Identification, Characterization And Anti-Fungal Activities Of Silk Proteins In Aspergillus Flavus Resistant And Susceptible Maize Inbreds

This study aimed to understand the mechanism of maize inbreds resistance to A. flavus by exploring the proteins that are differentially regulated in presence of pathogen. Silk has been hypothesized as one of the entry routes of fungal growth and so the proteome of silks was investigated by 1) performing a comparative proteomic study to identify silk proteins that are abundant in resistant maize inbreds and down-regulated or absent in susceptible inbreds, 2) identifying the up-regulated proteins in maize resistant and susceptible inbreds when challenged by A. flavus 3) by mapping the proteome of silk proteins in a A. flavus resistant inbred and 4) performing an antiungal assay to test antiungal activity of silk proteins extracted from resistant and susceptible maize inbreds. Using comparative proteomics, proteins that are contributing to the resistance phenotype and could be used for marker-assisted selection in breeding programs were identified from silks collected from resistant (Mp313E, Mp420) and susceptible (SC212m, Mp339) maize inbred 21 and 25 days after silk emergence (DAS) and also, from the silks of ears inoculated at 15 DAS and collected 6 days after inoculation (DAI). Silk proteins were extracted and analyzed by 2-dimensional gel electrophoresis (2-DE). Gel images were analyzed by PD Quest software (Bio-Rad) and proteins that were consistently different were identified using MALDI-TOF-TOF. Two candidate genes that were up-regulated in 21 and 25 DAS in resistant tissues were investigated for polymorphisms and their RNA expression was also studied. Nine proteins from all the differentially regulated proteins were mapped to chromosomes 1, 2, 4 and 6 which are known to have aflatoxin resistance QTLs. Proteome map of Mp313E silks was developed using 2-DE and multi dimensional identification technology (MudPIT) and approximately 971 identified proteins were functionally annotated from the sequences available at AgBase website. The reference map of Mp313E silks could also be used to link proteomics with trancriptomics, metabolic mechanisms and genomics. Antifungal assays using GFP-tagged A.flavus and chitinase assay on silk proteins from resistant and susceptible corn inbreds showed significant activity in the resistant line compared to the susceptible line (p\u3c0.01). A model describing the role of silk proteins in fungal resistance is proposed