![The role of the polymorphic ARC2 positions 401 and 403 in elicitor-dependent and elicitor-independent activation. A, Alignment of the amino acid sequences of Gpa2 and Rx1 in sequence exchange fragments 3a, 3b, and 3c. The predicted secondary structure is shown above the sequences. Bars represent α-helices, and arrows denote β-strands. Polymorphic positions are highlighted by gray boxes. Positions 401 and 403 are indicated by stars. The identities of these positions were exchanged between Gpa2 and Rx1 via targeted mutagenesis and introduced in constructs either as single substitutions or as a pair. B, Agroinfiltration assay of Gpa2 or the autoactive Gpa2/Rx1 construct G13R45 and mutant versions thereof. The Gpa2 variants were coexpressed with its elicitor RBP1 from Globodera pallida. The G13R45 variants were expressed in the absence of an elicitor to assess the elicitor-independent cell-death response. C, The effect of exchanging the residues at positions 401 and 403 between Rx1 and Gpa2 on the CC-NB-ARC/LRR interaction. In c-Myc-tagged versions of the CC-NB-ARC domains of Gpa2 and Rx1, the residues at positions 401 (Gpa2 R and Rx1 Q) and 403 (Gpa2 Y and Rx1 S) were exchanged. All possible combinations of Gpa2 and Rx1 CC-NB-ARC and LRR domains were tested for interaction in an anti-c-Myc coimmunoprecipitation assay. The immunoblots (bottom) show the coimmunoprecipitated 4×HA-tagged LRR domains. The LRR of Gpa2 binding the CC-NB-ARC R401Q of Gpa2 is indicated by a chevron. D, Elicitor-dependent weakening of the CC-NB-ARC/LRR interaction. The CC-NB-ARC and LRR domains of either Gpa2 or Rx1 were coexpressed in the absence and presence of their elicitors (G. pallida RBP1 and PVX CP106, respectively). Protein was extracted 32 hours post-infiltration, and the c-Myc-tagged CC-NB-ARC domains were immunoprecipitated. E, Interaction of the wild-type and R401Q version of the CC-NB-ARC of Gpa2, with the FLAG-tagged LRR in the presence of the coexpressed elicitor (G. pallida RBP1). The c-Myc-tagged CC-NB-ARC domains were immunoprecipitated. [See online article for color version of this figure.]](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/plphys/162/3/10.1104_pp.113.218842/3/plphys_v162_3_1510_f3.jpeg?Expires=1749948290&Signature=V4kPy59dp-EnjXQmy4KFvPkNvHyTar3M-qeNu2wHz~sgfi54VvlnxfZzr5EkTS6SU5obyepO0bSkPpEK9ae3ABNde5Lm00e2eLzoCCCQeqwSsQ5ePmWQk~YW5eOY0m8WTit7LOnHRC85PAPvyYD6X3T7a0FA-8dEf~tvqPkrMSpwL010zr9kOSMWToDhvLhbWe6uyaq3TiFvWP-UCdL44BlOuPYHxwrgNMk0ah9RFdc6oPDZbxNawDlpp1YlB42I7PfIc~pEa9oqnxUO7PxqXOTJhAQ5zWPRX3R~WhbHZMMfvekNV1Uqco~NJ~d~outY02yCB86ALoEuUlR6VRtzjQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The role of the polymorphic ARC2 positions 401 and 403 in elicitor-dependent and elicitor-independent activation. A, Alignment of the amino acid sequences of Gpa2 and Rx1 in sequence exchange fragments 3a, 3b, and 3c. The predicted secondary structure is shown above the sequences. Bars represent α-helices, and arrows denote β-strands. Polymorphic positions are highlighted by gray boxes. Positions 401 and 403 are indicated by stars. The identities of these positions were exchanged between Gpa2 and Rx1 via targeted mutagenesis and introduced in constructs either as single substitutions or as a pair. B, Agroinfiltration assay of Gpa2 or the autoactive Gpa2/Rx1 construct G13R45 and mutant versions thereof. The Gpa2 variants were coexpressed with its elicitor RBP1 from Globodera pallida. The G13R45 variants were expressed in the absence of an elicitor to assess the elicitor-independent cell-death response. C, The effect of exchanging the residues at positions 401 and 403 between Rx1 and Gpa2 on the CC-NB-ARC/LRR interaction. In c-Myc-tagged versions of the CC-NB-ARC domains of Gpa2 and Rx1, the residues at positions 401 (Gpa2 R and Rx1 Q) and 403 (Gpa2 Y and Rx1 S) were exchanged. All possible combinations of Gpa2 and Rx1 CC-NB-ARC and LRR domains were tested for interaction in an anti-c-Myc coimmunoprecipitation assay. The immunoblots (bottom) show the coimmunoprecipitated 4×HA-tagged LRR domains. The LRR of Gpa2 binding the CC-NB-ARC R401Q of Gpa2 is indicated by a chevron. D, Elicitor-dependent weakening of the CC-NB-ARC/LRR interaction. The CC-NB-ARC and LRR domains of either Gpa2 or Rx1 were coexpressed in the absence and presence of their elicitors (G. pallida RBP1 and PVX CP106, respectively). Protein was extracted 32 hours post-infiltration, and the c-Myc-tagged CC-NB-ARC domains were immunoprecipitated. E, Interaction of the wild-type and R401Q version of the CC-NB-ARC of Gpa2, with the FLAG-tagged LRR in the presence of the coexpressed elicitor (G. pallida RBP1). The c-Myc-tagged CC-NB-ARC domains were immunoprecipitated. [See online article for color version of this figure.]
