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.
An analysis of disease resistance networks in maize is being conducted by identifying protein-protein interactions. Several kernel proteins associated with the resistance of kernels to Aspergillus flavus infection/aflatoxin contamination have already been identified. Some of these proteins were also shown to have fungicidal or fungistatic properties. To further analyze these resistance-associated proteins (RAPs), to shed light on their co-regulation, as well as to better understand their role in resistance, a predicted interactome (graphical modeled all protein-protein interactions) for maize based on protein orthology (genes of the same origin in different species) of Arabidopsis thaliana was constructed. This interactome facilitates the integration and interrogation of transcript 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. To improve the resolution of this interactome, it was recently re-constructed using the newly released completed genome sequence of corn. The new interactome is being validated by identifying maize proteins that interact with ZmCup1 [a protein with a cupin domain (a family of proteins with the same conserved deoxyribonucleic acid (DNA) region)]. Yeast two hybrid (widely accepted method of verifying a protein-protein interaction) screens are being used to identify ZmCup1 interacting partners. The list of identified partners will be compared and contrasted to that predicted by the interactome. ZmCUP1, whose corresponding gene, ZmCup1, was identified as a RAP in maize, inhibited the germination of Aspergillus flavus conidia by 75%. We have produced ZmCup1 protein in high amounts, and its anti-fungal activity against several other plant pathogens is currently being assessed. Efforts to investigate other protein-protein interactions using the maize interactome will be under way once identity of proteins interacting with ZmCUP1 are validated. Some of the findings of this investigation have been reported in a Master’s thesis by a former Southern Illinois University graduate student who performed most of the research. A manuscript is being written reporting these results as well. The results of this research, by further characterizing RAPs, aid in our pursuit of identifying and characterizing novel endogenous sources of resistance to aflatoxin accumulation in corn and to ear rot. Research progress was monitored through phone calls, emails and reports.