A. flavus-secreted proteins during an in vivo fungal-cotton carpel tissue interaction

Authors: Mellon, Jay; Mattison, Chris; Grimm, Casey

Submitted to: Physiological and Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/4/2015
Publication Date: 9/17/2015
Citation: Mellon, J.E., Mattison, C.P., Grimm, C.C. 2015. A. flavus-secreted proteins during an in vivo fungal-cotton carpel tissue interaction. Physiological and Molecular Plant Pathology. 92:38-41.

Interpretive Summary: Aflatoxin is a very potent carcinogen and toxin that is produced by the fungus Aspergillus flavus. When this fungus infects target crops (corn, cotton, peanuts, tree nuts), the developing seed can be contaminated with this toxin, rendering the product unfit for additional agricultural uses. Cottonseed is particularly susceptible to aflatoxin contamination. These fungi are capable of producing a large number of hydrolytic enzymes that are crucial to breaking down plant cell walls. These hydrolase activities help the fungus breach plant cell walls and gain access to potential nutrient sources that include free sugars, polysaccharides, lipids, and storage proteins. Cotton carpellary tissue is a tough, fibrous tissue that helps prevent the spread of potential microbial pathogens between sections (locules) of a cotton fruit (boll). Carpel tissue cell walls contain a complex array of cellulose, additional supportive polysaccharides and structural proteins. In order to better understand the roles of these enzymes in fungal virulence, developing cotton bolls were inoculated with different strains of A. flavus and incubated for 7 days. Proteins were extracted from fungal-inoculated carpel tissue and subjected to a controlled proteolysis to produce peptide libraries. Mass spectral analysis of a peptide library from a 35-kD fungal protein revealed a match to A. flavus oryzin precursor protein. Oryzin displays a proteinase activity and may aid the fungus in breaching plant cell walls by digesting structural wall proteins. This research will benefit cotton breeders, producers and pathologists, and will aid in the formulation of methods to prevent aflatoxin contamination of target crops.

Technical Abstract: Cotton bolls (Gossypium hirsutum), 20 days-postanthesis in age, were inoculated with several different strains of Aspergillus flavus and allowed to incubate for 7 days in order to investigate extracellular hydrolases produced during in vivo host-fungal interactions. Carpellary tissue from the inoculated bolls was extracted with a MES buffer to isolate fungal proteins from the infection site. Protein composition of the carpellary tissue extracts was analyzed by 1-D denatured protein gel electrophoresis (SDS-PAGE). Protein bands were excised from gels and treated with trypsin to generate peptide libraries for fungal protein samples. Analysis of peptide samples was accomplished by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two peptides from a 35-kD fungal protein provided a match with strong confidence to A. flavus oryzin (AFL2G_01995) precursor protein. Oryzin is an A. flavus protein with protease activity. Since cotton carpel tissue is a defensive barrier surrounding developing seed, oryzin may aid the fungus in wall penetration by digesting plant cell wall structural proteins.