Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSsuch as Lolium perenne and L. arundinaceum. Colonization of host seedlings by E. festucae occurs when hyphae in the shoot apex invade developing hostleaves and extend via intercalary hyphal growth, a highly unusual mechanism of division and extension in non-apical compartments. We hypothesise thatintercalary hyphal growth is stimulated by mechanical stretch imposed by attachment of hyphae to elongating host cells, and that this stress is sensed bymechano-sensors located on the hyphal membranes. Genome analysis revealed that homologues of known mechano-sensors in Saccharomyces cerevisiaesuch as Mid1 (a stretch activated calcium ion channel), Wsc1 and Mid2 (cell wall integrity sensors) are present in the E. festucae genome. Genereplacement studies of mid1 and wsc1 in E. festucae reduced radial growth rate in axenic culture confirming the role of both genes in hyphal growth. Inaxenic culture both Dwsc1 and Dmid1 mutants were sensitive to fungal cell wall modifiers such as Calcofluor White, supporting their role in cell wallintegrity. Preliminary plant infection studies with Dwsc1 and Dmid1 mutants revealed a hyper-branched unsynchronized growth pattern within the host(Lolium perenne), and Dwsc1 also caused severe stunting in most plants suggesting a disruption in the symbiosis. A technique to stimulate intercalarygrowth under in-vitro conditions through mechanical stretch is being optimised to test the ability of Mid1, Wsc1 and Mid2 to sense mechanical stress andinitiate intercalary growth.566. Aspergillus flavus hypertrophy and hyphal entry by Ralstonia solanacearum is mediated by bacterial type three secretion system function. Joe ESpraker 1 , Nancy P Keller 2 . 1) Plant Pathology, University of Wisconsin Madison, Madison, WI; 2) Bacteriology, University of Wisconsin Madison, Madison,WI.Fungi and bacteria are two of the primary pathogens of plants, often infecting the same crops, however shockingly little is known of how theseorganisms interact independently of plant hosts. In examining the interaction between two economically important pathogens of peanut, Aspergillusflavus and Ralstonia solanacearm, a fungus and bacterium, respectively, we’ve shown that fungal hypertrophy is induced and that the bacterium is capableof entering these cells. The hypertrophic cells were imaged using calcofluor staining to show chitin cell wall structure. Bacterial invasion of these structureswas demonstrated using confocal microscopy of GFP labeled bacteria. Further, we demonstrate that bacterial mutants deficient in type three secretionsystems are incapable of eliciting the fungal hypertrophic response by culturing virtually isogenic bacterial type three secretion mutants. This is the firstreport of a well-known plant pathogenic bacterium eliciting fungal hypertrophy and invading hyphal cells. Current research is aimed at finding bacterialeffectors that may be facilitating this interaction and elucidating their mode of action.567. Vegetative hyphal fusion in epichloae endophytes. Jun-ya Shoji, Nikki D. Charlton, Sita R. Ghimire, Jin Nakashima, Kelly D. Craven. Plant BiologyDivision, The Samuel Roberts Noble Foundation, Ardmore, OK.Vegetative hyphal fusion establishes the interconnection of individual hyphal strands into an integrated network of a fungal mycelium. It is suspectedthat vegetative hyphal fusion plays many important roles such as in nutrient translocation, intramycelial signaling, and emergence of genetic diversity viahorizontal gene / chromosome transfer or interspecific hybridization. However, experimental support for these suspected roles is still largely lacking. Toinvestigate the role of hyphal fusion in fungal endophytes of epichloae, which form mutualistic symbiosis with grass hosts, we generated mutant strainslacking sftA, an ortholog of the hyphal fusion gene so in Epichloë festucae. The E. festucae DsftA mutant strains grew like the wild-type strain in culture butwith reduced aerial hyphae, and completely lacked hyphal fusion. The most striking phenotype of the E. festucae DsftA strain was that it failed to establisha mutualistic symbiosis with the tall fescue plant host (Lolium arundinaceum), and instead, killed the host plant within two months after initial infection.This suggests that hyphal fusion may have an important role in the establishment / maintenance of fungal endophyte-host plant mutualistic symbiosis. Tofurther investigate the importance of hyphal fusion in epichloae, frequency of hyphal fusion was quantified in different epichloae endophytes includingsexual isolates, asexual interspecific hybrids and asexual non-hybrids. A majority of sexual epichloae underwent frequent hyphal fusion, whereas hyphalfusion was less frequently found in asexual epichloae. Moreover, hyphal fusion was less common in asexual non-hybrid epichloae compared to asexualhybrids. Thus, it appears that the ability to undergo hyphal fusion correlates with the presence of the sexual cycle, and the hybrid status of epichloaeendophytes. Overall, our data provide evidence for the importance of hyphal fusion in establishment / maintenance of mutualistic symbiosis, andevolution of epichloae endophytes.568. Oxygen and the stomatal cue: Dissecting stomatal tropism in Cercospora zeae-maydis. R. Hirsch, B. Bluhm. Department of Plant Pathology,University of Arkansas Division of Agriculture, Fayetteville, AR.Cercospora zeae-maydis causes grey leaf spot of maize, one of the most widespread and destructive foliar diseases of maize in the world. Stomatalinfection is a critical, yet poorly defined, component of pathogenesis in C. zeae-maydis. At the onset of infection, the fungus senses and grows towardsmaize stomata, and then breaches the leaf surface by producing appressoria over stomatal pores. Directed growth toward distant stomata during infectionled us to hypothesize that C. zeae-maydis responded to an unknown chemical cue emanating from stomata. To elucidate mechanisms underlyinginfectious development in C. zeae-maydis, particularly stomatal tropism, a series of histological experiments were performed with epi-florescent andconfocal microscopy. Upon sensing maize stomata, C. zeae-maydis either reoriented hyphal tip growth towards stomata, or initiated new hyphaeoriginating from right-angle branches in close proximity to stomata. Hyphae exhibiting stomatal tropism were linear and lacked branches. Ontopographically accurate acrylic leaf replicas, C. zeae-maydis did not display stomatal tropism and failed to form appressoria upon encountering artificialstomata, which indicated that thigmotropic cues were not sufficient to elicit pre-penetration infectious development. However, in non-host interactions,C. zeae-maydis exhibited stomatal tropism and retained the ability to form appressoria over stomata, which suggested that a chemical cue emanating fromstomata elicited a chemotropic response in the fungus. Stomatal tropism and appressoria formation in C. zeae-maydis were impaired when atmosphericoxygen levels were disturbed, implicating the role of oxygen sensing in pathogenicity. This study characterized stomatal tropism during infection of maizeby C. zeae-maydis, directly implicated oxygen sensing as a component of pathogenicity, and provides a quantitative framework through which to studyfoliar pathogenesis and host/pathogen interactions in related systems.569. Host colonisation processes by symbiotic epichloid fungi are regulated through cAMP. Christine R. Voisey 1 , Damien J. Fleetwood 2 , Linda J. Johnson 1 ,Gregory T. Bryan 1 , Wayne R. Simpson 1 , Michael J. Christensen 1 , Suzanne J.H. Kuijt 1 , Kelly Dunstan 1 , Richard J. Johnson 1 . 1) Forage Biotechnology,AgResearch, Palmerston N, New Zealand; 2) School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand.The fungal symbiont, Epichloë festucae, colonises leaves of host grasses by ramifying through the shoot apical meristem (SAM) of the seedling, and theninfecting the leaf primordia. Hyphal infection of the SAM is dependent on apical growth, however after primordia have formed, leaf tissues undergo aphase of intercalary expansion, which the fungus, attached to host cells, must recapitulate to remain intact. E. festucae hyphae entering the leaf expansionzone switch from apical to intercalary growth, and extend in synchrony with the host until the leaf tissues mature. How colonising symbiotic fungiaccommodate the complexities of the plant developmental programme is currently unclear. Since cAMP signalling is often required for host colonisationby fungal pathogens, we disrupted the cAMP cascade by insertional mutagenesis of the E. festucae adenylate cyclase gene (acyA). Consistent with reports260