Pioneer Hi-Bred International Inc., Johnston, Iowa 50131, USA

Maize Helminthosporium leaf spot and ear mold is caused by Cochliobolus carbonum race 1, a fungal pathogen for which infection is dependent upon production of the cyclic tetrapeptide HC-toxin. Compatible interaction and disease occur only in maize lines homozygous for a mutation in the Hml gene; the encoded wild-type enzyme is an NADPH-dependent redtictase that converts HC-toxin to an inactive form. The observation that the toxin is a specific inhibitor of maize histone deacetylases has led to the hypothesis that the molecular basis for compatibility in this system is maintenance of a chromatin state in which induced expression of disease defense response genes is prevented without lethal repression of all transcription. Such an effect would explain the cytostasis often observed in cells exposed to pure HC-toxin and would produce metabolically active yet undefended host tissue for fungal infection.

Results and discussion
Indicators for defense responses were assayed in susceptible (hml/hml) maize plants after exposure to UV-C light and/or C. carbonum conidia from a toxin-deficient strain of race 1. The markers included salicylic acid (SA), PRm2 and a B-glucanase, and each was clearly induced upon exposure to the fungus or UV light. The level of expression for SA was significantly less, as was accumulation of the indicator proteins, when the elicitors were combined with HC-toxin at 0.1 mg/l. To investigate the specificity of suppression, similar experiments were conducted on other inducible genes that do not have direct roles in fungal pathogen defense. The constitutive mitochondrial chaperonin cpn60 (Hsp60) is induced two-fold by heat shock, but heat shock in the presence of HC-toxin showed repressed expression. Non-heat-treated plants, however, exhibited no change in cpn60 at any toxin level tested. Expression of maize proteinase inhibitor (MPI) is normally triggered by wounding or the hormone jasmonate (JA). MPI expression was induced by JA, UV light and C. carbonum inoculation as described above and this induction was also prevented by HC-toxin. Treatment with HC-toxin but no elicitors gave a strikingly different result. In the absence of hormone and with toxin present at 0.5 mg/l, MPI was measured at levels equal to those seen upon JA induction. Five-fold more MPI was present after exposure to HC-toxin at 1.0 mg/l. Marker accumulation normally seen in response to pathogenic attack is attenuated by HC-toxin. Work is now under way to extend these results and determine whether they can be quantified at the level of RNA transcripts. It would appear that as an inhibitor of histone deacetylase, the toxin prevents inducible transcription of genes required for an effective defense response. RNA profiling is being employed to identify these genes using susceptible maize seedling tissue collected 3, 6 and 22 h after three experimental treatments: exposure to HC-toxin, C. carbonum tox- spores, or spores and toxin. Comparing these to control samples will allow quantification of changes in transcription upon HD inhibition, during the resistant defense response, and during a defense response insufficient to confer resistance. RNA derivatives from each treatment will be hybridized to an Affymetrix microarray of about 700 maize ESTs with homology to genes that may be involved in disease defense. Results will be compared to a similiar approach using the Molecular Dynamics/Amersham MicroArray System. In collaboration with CuraGen Corporation the same RNA samples will also be analysed by a PCR-based profiling methodology. This technique does not require previous sequence information to identify transcripts with altered expression levels and thus provides a tool for gene discovery as well as transcription quantification. The isolation of defense-specific promoters regulated by chromatin remodeling is complemented by current studies on a number of new cDNAs for maize histone deacetylases. Perhaps identification of both the enzyme and the promoters targeted by HC-toxin will help advance characterization of disease resistance signalling pathways.