Open Access Research article

Homo-dimerization and ligand binding by the leucine-rich repeat domain at RHG1/RFS2 underlying resistance to two soybean pathogens

Ahmed J Afzal12, Ali Srour12, Abhishek Goil12, Sheeja Vasudaven13, Tianyun Liu4, Ram Samudrala4, Navneet Dogra5, Punit Kohli5, Ayan Malakar12 and David A Lightfoot12*

Author Affiliations

1 Department of Molecular Biology, Microbiology and Biochemistry and Center for Excellence the Illinois Soybean Center, Southern Illinois University at Carbondale, IL 62901, USA

2 Genomics Core Facility; Department of Plant Soil and Agricultural Systems, Southern Illinois University at Carbondale, Carbondale, IL 62901-4415, USA

3 Department of Molecular Biology and Biochemistry, University of California at Irvine, Irvine, CA 92697-4560, USA

4 Department of Microbiology Box 357242, University of Washington, Seattle, WA 98195-7242, USA

5 Department of Chemistry, Southern Illinois University at Carbondale, IL 62901, USA

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BMC Plant Biology 2013, 13:43  doi:10.1186/1471-2229-13-43

Published: 15 March 2013

Additional files

Additional file 1: Figure S1:

Effect of exogenous treatments of roots of X5 and Westag 97 with CLE peptides a root dip assay. SCN susceptible plants that had been infested with HgType 0 (isolate JB3) were depotted at 10 dai (24 dag) and a 0.5g root sample taken. Roots were then dipped in CLE peptides TGIF or HgCLE in water at 50 pM concentration and returned to the infested soil. Wilting and stem bending was more severe in X5 (panel B-D) plants that Westag 97 (panel F-H) plants. Panels A and E were before depotting. Panels B and F were 2 min after dipping and repotting. Panels C and G were 15 minutes after treatment. Panel D and G were at root harvest 28 dai (42 dag). Cyst were abundant on the water treated plants but 10±6 were found on each of the 5 plants dipped in HgCLE or GmTDIF (Table 2).

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Additional file 2: Table S1:

PROCHECK analysis of main chain parameters for the modeled GmRLK18-1 LRR structure. Stereochemical parameters such as percentage residues in allowed regions, omega angle standard deviation, the hydrogen bond standard deviation and the overall quality of the models are shown for the modeled GmRLK18-1-LRR and typical proteins resolved at 2.3 Å resolutions.

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Additional file 3: Figure S2:

Ramachandran plot of homology-modeled structure of GmRLK18-1 based on porcine ribonuclease inhibitor template. Each amino acid residue is represented by a black dot. Red shows the most favored residue positions, yellow additionally allowed residue positions, beige residue generously allowed residue positions and white disallowed residue positions.

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Additional file 4: Table S2:

Ramachandran plot statistics for GmRLK18-1 calculated from PROCHECK. Approximately 71 percent of the amino acid residues within the modeled protein were in allowed regions of the Ramachandran plot. An additional 26 percent of the amino acid residues were in additional allowed regions whereas 2.4 percent of the amino acids fell in generously allowed regions of the Ramachandran plot. Less than one percent residues (3) were in disallowed regions.

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Additional file 5: Table S3:

RAPDF scores for 3 LRR mutants and the wild type GmRLK18-1-LRR. The RAPDF scores suggested that these mutations may affect the stability of the homodimeric protein although these residues were not directly implicated in the homodimer interface.

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Additional file 6: Figure S3:

Sequence diversity among the seven GmRLK18-1 allotypes. Alanine at position 87 is only present in the ‘Peking’ sequence. Two additional changes Q to K at position 115 and H to N at position 274 are not exclusive to resistance type I.

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Additional file 7: Table S4:

Effect of three non-synonymous substitutions on protein stability calculated from the Fold X algorithm (http://fold-x.embl-heidelberg.de). The computed free energy of folding and the change in free energy between the wild type protein (Peking allele) and the mutant proteins is shown. The H to N and Q to K change increase the free energy of folding, whereas the alanine to valine change results in a significant decrease in the free energy. (DOC 45 kb)

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Additional file 8: Figure S4:

The 3D structures of LRR containing proteins showing high sequence homology to RHG1. Panel (a) shows the crystal structure of BRI1 (pdb id: 3RGX). Brassinosteroid recognition is mediated through the LRR domain of BRASSINOSTEROID-INSENSITIVE 1 (BRI1). BR1I exists as a monomer. The protein exists as a helical solenoid structure. (b) crystal structure of the Polygalacturonase inhibiting protein (PGIP). The PGIP protein (PDB id: 1ogq) is a cell wall localized protein that interacts with fungal endopolygalacturonases. (c) X-ray structure of the porcine ribonuclease inhibitor (PRI). The Leucine rich repeat of PRI (PDB id: 2BNH) forms tight complexes with ribonucleases thereby regulating RNA levels. PRI adopts a horseshoe configuration with the LRR motif composed of repeat beta-loop helix units.

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