Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSis a nucleotide derived from adenosine triphosphate that acts as a second messenger throughout all kingdoms. Intracellular cAMP levels are subject to alarge membrane-bound protein, the adenylate cyclase. In order to analyze the function of this gene and the importance of cAMP in the life cycle of F.graminearum, the adenylate cyclase gene (FGSG_01234) was deleted from the genome (DFgac1). DFgac1 displayed a drastically reduced growth oncomplete medium. This reduction in growth could partially be complemented by addition of a cAMP analog. Furthermore, the mutant was unable toproduce perithecia on detached wheat nodes but more artificial conditions like carrot agar allowed perithecia development. Possibly, this points to asensing problem of DFgac1. Although growth on agar was reduced, conidia production was increased. Pathogenicity towards wheat was drasticallyreduced in DFgac1 compared to the wild type. Point-inoculated spikelets showed only small lesions even after 21 days post inoculation. No deeperinfection occurred and mycelial growth was never detectable near the rachis. Thus, fungal hyphae never grew from the inoculated spikelet to the adjacentone. Fluorescence microscopy using a DFgac1-strain expressing dsRed constitutively in the cytosol revealed that FgAC1 controls the development ofinfection structures like lobate appressoria and infection cushions. Removal of hyphae superficially colonizing flower leaves and subsequent analysis byscanning electron microscopy demonstrated the lack of any fungal penetration holes. Instead, hyphae on flower leaves produced massively new conidia,thereby circumventing the infection cycle, something never observed in the wild type. DFgac1-strains are unable to produce the mycotoxin deoxynivalenolboth in vitro and during wheat infection. In this study, for the first time, we implicate the cAMP signaling pathway to important processes in F.graminearum like development of infection structures, pathogenicity, secondary metabolite production and sexual reproduction.546. The ATF/CREB transcription factor Atf1 is essential for full virulence, deoxynivalenol production and stress tolerance in the plant pathogenFusarium graminearum. Thuat Van Nguyen, Birgit Hadeler, Cathrin Kröger, Wilhelm Schäfer, Jörg Bormann. Molecular Phytopathology, UniversityHamburg, Hamburg, Germany.The filamentous ascomycete Fusarium graminearum is a highly organ specific pathogen that resides on small grain cereals like rice, wheat, barley, andmaize. Grains infected with F. graminearum accumulate high amounts of mycotoxins, most prominent of which are deoxynivalenol (DON) and zearalenone(ZEA). The stress-activated MAP-kinase FgOS-2 (Saccharomyces cerevisiae HOG1) is a central regulator in the life cycle of F. graminearum (Nguyen et al.,2012. MPMI 25:1142-1156). FgOS-2 regulates, among others, virulence to wheat and maize, and DON- and ZEA-production. Here, we present data on thefunctional characterization of a putative downstream regulator, the ATF/CREB activating transcription factor FgAtf1. We created deletion and overexpressionmutants of Fgatf1, the latter one also in an FgOS-2 deletion mutant. Like FgOS-2, FgAtf1 is mainly involved in osmotic stress response.Bimolecular fluorescence complementation demonstrates an interaction of both proteins under osmotic stress conditions. Deletion mutants in Fgatf1(DFgatf1) are more sensitive to osmotic stress (e.g. mediated by NaCl) and less sensitive to oxidative stress mediated by H 2O 2 compared to the wild type.Furthermore, sexual reproduction is delayed: perithecia develop much slower and some remain immature even after prolonged incubation. DFgatf1strains show an increased DON-production under in-vitro induction conditions compared to the wild type. However, during wheat infection, DONproductionis strongly reduced. Expression of genes encoding for key enzymes in the DON-biosynthesis pathway is regulated accordingly. In infectionassays on wheat and maize, the DFgatf1 strains show a reduced virulence compared to the wild type. Interestingly, constitutive expression of Fgatf1 leadsto hypervirulence on wheat, maize and Brachypodium distachyon. Moreover, constitutive expression of Fgatf1 in a DFgOS-2 mutant background partiallycomplements DFgOS-2-phenotypes regarding growth on osmotic-stress medium, sexual reproduction, and virulence towards wheat and maize.Furthermore, FgAtf1 is involved in the regulation of light-responsive genes. Taken together, these results provide new insights in the stress responsesignaling cascades of F. graminearum and assign the transcription factor FgAtf1 a central role in pathogenic development and secondary metabolism.547. The stress-activated protein kinase FgOS-2 is a key regulator in the life cycle of the cereal pathogen Fusarium graminearum. Thuat Van Nguyen,Birgit Hadeler, Cathrin Kröger, Wilhelm Schäfer, Jörg Bormann. Molecular Phytopathology, University Hamburg, Hamburg, Germany.Fusarium graminearum is one of the most destructive pathogens of cereals and a threat to food and feed production worldwide. It is an ascomycetousplant pathogen and the causal agent of Fusarium head blight disease in small grain cereals and of cob rot disease in maize. Infection with F. graminearumleads to yield losses and mycotoxin contamination. Zearalenone (ZEA) and deoxynivalenol (DON) are hazardous mycotoxins; the latter is necessary forvirulence towards wheat. Deletion mutants of the F. graminearum orthologue of the Saccharomyces cerevisiae Hog1 stress-activated protein kinase, FgOS-2 (DFgOS-2), showed drastically reduced in planta DON and ZEA production. However, DFgOS-2 produce even more DON than the wild type under in vitroconditions, whereas ZEA production is similar to that of the wild type. These deletion strains are dramatically reduced in pathogenicity towards maize andwheat. We constitutively expressed the fluorescent protein dsRed in the deletion strains and the wild type. Microscopic analysis revealed that DFgOS-2 isunable to reach the rachis node at the base of wheat spikelets. During vegetative growth, DFgOS-2 strains exhibit increased resistance against thephenylpyrrole fludioxonil. Growth of mutant colonies on agar plates supplemented with NaCl is reduced but conidia formation remained unchanged.However, germination of mutant conidia on osmotic media is severely impaired. Germ tubes are swollen and contain multiple nuclei. The deletion mutantscompletely fail to produce perithecia and ascospores. Furthermore, FgOS-2 also plays a role in reactive oxygen species (ROS)-related signalling: FgOS-2deletion mutants are more resistant against oxidative stress mediated by H 2O 2. We found that the transcription and activity of fungal catalases ismodulated by FgOS-2. Among the genes regulated by FgOS-2 we found a putative calcium-dependent NADPH-oxidase (noxC) and the transcriptionalregulator of ROS metabolism, Fgatf1. The present study describes new aspects of stress-activated protein kinase signalling in F. graminearum.548. Innate Immunity in Fusarium graminearum. Vong shian Simon Ip Cho 1,2 , Gitte Erbs 3 , Thomas Sundelin 3 , Peter Busk 4 , Mari-Anne Newman 3 , StefanOlsson 1 . 1) Genetics and Microbiology, University of Copenhagen, Copenhagen, Denmark; 2) USDA-ARS Cereal Disease Laboratory, University ofMinnesota, Saint Paul, MN, USA; 3) Transport Biology, University of Copenhagen, Copenhagen, Denmark; 4) Dept. Biotechnology, Aalborg University,Copenhagen, Denmark.Fungi are often mostly recognized as plant pathogens that cause harm to important economical plants. In nature however, fungi are frequently victims ofbacterial parasitism but little is known about fungal defense mechanisms. The potential existence of fungal innate immunity was studied using Fusariumgraminearum as model organism and bacterial flagellin to mimic the presence of bacteria in an in vitro environment. The presence of flagellin triggered aninitial mitochondrial and cell membrane hyperpolarization which was detected using the florescent dye DiOC 7(3). This was followed by the production ofthe secondary signalling molecule Nitric Oxide (NO), common to innate immunity signalling in other eukaryotes. NO was monitored using the fluorescentdye DAF-FM. NO appears to be produced by an inducible enzyme that is regulated by complex mechanisms but centrally modulated byCalcium/Calmodulin. Inhibition studies suggest the presence of a Nitric Oxide Synthase (NOS), but no typical arginine utilizing NOS was identified withinthe F. graminearum’s genome by homology search. Various genes bearing resemblance to the archetypal NOS, as well as argininosuccinate lyase weredeleted. However, the mutants still produced NO. The presence of alternative pathways contributing towards the production of NO was investigated byadding a variety of potential substrates to challenged cultures. Various reactions were observed suggesting that several pathways are present. Inconclusion, F. graminearum reacts strongly to the presence of the bacterial Microbial Associated Molecular Pattern (MAMP) flagellin with an up-regulation27th Fungal Genetics Conference | 255