Phytochrome and pheromone signalling during the induction of a new cell type involved in sexual fusion in filamentous fungi

The asexual spore produced by filamentous ascomycete fungi is called a conidium. The ascomycetes are the largest group of fungi and contain the majority of crop and human fungal pathogens. Conidia are the main reproductive cells and dispersal agents of these organisms, but can also act as male fertilizing agents during sexual reproduction. We have discovered that conidia of the model ascomycete fungus, Neurospora crassa, can produce three types of filamentous cells: (a) the conidial germ tube which is involved in colony establishment, (b) the conidial anastomosis tube which is involved in forming complex cellular networks by undergoing cell fusion, and (c) the most recently discovered conidial sex tube which is involved in sexual fusion with cells of the opposite sex. The mechanism by which conidial sex tubes are produced, and particularly how they are regulated by sex pheromones and by light, is the focus of the research in the present proposal. One interesting aspect is that the conidial sex tubes are formed on the side closest to the light source (i.e. they exhibit a positive phototropism). The first objective of our research proposal will be to analyse the role of pheromone signalling. In particular, we will determine whether a synthetic sex pheromone can induce conidial sex tubes to form. We have found that conidial sex tubes are formed in response to red light and this involves two red light photoreceptors called phytochromes. Our second objective will be to analyse how these phytochrome receptors respond to red light, and in doing so, regulate the formation of conidial sex tubes. Neurospora crassa was the first filamentous fungus to have its genome completely sequenced and as a result has been shown to possess ~ 10,000 genes. Each of Neurospora's ~ 10,000 genes is being mutated to produce 'knockout mutants. Our third objective will be to screen several hundred of these knockout mutants to determine if they are defective in conidial sex tube formation and/or phototropism. We have also obtained evidence that two other photoreceptors are involved in the formation of conidial sex tubes: a blue light photoreceptor called cryptochrome, and a green light photoreceptor belonging to the opsin family of proteins (which also includes the photoreceptor rhodopsin which is found in the human eye). The roles of these photoreceptors in fungi are currently unknown. Our fourth objective will be to analyse the roles of cryptochrome and two different opsins in the formation of conidial sex tubes and possibly their phototropic growth. The fifth objective will involve visualizing the phytochrome, cryptochrome and opsin photoreceptors by tagging them with fluorescent labels which allow them to be imaged in living cells with a fluorescence microscope. In this way we will be able to directly monitor whether they change their subcellular location in response to light exposure. Our final objective will be to be to determine if the different photoreceptors interact and act in concert in response to the light signals that result in the formation and phototropism of conidial sex tubes.

We have discovered a new cell type produced by macroconidia of Neurospora crassa that we have called the 'conidial sex tube'. The overall aim of the proposed project is to understand the mechanistic basis of signalling during conidial sex tube (CST) formation and phototropism. The first part will analyse the role of pheromone signalling in these processes, and particularly determine if synthetic sex pheromone of both mating types can induce CSTs. The second part of the project will involve a photo-physiological analysis of phy mutants to determine the response modes of Neurospora's two phytochromes, PHY-1 and PHY-2. In particular we will test whether the two phytochromes act as reversible light switches, and whether both have functional roles in both mating types. In the third part, knockout mutants of genes encoding signalling and photoregulatory proteins will be screened in both mating types to determine which are defective in CST induction and phototropism. We will be particularly interested in determining whether phytochrome signalling in N. crassa shares features in common with the biochemical machinery involved in phytochrome signalling in plants. In the fourth part, the roles in CST induction and phototropism of the putative blue photoreceptor, cryptochrome (CRY), and putative green light opsin photoreceptors, ORP-1 and NOP-1, will be analysed. In the fifth part, the phytochrome and cryptochrome receptors will be fused to GFP and whether they move between the cytoplasm and nucleus on exposure to light, will be assessed. In addition, we will also determine whether these photoreceptors and/or the opsin photoreceptors,become localized to the site of subsequent CST formation in response to unilateral light Finally, we will identify proteins which interact with the phytochromes, and image their interactions, during CST induction and phototropism.