Functional analysis of fungal nonself recognition

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The proposed research explores a fundamental characteristic of life – the ability of cells to identify and appropriately respond to nonself. Similar to other organisms, fungi use nonself recognition to reduce disease transmission, and to modulate intra- and interspecies interactions. We study a form of nonself recognition referred to as vegetative incompatibility, whereby cell fusion between different fungi results in Programmed Cell Death (PCD). We aim to learn how this process works, how it integrates into cellular networks, and how we can apply this knowledge to control fungi that have negative impacts on human affairs. We have four specific objectives for this grant cycle. Our first is to explore protein interactions that trigger nonself recognition in the filamentous fungi Neurospora crassa and Cryphonectria parasitica. We will use genetic and biochemical techniques to study how incompatibility proteins interact during nonself recognition to trigger PCD. For example, we have identified a small 63 amino acid segment of the ‘PA’ form of UN-24 that causes cell death when it interacts with the OR form of this protein. This is interesting since, in addition to incompatibility function, the un-24 gene encodes the large subunit of ribonucleotide reductase (RNR), an essential enzyme that synthesizes the nucleotides needed for DNA synthesis. RNR is thus a prime chemotherapeutic target and understanding how to specifically inhibit this enzyme has applications in cancer therapy and antimicrobial drug development. Second, we aim to understand the basis of ‘escape’ from self-incompatibility in N. crassa, a process that results in mutagenesis of the ‘het-6’ nonself recognition gene. This mutational process resembles hypermutation of antibody genes in vertebrates – an adaptive response to enhance binding by antibodies to foreign substances. We have developed the protocols that allow us to identify what genes are involved in hypermutation of het-6 in N. crassa and show that the process requires genes that play a role in DNA damage signaling. This study of escape will provide novel information on how nonself recognition systems rapidly evolve. For our third objective we will transfer incompatibility genes from filamentous fungi into brewer’s yeast to further study the biochemical pathways affected by these genes. Yeast is ideal for this; it offers a powerful experimental system and does not have an endogenous vegetative incompatibility system. Therefore, with yeast we can more efficiently study how incompatibility proteins interact with each other, and how these interactions perturb biochemical processes to bring about PCD. This provides a platform to study protein-based growth inhibitors using methods that we currently employ to determine mode-of-action of novel, plant-derived antifungals. Finally, we will investigate how C. parasitica responds to nonself recognition using high-throughput sequencing of the ‘transcriptome’, or all the RNA molecules in the cell. We will also investigate how a naturally occurring virus of C. parasitica, CHV1, and a suppressor mutation, dcl2-, are able to attenuate incompatibility-associated PCD. This work will take advantage of our recent completion of the genome sequence of C. parasitica, the causal agent of the blight that decimated our chestnut trees. The study will provide insights into biological control strategies of this and other plant pathogens. Overall, this research will help train 6 PhD, 7 MSc, and over 30 undergraduate students in highly marketable areas of genetics, microbiology and biochemistry. This student-centered research will advance our knowledge in areas of both basic and applied science.