Identification of hydrolytic activities expressed by Aspergillus flavus grown on cotton carpel tissue

To identify proteins important for invasion into host plant tissue, Aspergillus flavus was cultured on medium containing cotton carpel tissue as the sole carbon source. We identified several hydrolases suggesting they are important as A. flavus virulence factors for plant colonization. Specifically, Aspergillus flavus AF13 secreted at least two endoxylanase activities and a pectolytic activity when grown on the cotton carpel tissue medium. A concentrated sample derived from the A. flavus growth medium (6-day) was subjected to gel filtration chromatography on a BioGel P-30 column. A major endoxylanase activity was separated from the other fungal-secreted proteins. Additional fungal secreted proteins were partially resolved by gel filtration chromatography on a BioGel P-60 column. Multiple proteins with molecular weights in the 20 to 70 kD range were present in the harvested fungal growth medium. Analysis of these fungal-secreted proteins by liquid chromatography/tandem mass spectrometry identified both endo- and exo-glucanase proteins, α-l-arabinofuranosidase, glucoamylase, α-amylase A, pectate lyase A, xylanase F1, acetylxylan esterase, glutaminase A, as well as conserved hypothetical proteins of unknown function. These proteins likely assist A. flavus in the maceration of plant cell walls, allowing for pathogenic entry and accession of host nutrient resources. Pectolytic and xylanolytic hydrolases, as well as glucanases, appear to be important A. flavus virulence factors.     

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

Cotton carpel tissue (35–45 days post-anthesis) 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, although penetration of the tissue was not as dramatic. 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. Interestingly, the pectins could only be detected in the samples treated with xylanolytic enzymes, indicating that the pectins were being masked by xylans. 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, in addition to pectolytic hydrolase activities.     

Influence of environmental pH modulation on efficiency of apoplastic PR proteins during Fusarium culmorum– wheat seedling interaction

Variation of apoplastic pH by Fusarium culmorum and its influence on the production, activity and isoenzymes patterns of the pathogenesis‐related (PR) proteins β1,3‐glucanase, chitinase and peroxidase enzymes were detected in apoplastic fluids (AFs) from infected wheat seedlings. The time course in the 24–48 h interval post infection was characterized by an increase in activity and isoenzymatic differential induction of the selected PR proteins and by a concomitant rise of apoplastic pH. Chitinase attained maximum activity at pH 8·0 in the case of inoculated seedlings. Optimal β1,3‐glucanase activity in the pH range 6·0–8·0, was observed at pH 7·0. Peroxidase was strongly affected by pH, with enzyme activity having a maximum rate at pH 6·0 and thereafter rapidly declining at higher pH. Maximum peroxidase activity paralleled the appearance of the complete isoenzymatic pattern. In order to investigate the biological role of PR proteins in AFs, the in vitro antifungal activity was evaluated. In the interval 0–6 h, pH of macroconidia suspensions rose up to 7·2. AFs revealed inhibitory activity against germinating macroconidia of F. culmorum by decreasing the germination efficiency of macroconidia apical compartments, while this effect was compensated by an increased germination capacity of middle compartments. Present results suggest that during infection of wheat seedlings by F. culmorum the pH modulation favours host colonization by enhancing the activity of pectin lyase, and simultaneously inhibits the capacity of the host to oppose the pathogen by interfering with peroxidases which represent an important component of the defence arsenal.