Immunohistochemical investigation of cotton carpel tissue exposed to xylanolytic hydrolases of Aspergillus flavus

Authors: Mellon, Jay; Vaughn, Kevin

Submitted to: Physiological and Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/24/2011
Publication Date: 9/6/2011
Citation: Mellon, J.E., Vaughn, K.C. 2011. Immunohistochemical investigation of cotton carpel tissue exposed to xylanolytic hydrolases of Aspergillus flavus. Physiological and Molecular Plant Pathology. 76:34-38.

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. Xyloglucans and xylans are plant polysaccharides that are important structural components of plant cell walls. These fungi are capable of producing a number of hydrolytic enzymes that are crucial to breaking down xylans. 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. In order to better understand the roles of these enzymes in fungal virulence, A. flavus was grown in a medium containing larch xylan as a carbon substrate. The fungus secreted multiple xylanolytic activities into the growth medium. 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). Cotton carpel tissue was exposed to a mixture of A. flavus xylanolytic hydrolases. Treated carpel tissue was examined microscopically after visualization by multiple antibodies possessing specificities to different plant cell wall components. This tissue analysis revealed that the secondary wall structure of carpel cells was highly eroded and disrupted. These results suggest that this mixture of hydrolase enzymes is an important virulence factor for the fungus, aiding in fungal penetration of plant host defense barriers. This research will benefit oilseed breeders, producers and pathologists, and will aid in the formulation of methods to prevent aflatoxin contamination of target crops.

Technical Abstract: Cotton carpel tissue (35-45 dpa) that had been treated with a mixture of xylanolytic hydrolases derived from Aspergillus flavus was subjected to immunocytochemical analysis. Microscopic examination of treated tissues revealed severe degradation of the secondary wall structure. Control tissue cells revealed the presence of high concentrations of xylans/arabinoxylans throughout the cell wall, as well as significant concentrations of arabinogalactan proteins in secondary wall structure. Carpel cells treated with a mixture of A. flavus-produced xylanolytic hydrolases showed a much reduced presence of labeling by xylan-specific antibodies on the inner wall surface, suggesting a severe loss of these plant polysaccharides in the secondary wall structure. Carpel exposure to a purified 14-kD endoxylanase from A. flavus also resulted in a severe reduction of xylans from secondary wall structure. Arabinogalactan proteins were not as severely affected by the xylanolytic hydrolases. Comparison of control tissue with hydrolase-treated tissue stained with toluidine blue revealed an apparent reduction in wall thickness, supporting the conclusion of secondary wall structure degradation. Hydrolase-treated carpel cells did not reveal degradation of pectin material (homogalacturonan) within wall structure. These results are consistent with the conclusion that the xylanolytic hydrolase complex of A. flavus is a critical factor for host cell wall maceration and may represent another important fungal virulence factor.