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item Glenn, Anthony - Tony

Submitted to: Mycopathologia
Publication Type: Proceedings
Publication Acceptance Date: 10/21/2002
Publication Date: 1/1/2003
Citation: Glenn, A.E. 2003. Identification of fusarium verticillioides genes differentially expressed in response to a corn antimicrobial compound (boa). Mycopathologia. v.155.

Interpretive Summary: As a normal process of their development, corn seedlings produce a group of small molecular weight compounds (known as DI MBOA, DIBOA, MBOA, and BOA) that serve as insect feeding deterrents and as toxic defensive compounds against a wide range of microbes. However, the fungus Fusarium verticillioides, which is the most common fungus found associated with corn throughout the world, is resistant to these compounds. This fungus has the capacity to detoxify these compounds by metabolically transforming them into non-toxic compounds (a process referred to as biotransformation). This ability to detoxify these compounds may enhance the survival of the fungus in the cornfield and thus lead to subsequent infection and colonization of corn plants. Fungal genes involved in this process of biotransformation are of interest, and those genes up-regulated in response to the BOA compound were identified using a technique called suppression subtractive hybridization (SSH). Among the genes identified so far, those with similarity to an amidase and an arylamine N-acetyltransferase were of particular interest, since these enzymes catalyze chemical modifications similar to those believed to be involved in the detoxification of BOA. We hypothesize that an amidase partially degrades BOA to an intermediate compound, which is then modified by addition of a chemical group by a transferase enzyme to produce the final non-toxic compound. The combination of molecular biology along with available mutant strains of the fungus has allowed us to thoroughly examine the process of detoxification.

Technical Abstract: Corn produces DIMBOA and DIBOA, small molecular weight, highly reactive preformed compounds that are implicated in resistance to microbial diseases and insect feeding. Due to the inherent instability of these compounds, they chemically transform into the more stable antimicrobials MBOA and BOA, respectively. Fusarium verticillioides, the most common fungal pathogen associated with corn, has the physiological capacity to biotransform MBOA and BOA into non-toxic metabolites. Thus, the antimicrobials are not effective deterrents of this fungal endophyte of corn. While data suggest biotransformation of these compounds is not a major virulence factor, such metabolic capacity may enhance the ecological fitness of F. verticillioides in a cornfield environment. Genetic analyses of F. verticillioides showed at least two loci, FDB1 and FDB2, are necessary for biotransformation. The biotransformation pathway is suggested to involve hydrolysis of BOA (Fdb1p) to produce 2-aminophenol, which is subsequently modified by addition of a malonyl group (Fdb2p) to produce N-(2-hydroxyphenyl) malonamic acid (HPMA). If either gene is mutated, detoxification does not occur and the fungus cannot grow on BOA-amended medium. In an effort to molecularly characterize FDB1 and FDB2 as well as other genes involved in biotransformation, we employed suppression subtractive hybridization (SSH), which would target genes up-regulated in response to BOA. The BOA-subtracted cDNA library (384 clones) was sequenced, providing 182 clones with homology to >30 proteins (BLASTX; =1E-04). Among the clones identified, those with similarities to amidase and arylamine N-acetyltransferase were of particular interest, since these enzymes catalyze chemical modifications similar to those postulated for Fdb1p and Fdb2p. Genomic cosmid clones were identified for each using the respective cDNAs as probes. The putative amidase cosmid clone genetically complemented an fdb1 mutation, while the putative N-acetyltransferase (=N-malonyltransferase) cosmid clone complimented an fdb2 mutation. Thus, the proposed chemical modifications and the putative proteins involved are mutually supported. These results demonstrate the utility of SSH for cloning genes previously characterized by forward genetics.