Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSIdentification and functional assay of Phytophthora sojae avirulence effectors. Yuanchao Wang, Suomeng Dong, Weixiao Yin. Plant Pathology Dept,Nanjing Agri Univ, Nanjing, China.Phytophthora sojae is a notorious oomycete pathogen producing a great loss on global soybean production annually. The disease outcome betweensoybean and P. sojae depends on whether hosts could recognize pathogen avirulence effectors. Recently identified oomycete avirulence effectors arecharacterized by N-terminal host entry motif (RxLR motif), sequence and transcriptional polymorphisms between virulent and avirulent strains. Benefitfrom 454 genome sequencing and solexa transcriptome sequencing of P. sojae strains, eight RxLR effectors are bioinformatically identified, geneticmapping suggested that two of them perfectly matched Avr3b and Avr1d phenotype respectively. Transient expression of the ORF from avirulence strainon soybean specifically triggered Rps3b and Rps1d mediated program cell death, respectively. confirming that they encodes avirulence effector Avr3b andAvr1d. Transient expression of Avr3b and Avr1d on Nicotiana benthamiana could promote the infection of Phytophthora capasici, suggesting bothavirulence effectors could suppress plant immunity and contribute to pathogen infection. Silencing of Avr3b impaired the virulence of Phytophthora sojae.Our progress in elucidating the mechanism under the inhibiting plant immunity by these effectors will be presented.Fungal lipoxygenases: a novel instigator of asthma? Gregory J. Fischer 1 , Katharyn Affeldt 3 , Erwin Berthier 2 , Nancy P. Keller 1,2,3 . 1) Department of Genetics,University of Wisconsin-Madison, Madison, WI; 2) Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI;3) Department of Bacteriology, University of Wisconsin-Madison, Madison, WI.Statement of Purpose: Fungi have long been associated with asthmatic diseases, yet the exact mechanism(s) by which fungi induce asthma is unknown.We propose that fungal lipoxygenase enzymes and their eicosanoid products are involved in asthmatic diseases. Human 5-lipoxygenase derivedleukotrienes induce inflammation, mucus secretion, vasodilation, and bronchial constriction. We hypothesize that the fungal pathogen Aspergillusfumigatus is capable of secreting a 5-lipoxygenase homolog, LoxB, that participates in eicosanoid production, including leukotrienes. This secretedhomolog is translocated into lung epithelial cells, participates in the production of leukotriene and other eicosanoids, and exacerbates asthmaticresponses, such as bronchoconstriction. Together, this work will help delineate the role fungal products play in asthmatic diseases. Methods: We areassessing fungal interactions with lung epithelial cells using a microfluidic in-vitro platform followed by murine asthma model research. To assess theeffects of LoxB overexpression, mass spectrometry was used to identify eicosanoid oxylipins within culture supernatants. Results: We have identified anAspergillus fumigatus lipoxygenase, LoxB, with high identity to human 5-lipoxygenase. Moreover, we have identified a motif in LoxB that may mediateentry into lung epithelial cells. To fully understand the impact of LoxB in asthma, we have developed an Aspergillus fumigatus strain that overexpressesLoxB. Overexpression of LoxB results in increased levels of various eicosanoids that are known to cause airway hyperresponsiveness and increased mucusproduction. Future work will focus on characterizing the effect these eicosanoid products have on the airway and whether fungal effector translocationresult in increased leukotriene levels.Magnaporthe oryzae has evolved two distinct mechanisms of effector secretion for biotrophic invasion of rice. Martha C. Giraldo 1 , Yasin F. Dagdas 2 ,Yogesh K. Gupta 2 , Thomas A. Mentlak 2,4 , Mihwa Yi 1 , Hiromasa Saitoh 3 , Ryohei Terauchi 3 , Nicholas J. Talbot 2 , Barbara Valent 1 . 1) Plant Pathology, KansasState University, Manhattan, KS. USA; 2) School of Biosciences, University of Exeter, EX4 4QD, UK; 3) Iwate Biotechnology Research Center, Kitakami,Iwate, 024-0003 Japan; 4) Cambridge Consultants Ltd, Cambridge, CB4 0DW, U.K.Pathogens secrete effector proteins into host tissue to suppress immunity and cause disease. Pathogenic bacteria have evolved several distinct secretionsystems to target specific effector proteins during pathogenesis, but it was not previously known if fungal pathogens require different secretorymechanisms. We present evidence that the blast fungus Magnaporthe oryzae possesses distinct secretion systems for delivering effector proteins duringbiotrophic invasion of rice cells. M. oryzae secretes cytoplasmic effectors targeted for delivery inside rice cells and apoplastic effectors targeted to theextracellular space. Cytoplasmic effectors preferentially accumulate in the biotrophic interfacial complex (BIC), a novel in planta structure located besidethe tip of the initially filamentous invasive hypha and then remaining next to the first differentiated bulbous invasive hypha cell. In contrast, apoplasticeffectors remain in the extracellular compartment uniformly surrounding the invasive hypha inside the invaded cell. Disruption of the conventional ER-Golgi secretion pathway by Brefeldin A (BFA) treatment blocked secretion of apoplastic effectors, which were retained in the ER, but not secretion ofcytoplasmic effectors. Fluorescence Recovery After Photobleaching experiments confirmed that cytoplasmic effectors continued to accumulate in BICs inthe presence of BFA. Analysis of mutants showed that the BIC is associated with a novel form of secretion involving exocyst components, Exo70 and Sec5,and the t-SNARE Sso1, which are required for efficient delivery of effectors into plant cells and are critical for pathogenicity. By contrast, effectors whichfunction between the fungal cell wall and plant plasma membrane are secreted from invasive hyphae to the apoplast by the ER-Golgi secretory pathwayconserved in eukaryotes. We propose a model for the distinct secretion systems that the rice blast fungus has evolved to achieve tissue invasion.Domains for plant uptake of Ustilago maydis secreted effectors. Anupama Ghosh, Armin Djamei, Shigeyuki Tanaka, Regine Kahmann. Max PIanckInstitute for Terrestrial Microbiology, Department of Organismic Interactions, Karl-Von-Frisch-Strasse 10, D-35043 Marburg, Germany.The genome of the corn smut fungus Ustilago maydis codes for a large repertoire of secreted effectors. Some of them play crucial roles for virulence andestablishment of the biotrophic phase. The chorismate mutase Cmu1 is one such secreted translocated effector of U. maydis. cmu1 deletion strains areattenuated in virulence that is attributed to higher salicylate levels in plants infected with the mutant strain, most likely through alterations in thechanneling of chorismate from the plastids to the cytosol. Here we identify the motif in Cmu1 that is necessary for the translocation of the protein acrossthe plant plasma membrane and present a mutational analysis of this region. To test for uptake we assayed the ability of mutant proteins to complement acmu1 mutant strain as well as the retained ability to complement the growth defect of a Daro7 strain of S. cerevisiae in minimal medium. By deletionanalysis a region of 20 amino acids adjacent to the signal peptide was shown to be essential for the translocation. Microscopic analysis of maize tissueinfected with U. maydis strains expressing Cmu1-mcherry fusion proteins with or without the probable uptake motif revealed that the 20 amino acid motifallows binding of the protein to an as yet unknown plant plasma membrane component. We hypothesize that the translocation of Cmu1 across the plantplasma membrane is a two step process; initiated by binding followed by translocation across the membrane. In addition, we present results where the 20amino acid motif is substituted by motifs from other effectors.27th Fungal Genetics Conference | 65