Investigating the functional regions of amino acids (AAs) from 1170–1200 and the C-terminal end of Rp1-D21.

<p>(A) Investigating the point mutations in the region of AAs 1170–1200 which are important for inducing hypersensitive response (HR). Top: Multiple sequence alignment of Rp1-D, Rp1-D21, Rp1-dp2, V1 and V2. The black, red and light blue shaded regions represent 100%, 75% and above 50% similarity of the amino acids, respectively. The polymorphic amino acids are boxed, and the position of 1184 (corresponding to 1182 of Rp1-D21) is labeled above the sequences. Bottom left: The HR phenotype of V1, V2 and V1-derived point mutants as indicated. A representative leaf was photographed at 3 days post infiltration (dpi). Bottom right: Total protein was extracted from agro-infiltrated leaves at 30 hours post infiltration (hpi), and anti-HA antibody was used to detect the expression of the fused proteins. Equal loading of protein samples was shown by Ponceau-S staining of Rubisco subunit. (B) Investigating the phenotype conferred by various recombinants and point mutants at the C-terminal end of Rp1-D21. Top: Multiple sequence alignment of the C-terminus of Rp1-D, Rp1-D21 and Rp1-dp2. The polymorphic amino acids are boxed. Bottom left: Schematic diagram of the constructs used. The K1184N mutation is indicated by a red vertical bar in Rp1-dp2(K1184N), V1(K1184N) and V18. Bottom middle: The HR phenotype of the constructs transiently expressed in <i>N</i>. <i>benthamiana</i>. A representative leaf was photographed at 3 dpi. Bottom right: Total protein was extracted from agro-infiltrated leaves at 30 hpi, and anti-HA antibody was used to detect the expression of the fused proteins. Equal loading of protein samples was shown by Ponceau-S staining of Rubisco subunit. The experiments were repeated three times with the same results. (C) The final model presenting the functional regions important for autoactivity. In this model, white represents regions derived from Rp1-dp2 and black the region from Rp1-D that need to be present in the same construct to result in the autoactive HR in <i>N</i>. <i>benthamiana</i>. Regions in grey can be derived from either paralog. The combination of AA 458–651 from Rp1-dp2 and AA 1184–1292 (especially N1184 and the last 16 AAs in red vertical bars) from Rp1-D are important for HR induction.