Research Projects in Gene Regulation

We propose to identify all of the plant cell wall degrading enzymes produced by A. nidulans while growing on plant biomass. This will involve developing a negative genetic screening method designed to yield cDNAs for all NEW proteins the fungus produces when it is shifted from growth on glucose to a medium containing one or all of the whole range of plant cell wall polysaccharides including pectins, cellulose, and hemicelluloses. cDNAs prepared from RNA extracted from glucose-grown cultures as a template will be labeled and used to screen a cDNA library made from RNAs originating from polymer-containing cultures. Negative selection of all NEW proteins will be done by DNA/DNA hybridization between the labeled "glucose-grown" probe and membrane lifts from the "polymer-grown" library carried in E. coli as plasmids. Transcripts present in both the cDNA probe and the library being screened will appear as positives. However, transcripts present only in the library that is being screened will appear as negatives. Negatives will be selected for further analysis. Since this experimental approach is independent of gene function, we do not have to know anything about the encoded protein in order to detect its cDNA. Moreover the genes identified under negative selection are all the ones specifically induced as a consequence of the physiological shift. Thus, our approach is comprehensive because a whole set of genes can be isolated. If successful and robust enough, this method should be a powerful tool that will allow us to survey quickly a large set of nutritional circumstances and assemble a comprehensive set of genes involved in decomposition of plant cell walls. Many of the genes will be identified by homology to known sequences in the public databases. We will also produce gene products of interest in heterologous expression systems and determine their mode of action (function) by employing a range of structurally defined fluorescent substrates. In future projects we plan to employ these genetically well-defined cDNAs to study the key molecular events involved in induction and transcriptional control of plant cell wall degrading enzymes. Furthermore, availability of all activities genetically isolated will allow us to reconstruct the cell wall catabolic pathway in fungi, taking advantage of targeted genetic modifications of catalytic and regulatory functions.

The fungus Cochliobolus sativus causes plant disease on cultivated barley and related plants. Infection occurs through production of differentiated cell-types initiated by vegetative propagating hyphae. Cells specialized in invasion, produce a complex set of enzymes that depolymerize plant cell walls during the penetration process and require multiple activities that overlap functionally. The objective of this research is to test the biological neccessity of plant cell wall degrading enzymes during fungal plant infections, via the inactivation of regulatory genes that control expression of these activities. To address the problems associated with redundant substrate-enzyme interactions, we propose a suicide selection approach to isolate mutants defective in both, substrate- and infection-mediated induction loci. The idea is simple: Lethal-substrates are synthesized by covalently linking fungicide molecules to substrate fragments; wild-type strains degrade the substrate releasing the fungicide and die. Mutants that fail to degrade lethal-substrates survive, are selected, biochemically, genetically and molecularly characterized and the loss-of-function phenotype during plant infections determined. Suicide substrates prepared by covalently bonding hygromycin B to pectin (HY-Pectin) or xylan (HY-xylan) fragments function as predicted: they need to be enzymatically degraded before they are able to arrest vegetative growth. Moreover, a significant number of mutants that survive HY-pectin suicide selection have been isolated (0.14% of UV-irradiated survivors) and a subset is unable to assimilate other polysaccharides (i.e., cellulose and xylan) as well. This project should provide important insights into the role of plant cell wall degradation during fungal infections because at least some mutations in regulatory genes are likely to result in strains unable to produce any transcripts that encode cell wall degrading activities. In future work, these mutants will provide a powerful genetic background to further define other molecular interactions that control genes that cause disease in plants.

Many fungi colonize plant tissues leading to problems such as spoilage of fruits and vegetables, contamination of foods with toxic or carcinogenic compounds and infections that cause production losses. Plant cell walls are forceful physical barriers to the invading fungus and one essential step in most plant-fungus interactions is enzymatic breakdown of plant cell wall polymers.

In early 1996 we launched a collaborative research program with the aim of understanding of the regulatory biochemistry that controls the genetic expression of these enzymes. The potential of this research is best illustrated by the prospect that plants can be genetically engineered to produce a factor, most likely a protein, which blocks the expression of cell wall degrading enzymes in fungi, resulting in natural resistance against a wide range of fungal infections. However, inactivating biosynthesis or inhibiting enzymatic activity of one or a limited group of enzymes is insufficient to prevent cell wall degradation and fungal growth on plant substrates. Thus, our hypothesis is that if we prevent induction of all cell wall degrading enzymes, by affecting the action of a global regulator, life of the fungi on the plant would be impossible.

Our current interdisciplinary research focuses on the identification and cloning of fungal genes that control the expression of these enzymes. We have developed, and successfully tested, a positive selection method that identifies mutants unable to cause plant cell wall polymer breakdown. An antibiotic is attached to a cell wall polymer fragment. in such a way that the resulting suicide substrate cannot directly be taken up by the fungal cell. However, if the fungus produces enzymes that degrade the suicide molecule, i.e. cell wall hydrolases, the antibiotic is converted to a form in which it can enter the cell and kill the fungus. Since production of any enzyme capable of converting the inactive toxic oligomer into an active form is lethal, mutants which survive the suicide screen should be severely affected in cell wall degrading functions. Because the selection is based on the interaction between the substrate and enzymatic activity, survivors are likely to be affected in the ability to activate the expression of multiple enzymes. From all the possible kinds of mutants, the most likely to be identified are the ones defective in substrate recognition, signal transduction and specific activation of gene expression.

The plan is to create a large collection of Aspergillus nidulans mutants that survive the above described suicide selection screen Mutants will be grouped, into genetic complementation groups (loci) and classified according to biochemical (i.e., presence or absence of cellulases, pectinases and xylanases) and physiological (i.e., growth on different polysaccharides) characteristics. We expect to identify several genes and will choose one for molecular complementation using an A. nidulans genomic (cosmid) DNA library. Once the mutation has been successfully complemented, we will rescue the cosmid DNA, subclone and sequence the minimal DNA fragment that functionally complements the mutation. The DNA sequence information should reveal the biochemical activity required for regulation of the cell wall degrading activity.

This project should provide important insights into the regulatory biochemistry of plant cell wall degrading enzymes. In future work, we will clone and sequence additional mutants to identify other activities involved in the activation pathway, isolate homologs from fungal plant pathogens and test deletion mutants in the ability to infect, spoil or contaminate food products.