Dong Qi, Sang-Hee Kim, Thomas P Burke, Roger W Innes
Affiliations

Activation of a plant nucleotide binding-leucine rich repeat disease resistance protein by a modified self protein

Brody J DeYoung et al. Cell Microbiol. 2012 Jul.

Abstract

Nucleotide binding-leucine rich repeat (NB-LRR) proteins function as intracellular receptors for the detection of pathogens in both plants and animals. Despite their central role in innate immunity, the molecular mechanisms that govern NB-LRR activation are poorly understood. The Arabidopsis NB-LRR protein RPS5 detects the presence of the Pseudomonas syringae effector protein AvrPphB by monitoring the status of the Arabidopsis protein kinase PBS1. AvrPphB is a cysteine protease that targets PBS1 for cleavage at a single site within the activation loop of PBS1. Using a transient expression system in the plant Nicotiana benthamiana and stable transgenic Arabidopsis plants we found that both PBS1 cleavage products are required to activate RPS5 and can do so in the absence of AvrPphB. We also found, however, that the requirement for cleavage of PBS1 could be bypassed simply by inserting five amino acids at the PBS1 cleavage site, which is located at the apex of the activation loop of PBS1. Activation of RPS5 did not require PBS1 kinase function, and thus RPS5 appears to sense a subtle conformational change in PBS1, rather than cleavage. This finding suggests that NB-LRR proteins may function as fine-tuned sensors of alterations in the structures of effector targets.

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Figures

Fig. 1
Fig. 1. Both PBS1 cleavage products are necessary and sufficient for activating RPS5 and associate with RPS5
A. Activation of RPS5 by co-expression with both PBS1 cleavage products. The indicated proteins were transiently co-expressed in N. benthamiana. Leaves were photographed 24 h after transgene induction. N-term, PBS1 N-terminal engineered cleavage product; C-term, PBS1 C-terminal engineered cleavage product; EV, empty vector. B. Quantification of HRs induced by co-expression of RPS5 and PBS1 cleavage products. The HR was quantified as described in Experimental Procedures, with sample sizes of 5 injections. Error bars indicate standard error. C. Co-immunoprecipitation of PBS1 cleavage products with RPS5. The indicated PBS1 constructs were transiently co-expressed with RPS5:Myc or empty vector. D. AvrPphB and RPS5K189N:Myc in N. benthamiana. Proteins were immunoprecipitated with anti-cMyc (IP). As a control for expression, a portion of each sample was taken prior to immunoprecipitation (input). Immunoblots were performed with the antibodies indicated on the right. The slower migrating band in panel C detected in the anti-HA blot corresponds to full-length PBS1 due to incomplete cleavage by AvrPphB. E. Co-IP of engineered PBS1 cleavage products with each other. The indicated constructs were transiently co-expressed in N. benthamiana and immunoprecipitated with anti-cMyc. sYFP is used as a negative control and is targeted to the plasma membrane by an N-terminal fusion with the first 20 amino acids of RPS5 (Qi et al., 2012).
Fig. 2
Fig. 2. The amino- and carboxy-terminal non-kinase domain portions of PBS1 are dispensable for RPS5 activation
A. Schematic representation of the PBS1 protein showing relevant mutations and truncation constructs. The kinase domain is indicated by gray shading and the full-length protein is 456 amino acids long. The AvrPphB recognition sequence (GDK) is indicated in the kinase domain. Cleavage occurs after lysine 243 and is marked with a zigzag line. Missense substitutions are indicated above the protein diagram and are marked by a straight line. C-terminal and N-terminal truncation constructs are indicated by rectangles with numbers indicating the position of the terminal amino acid residue. Ability to induce visible HR when co-expressed with AvrPphB and RPS5 in N. benthamiana (panel B) is indicated by ‘+’ and ‘−’ symbols inside the rectangles. B. The indicated PBS1 constructs were transiently expressed with RPS5 and AvrPphB in N. benthamiana and assessed for HR elicitation. PBS1 constructs were named according to the position of the amino acids present (e.g 69-371 contains amino acids 69 through 371). For amino-terminal truncations, a methionine was added for translation initiation. For carboxy-terminal truncations, a stop codon was added for translation termination. C. Quantification of the HR using electrolyte leakage. Error bars indicate standard deviation. Conductivity was measured after floating leaf disks in deionized water for 14 hours following transgene induction.
Fig. 3
Fig. 3. Kinase inactive variants of PBS1 activate the HR and interact with RPS5
A. The indicated PBS1 constructs were transiently expressed with RPS5 and AvrPphB in N. benthamiana and assessed for HR elicitation. A representative leaf for each construct was photographed 24 h after transgene induction. EV, empty vector. B. The HR was quantified as described in Experimental Procedures, with sample sizes of 56–58 injections. Error bars indicate standard error. C. The indicated constructs were transiently co-expressed in N. benthamiana. Proteins were immunoprecipitated with anti-cMyc (IP). Immunoblots were performed with the antibodies indicated on the right. EV, Empty Vector; K115N, PBS1K115N; G252R, PBS1G252R.
Fig. 4
Fig. 4. Loss of PBS1 function by truncation corresponds to loss of cleavage by AvrPphB
PBS1 constructs were transiently expressed in N. benthamiana in the absence or presence of AvrPphB. A. Immunoblot of PBS1 truncation products expressed in the absence of AvrPphB showing that all expressed and migrated at the expected sizes. B. Immunoblot of PBS1 truncation products expressed in the presence of AvrPphB. Only a subset of PBS1 truncations were cleaved by AvrPphB as evidenced by appearance of a faster migrating band (indicated with arrows). EV, empty vector.
Fig. 5
Fig. 5. Insertion of alanine residues at the PBS1 cleavage site activates RPS5
A. Schematic representation of synthetic PBS1 constructs. The kinase domain (amino acids 78-360) and activation segment (amino acids 231-260) of PBS1 are represented by a light blue box and dark blue box, respectively. Synthetic PBS1 constructs containing the indicated alanine insertions after lysine 243 are shown above the diagram. B. HR phenotypes of Agrobacterium-infiltrated N. benthamiana leaves. The indicated synthetic and wild type PBS1:HA constructs were transiently co-expressed with RPS5-Myc or with RPS5-Myc and AvrPphB. 50 μM dexamethasone was sprayed 40 hours after injection and photographs were taken 24 hr later. This experiment was repeated three times with similar results. C. Quantification of the HR triggered by the PBS1 constructs described in (B) using electrolyte leakage. Error bars indicate standard deviation. Conductivity was measured at the indicated time points after dexamethasone treatment. This experiment was repeated once with a similar result. D. Expression of PBS1 constructs described in (B) was confirmed by immunoblot analysis with anti-HA antibody. The same blot was stained with Ponceau S solution to show equal loading of protein samples. Samples were taken 4 hr after dexamethasone treatment.
Fig. 6
Fig. 6. Predicted structure of the PBS1 kinase domain aligned with the known structure of Pto
A. Ribbon diagrams are shown for both PBS1 (yellow) and Pto (white). The activation segment of PBS1 is colored in red, with the AvrPphB cleavage site (lysine 243) indicated in green. B. Wild-type PBS1 is shown in yellow and PBS1 with an insertion of five alanines at the AvrPphB cleavage site in magenta. Note that the computer model attempts to maximize the alignment between the two forms of PBS1, but it is likely that the five alanine insertion would cause greater conformational changes than indicated in the model.
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References

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