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Abstract Detail


Plant-Pest Interactions

Yu, Hyun Young [1], Kittur, Farooqahmed S. [2], Bevan, David R. [3], Esen, Asim [2].

Mapping of β-Glucosidase Aggregation Factor Binding Regions and Identification of Specific Amino Acids on the Maize β-Glucosidase Isozyme Glu1 that are Involved in Glu1-BGAF Interaction.

We have shown that a lectin called β-glucosidase aggregating factor (BGAF) is responsible for β-glucosidase aggregation in maize. Furthermore, we have mapped the BGAF-binding regions on β-glucosidase by domain swapping between maize β-glucosidase isozymes Glu1 and Glu2 (binder) and sorghum β-glucosidase (dhurrinase) isozymes Dhr1 and Dhr2 (non-binder). Results of gel-shift assays suggest that the amino acids critical for BGAF binding are located in the N-terminal region (Ile72-Thr82) and the C-terminal region (Phe466-Ala512) plays a minor role in the interaction. To determine more precisely the contribution of these regions, frontal affinity chromatography (FAC) was used to determine the dissociation constants (kd). The kd value for wild type Glu1 was 240 nM, whereas chimera C2 (C-terminal region of Glu1 swapped with the corresponding region of Dhr1) gave a kd of 550 nM. In contrast, chimera C43 (N-terminal region of Glu1 swapped with that in Dhr1) showed no affinity for BGAF. Chimeras constructed by swapping shorter segments within the 47 amino acids long extreme C-terminal region showed higher kd values. The above results indicate that the critical amino acids involved in BGAF-binding are in the N-terminal peptide spanning Ile72-Thr82 and that the C-terminal region (Phe466-Ala500) is required for BGAF-Glu1 complex stability. To identify specific amino acids, we mutated the unique amino acids in the N-terminal region of Glu1 (Ile72, Asn75, Lys81, and Thr82) to those in Dhr2 (Val72, Asp75, Glu81, and Glu82). Of these single amino acid substitutions, the replacement of Lys81 or Thr82 in Glu1 with the corresponding residue (Glu81 or Glu82) in Dhr2, respectively, completely abolished binding to BGAF, suggesting that these residues play critical role in BGAF binding.


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1 - Virginia Polytechnic Institute and State University, Biological Sciences, 5028, Derring Hall, Department of Biological Sciences, Virginia Tech, Blacksburg, VA, 24061, U.S.A.
2 - Virginia Polytechnic Institute and State University, Biological Scienc
3 - Virginia Polytechnic Institute and State University, Biochemistry
4 - Virginia Polytechnic Institute and State University, Biological Scienc

Keywords:
none specified

Presentation Type: Plant Biology Abstract
Session: P
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM
Number: P14019
Abstract ID:1660


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