5.3.27
CLONING OF FUMONISIN-METABOLIZING ENZYMES AND THEIR EXPRESSION IN TRANSGENIC MAIZE

J DUVICK, J MADDOX, J GILLIAM, X WANG, T ROOD, W MARSH, B NORMAN and A TOMAS

Department of Crop Protection, Pioneer Hi-Bred, Anti, Inc., Box 1004 Johnston, Iowa 50131, USA

Background and objectives
The widespread occurrence in maize of fumonisins, a family of mycotoxins produced by the ear-rot fungus Fusarium moniliforme, is a concern worldwide. Although often associated with visibly mouldy grain, fumonisins can also be present at significant levels in asymptomatic but nevertheless infected grain. Fumonisins mimic sphingolipid precursors and disrupt sphingolipid biosynthetic pathways in both animal and plant cells, with potentially profound consequences for cellular metabolism. Fumonisins are acutely toxic to certain livestock, especially horses and swine, and have carcinogenic properties in rats; hence minimizing fumonisin levels in feed and food grain is a worthwhile goal [1]. This, together with the possibility that fumonisins might contribute to the virulence of F. moniliforme in corn, led us to adopt a strategy of detoxification of fumonisin in planta using a transgene approach.

Results and conclusions
We have identified several microbes (two species of black yeast fungus and a Gram-negative bacterium) that colonize mouldy corn and metabolize fumonisins to CO2 in liquid culture [2], and have used these fumonisin-metabolizing microbes (FMMs) as a source for genes for detoxification of fumonisin in transgenic maize. The initial steps of metabolism by FMMs consist of de-esterification of two tricarbanylic acid (TCA) esters by a soluble esterase, followed by oxidative deamination of the resulting amine alcohol backbone by a wall- or membrane-associated deaminase. Prior hydrolysis of TCA groups is required for deamination by the microbial deaminases. Together, these two catabolic steps are likely to inactivate fumonisin. We hypothesize that the esterase, deaminase and other downstream enzymes together constitute a previously undescribed catabolic pathway that provides a unique carbon-source niche for FMMs. We have cloned genes for both fungal and bacterial fumonisin esterases and shown them to be members of a diverse group of serine hydrolases. Cloning of other pathway enzymes is in progress. We have expressed both esterases separately in transgenic maize plants under control of a constitutive maize ubiquitin promoter. Transgenic maize plants expressing fumonisin esterases were active in hydrolysing exogenously applied fumonisin in all tissues examined including mature seed. In greenhouse and field experiments, esterase-expressing maize plants inoculated with toxigenic F. moniliforme accumulated hydrolysed fumonisin in mature kernels, in contrast to non-esterase, Fusarium-inoculated plants which had very low levels of hydrolysed fumonisin. Fumonisin Bl (FB1) levels, although highly variable among samples, were lower on average in seed from esterase-positive plants. Complete loss of fumonisin was not expected because ears were from hemizygous plants and thus were segregating 1:1 for esterase expression in the non-maternal kernel tissues. Visual mould symptoms and internal colonization did not differ significantly between esterase and non-esterase ears, possibly because the hydrolysis product (AP1) still retains biological activity. Nevertheless these findings confirm the feasibility of enzymatic detoxification of a mycotoxin in maize.

References
1. Munkvold GP, Desjardins AE, 1997. Plant Disease 81, 556.
2.Dijvick JP, Rood TR, Maddox JR, Gilliam JT, 1997. In Molecular Genetics of Host-Specific Toxins in Plant Disease, Proceedings of the 3rd Tottori International Symposium on Host-Specific Toxins. Elsevier, The Netherlands, in press.