Location: Food and Feed Safety Research2010 Annual Report
1a. Objectives (from AD-416)
To identify and characterize novel endogenous sources of resistance to ear rot and aflatoxin accumulation in corn.
1b. Approach (from AD-416)
The focus of this research will be on a subset of the proteins that have been identified in proteomic screening conducted at the USDA/ARS/SRRC lab, as putatively involved in aflatoxin accumulation and/or ear rot resistance. Identified proteins will be further characterized and the corresponding genes will be isolated and cloned. Proteins will be expressed in a suitable in vitro system and their possible physiological functions will be investigated. Knowledge of A. flavus functional genomics techniques will be used to identify/test proteins for target-specific interruption of aflatoxin biosynthesis. The information obtained through the above studies will be used to explore the potential of the identified genes to serve as markers in marker-assisted breeding programs.
3. Progress Report
Characterization of ZMCup1, a maize cupin (a type of protein) with antifungal properties against Aspergillus (A.) flavus, which produces the potent carcinogen, aflatoxin. Sequences of peptide fragments of the cupin protein, found to be more abundant in corn lines exhibiting resistance to A. flavus, were provided by Food & Feed Safety Research Unit, Agricultural Research Service. Upon analysis, the fragments were found to be part of a protein that belongs to the cupin (a diverse protein family containing a conserved structure) superfamily. Proteins in the cupin superfamily have diverse reported functions including seed storage, auxin binding (a protein that binds the plant hormone, auxin), sucrose binding, signaling functions and diverse enzymatic activity. The corresponding gene, ZMCup1, was identified in corn. The gene was expressed using a bacterial expression system. The expressed ZmCup1 inhibited the germination of A. flavus conidia (reproductory spores of the fungus) by 75%. Analysis of Disease Resistant Networks in Maize by Identification of Protein-Protein Interactions: Several kernel proteins associated with the resistance of kernels to ear rots have already been identified. Some of these proteins were shown to have fungicidal or fungistatic properties. To further analyze these resistance-related proteins, shed light on their co-regulation, and to better understand their role in resistance, we constructed a predicted interactome (a graphical model of all protein interactions) for maize based on protein orthology (genes in different species derived from a common ancestor) of Arabidopsis thaliana. This interactome allows the integration and interrogation of ribonucleic acid transcript (the product of gene expression) abundance and protein abundance expression data to investigate and confirm possible interactions among these proteins and to reveal and identify possible crosstalk between networks resulting in disease resistance. The interactome was used to predict interacting partners of ZmCUP1. Predicted interactions were validated through yeast two hybrid screens and confirmed through co-immunoprecipitation assays. The interaction of ZmCUP1 with a putative transcription factor and with a heat shock protein have been confirmed. Research progress was monitored through teleconferencing, frequent email communications, and reports.