I am a biologist with a traditional education in biochemical genetics applied to microorganisms. Accordingly, I learned how to simplify elaborate biological phenomena, and focus on elementary gene-protein subsets. My scientific interest emphasizes biological views of how and why simple eukaryotes decide to convert from one functional cell-type, or genetic condition into another. My current extramural funding, focuses research goals on molecular genetics of sensors and recognition mechanisms employed by fungi to assimilate pectin (a plant cell wall component) and respond to various stress conditions. Figure 1 summarizes the common theme of my research program. In this document I will try to outline some of my recent research accomplishments (last 4 years) and highlight some of my future goals.

Plant Cell Wall Polysaccharide Recognition in Fungi. The main focus has been identification of loci involved in recognition of plant cell wall polysaccharides. Utilization of pectin as a source of food requires the expression of a large number of proteins encoded by multiple genes with redundant functions (Prade, 1996). Historic evidence shows that polysaccharide recognition systems exist in fungi and other microorganisms, but nothing is known about them (Prade, et.al 1999). We developed a method to isolate mutants affected in pectin recognition. Figure 2 describes, in summary, the lethal substrate we synthesized and utilized to recover mutants defective in pectin recognition.

Pectin based lethal substrates (HPGP) function by releasing a fungicide, hygromycin B, covalently bound to the reducing end of pectin fragments (PGP) when degraded by pectinases. Because the mutants we are interested in, fail to recognize pectin they should also fail to release hygromycin B and survive the selective pressure. Cochliobolus sativus HPGP-resistant (HPGPR) mutants fall into three carbon-coupled phenotypic classes: one third of them fail to grow completely on PGP, another third grows at reduced levels and the remainder at near wild type rates (Prade and Mort 1999). Some of the HPGPR mutants unable to utilize PGP were also unable to grow on xylan and cellulose. Thus, our results indicate that pectin recognition is coupled at a higher level, to utilization of other carbohydrates present in plant cell walls (Prade and Mort 1999). Employing a similar gene-hunting approach, we were able to recover a similar mutant collection in Aspergillus nidulans (Muralla, et.al 2000). Furthermore, we were able to determine partial DNA sequences for ten tagged loci and found a fatty acid desaturase, a putative transmembrane protein and seven ORFs with unclear functions. The tenth strain showed homology to SIP3 a yeast gene that directly interacts with SNF1, the main carbon regulator in yeast. The A. nidulans SIP3 homolog is located terminally on the right arm of chromosome II (Muralla, et.al 2000).

In the coming years I plan to strengthen the funding for this research and allocate additional research personnel. The C. sativus studies are likely to focus on experiments aimed towards the establishment of a role for pectin degradation during pathogenesis and the identification of molecular targets suitable for drug development. The A. nidulans system is likely to be centered on large-scale and genome-wide functional studies in particular we plan to construct and decipher microarray produced gene expression profiles from selected HPGPR mutants. In addition, we also plan to study in molecular detail mutants such as SIP3 and isolate the A. nidulans SNF1 homolog.

So far we have identified about half of the 8,000 predicted A. nidulans genes, are in the process of producing microarrays and a database to display all the genetic and functional information associated with those genes. In the coming years my lab plans to increase the number of genes on the microarray to nearly 8,000 and improve the annotation of expression data using PipeOnline. My laboratory also plans to become a major user of microarray slides to be employed in the study of gene expression profile changes during salinity stress adaptations and mutants defective in pectin recognition. Further development of novel algorithms and online display methods are expected to constitute a major activity of my bioinformatics research group.

Functional Genomics of Stress Tolerance Finally, a major grant from the National Science Foundation (Bressan, et.al 1999) recently awarded is likely to spin-off several projects in my laboratory that will focus on the A. nidulans portion of the genome associated with stress adaptations (Prade and Bohnert 2001).