CONTROL OF TOXIC ENDOPHYTIC FUNGI WITH BACTERIAL ENDOPHYTES AND REGULATION OF BACTERIAL METABOLITES FOR NOVEL USES IN FOOD SAFETY
Location: Toxicology and Mycotoxin Research
Title: Arylamine N-acetyltransferases in mycotoxigenic and related fungi of agricultural significance
Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: May 1, 2010
Publication Date: September 3, 2010
Citation: Karagianni, E.P., Ormiston, C.B., Fakis, G., Glenn, A.E., Boukouvala, S. 2010. Arylamine N-acetyltransferases in mycotoxigenic and related fungi of agricultural significance. In: Proceedings of the 5th International Workshop on the Arylamine N-Acetyltransferases, September 1-3, 2010, Paris, France. p. 16.
Mycotoxigenic fungi are of worldwide concern, as they contaminate crops and compromise food safety. Many of these fungi are also aggressive plant pathogens with devastating effects on maize, and wheat. The host plants possess a variety of defensive mechanisms against those fungi, including the production of phytotoxins that kill or inhibit the growth of the invading pathogens. Maize, wheat and rye constitutively produce benzoxazinones, such as DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one) and DIBOA (2,4-dihydroxy-2H-1,4-benzoxazin-3-one), implicated in resistance to a number of fungal and other diseases. These compounds are synthesized as biologically inactive glucosides that upon pathogenic attack are liberated and spontaneously degrade to the corresponding benzoxazolinones, MBOA (6-methoxy-2-benzoxazolinone) and BOA (2-benzoxazolinone). These are effective fungistatic compounds that inhibit the growth of a wide range of microbes, including several Fusarium species. Interestingly, the mycotoxigenic maize-pathogenic fungus Fusarium verticillioides (teleomorph Gibberella moniliformis) has been demonstrated to tolerate MBOA and BOA at concentrations that inhibit growth of other fungi. This tolerance results from biotransformation of these benzoxazolinones into non-toxic metabolites, a pathway involving an N-acyltransferase enzymatic activity encoded by a 1038bp intronless NAT-homologous gene (Genebank ID: EU552489) residing at the FDB2 locus on chromosome 3 of F. verticillioides .
We have undertaken characterization and cloning of NAT-homologous genes in the mycotoxigenic F. verticillioides M3125 and Fusarium graminearum (teleomorph Gibberella zeae) NRRL 31084 strains, the related tomato pathogen Fusarium oxysporum f.sp. lycopersici NRRL 34936 strain, as well as in two more divergent filamentous ascomycetes, Aspergillus flavus NRRL 3357 (an aflatoxin-producing contaminant of grain and an agent responsible for 30% of human aspergillosis cases) and Aspergillus nidulans FGSC A4 (a facultative pathogen and an established model organism). Putative NAT gene open reading frames (ORFs) were predicted in silico, followed by amplification from genomic DNA and cDNA prepared from all five organisms. Sequencing of the amplified products was performed to experimentally annotate the NAT-homologous genes and determine their exon-intron structure. A total of 16 fungal NAT loci were characterized (4 in F. verticillioides, 3 in F. graminearum, 4 in F. oxysporum, 4 in A. flavus and 1 in A. nidulans), and their sequences were named according to the consensus nomenclature and deposited to the EMBL database. The transcripts of two loci [(GIBMO)NAT4 and (ASPFN)NAT4] were demonstrated to bear compromised ORFs and are likely to be the products of pseudogenes. There are 7 intronless and 4 segmented NAT loci in Fusaria, while all NAT loci of the Aspergilli contained introns within their coding region.
Conventional reverse transcription PCR confirmed the expression of all NAT genes under the culture conditions applied. We have further proceeded to assessing the expression levels of individual NAT transcripts in the presence or absence of BOA and 2,4-dichloroaniline in the growth medium of each strain. Our data so far indicate little or no effect of the xenobiotic compounds on NAT gene expression in A. nidulans, F. oxysporum and F. graminearum, while they appear to induce a decrease in the levels of certain NAT transcripts in A. flavus. The most prominent effect was evident in F. verticillioides, where xenobiotic exposure significantly increased the levels of all NAT transcripts. This is consistent with the postulated involvement of F. verticillioides NAT in detoxification pathways enhancing the tolerance of the fungus to host phytotoxins, and has potential implications on agricultural management practices. Refined quantitative PCR analyses are currently in progress in our laboratories, alongside with the expression of recombinant fungal NAT proteins.