Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS593. Characterization of the CoPRF1 mutant of Colletotrichum orbiculare defective in evasion of host defense responses. Kaoru Tanaka, Yasuyuki Kubo.Graduate School of Life and Environmenrtal Sciences, Kyoto Prefectural University, Kyoto, Japan.Plant pathogens have co-evolved with their host plants which have evolved the defense system against their pathogens. It is generally accepted thatplants express basal immunity by the recognition of the pathogen-associated molecular patterns, but compatible pathogens suppress the plant basaldefense by secreting effector proteins. In our previous study, we have obtained several pathogenicity deficient insertional mutants in Colletotrichumorbiculare by Agrobacterium tumefaciens-mediated transformation (AtMT). Among them, in the mutant named YK4524 it was shown that a T-DNAinsertion disrupted a gene which presumably encodes an extracellular protein with signal peptide sequence. And BLAST search of the predicted sequencefound no significant homologous genes in published databases, suggesting that it is unique to C. orbiculare. So we named this gene CoPRF1 (Pathogenesisrelatedfactor). Target gene disruption mutants of coprf1 obtained by AtMT showed significant reduction in virulence on the host leaves. However,characteristics such as germination, appressorium formation and penetration hyphal formation of coprf1 mutants in vitro were normal, indicating thatCoPRF1 is not essential for infection related morphogenesis. On the other hand, penetration ability of mutants was attenuated on intact cucumbercotyledons, and the elongation of its invasive hyphae was slower compared with the wild type. To confirm the possibility that decreased virulence ofcoprf1 mutants was involved in plant defense responses, we inoculated coprf1 mutant on cucumber cotyledons of which defense responses was disturbedby transient heat-shock. As expected, the pathogenicity of coprf1 mutant was restored. Furthermore, expression analysis of CoPRF1 by RT-PCR showedthat CoPRF1 expressed in planta, but not in vitro culture. From these results, it was suggested that CoPRF1 would engage in evasion of host basalresistance at the host infection.594. CPS1 mutants in Coccidioides are avirulent and act as an attenuated vaccine in the valley fever mouse model. Hema P. Narra 1,4 , Lisa F. Shubitz 2,3 , M.Alejandra Mandel 1,3 , Leslie Gunatilaka 5 , Hien Trinh 2,3 , Marc J. Orbach 1,3 . 1) School of Plant Sciences, Univ. of Arizona, Tucson, AZ; 2) Veterinary Sciences andMicrobiology, Univ. of Arizona, Tucson, AZ; 3) Valley Fever Center for Excellence, Univ. of Arizona, Tucson, AZ; 4) Department of Pathology, Univ. of Texas,Medical Branch, Galveston, TX; 5) School of Natural Resources and the Environment, Univ. of Arizona, Tucson, AZ.Coccidioides species are mammalian pathogens endemic to the Southwestern US as well as parts of Mexico, Central and South America. The disease theycause, coccidioidomycosis, or valley fever, is considered an emerging infectious disease due to increases in reported cases over the past 10 years. Toidentify virulence factors of this pathogen that may be targets for therapeutics, we have identified and disrupted genes that are important forpathogenicity in both plant pathogens and other animal pathogens. Based on the work of Liu et al. (2003), we disrupted the Coccidioides ortholog of C.heterostrophus CPS1 in C. posadasii strain Silveira. CPS1 was originally identified as a potential non-ribosomal peptide synthase component, because itencodes a polypeptide with two AMP binding domains related to the adenylation domains in bacterial non-ribosomal peptide synthases. However it alsocontains a putative N-terminal DMAP1b domain. In mammals, this domain binds the DMAP1 transcriptional co-repressor that has been shown to bindregulatory proteins and is proposed to act as a co-repressor of transcription. The C. posadasii cps1 deletion strain is non-pathogenic in susceptible mice butdoes initiate the formation of spherules, the infectious form of Coccidioides. The mutant also forms spherules in vitro. Whether Cps1 plays a role as aregulator of virulence via the DMAP1b domain, or via production of a potential toxin is not known. This is being explored via RNA-seq analysis and isolationof secreted metabolites from both the wild type strain Silveira and the cps1 mutant. The cps1 mutant appears to have great potential as an attenuatedvaccine since it protects from infection; when susceptible mice are challenged with wild type C. posadasii after inoculation with the cps1 mutant, nearly allexperience extended survival of at least four weeks and have low fungal burdens. inoculation with the cps1 mutant, they are completely resistant toinfection. Lu, S. W., S. Kroken, B. N. Lee, B. Robbertse, A. C. L. Churchill, O. C. Yoder, and B. G. Turgeon. 2003. A novel class of gene controlling virulence inplant pathogenic ascomycete fungi. Proceedings of the National Academy of Sciences of the United States of America 100:5980-5985.595. Elucidating the response of wheat to the exposure of Stagonospora nodorum effectors. Lauren A. Du Fall, Peter S. Solomon. Research School ofBiology, Australian National University, Canberra, ACT, Australia.The dothideomycete Stagonospora nodorum is a necrotrophic fungal pathogen of wheat and is the causal agent of Stagonospora nodorum blotch (SNB).This disease is responsible for over $100 million of yield losses in Australia annually. Recent studies have shown that this fungus produces a number ofeffector proteins that are internalized into host cells of susceptible wheat cultivars. The mechanism by which these effectors induce tissue necrosis insusceptible hosts is yet to be fully elucidated. We have applied a multi-omics approach to elucidate the cellular processes leading to disease and provideinsight into the mode-of-action of these effectors. Gas chromatography-mass spectrometry analysis of primary polar metabolites has been undertaken ontissue extracts and apoplastic fluid from SnToxA infiltrated wheat. Results illustrate widespread perturbations in primary metabolism and reveal the firstdirect evidence of an increase in energy production in response to a pathogen effector. To further understand the host response to SnToxA at thesecondary metabolism level, samples were also analysed using liquid chromatography-mass spectrometry. Our data indicate SnToxA causes an increase indefence-related secondary metabolites. The effect of these metabolites on Stagonospora nodorum growth and sporulation in vitro and in planta hasidentified several compounds with novel anti-fungal properties. These complementary approaches have provided a novel insight into the contribution ofthe SnToxA effector protein to SNB in wheat.596. Nep1-like proteins of the downy mildew Hyaloperonospora arabidopsidis trigger immunity, but not necrosis, in the Arabidopsis host. Stan Oome 1,2 ,Adriana Cabral 1 , Simon Samwel 1 , Tom Raaymakers 1 , Guido Van den Ackerveken 1,2 . 1) Plant-Microbe Interactions, Utrecht University, Utrecht, Netherlands;2) Centre for BioSystems Genomics (CBSG), Wageningen, Netherlands.The genome of the downy mildew pathogen Hyaloperonospora arabidopsidis, an obligate biotrophic oomycete, encodes several necrosis and ethyleneinducingpeptide 1 (Nep1)-like proteins (NLP). The NLPs of H. arabidopsidis (HaNLPs) constitute a family of 12 genes and 15 pseudogenes, most of whichform a species-specific clade separate from NLPs of related Phytophthora species, suggesting that the family has recently expanded. The secreted HaNLPswere found to be nontoxic when tested on Arabidopsis or tobacco, in contrast to known necrosis-inducing NLPs, e.g. the P. sojae PsojNIP protein that iscytolytic and induces a strong cell death response in dicot plant tissues. Even HaNLP3, which is most similar to necrosis-inducing NLP proteins of otheroomycetes, and which contains all amino acids that are known to be important for necrosis-inducing activity, did not induce necrosis. Chimerasconstructed between HaNLP3 and the necrosis-inducing PsojNIP protein demonstrated that most of the HaNLP3 protein is functionally equivalent toPsojNIP, except for an exposed domain that prevents necrosis induction. The HaNLP genes are mostly expressed early during infection, suggesting analternative function of noncytolytic NLP proteins during biotrophic infection of plants. To investigate if HaNLP production in the host affects susceptibilityto infection, transgenic Arabidopsis plants were generated. Surprisingly, overexpression of HaNLP3, 5, 6, 9, and 10 resulted in plants with severely reducedgrowth. To be able to monitor NLP-effects on pathogen infection, in the absence of growth reduction, an Arabidopsis line with an estradiol-inducibleHaNLP3 construct was generated. DNA microarray analysis revealed that plant immune responses were strongly activated upon estradiol-induced HaNLP3expression. Furthermore, resistance to H. arabidopsidis infection was activated, suggesting that the plant is able to recognize the pathogen-associated27th Fungal Genetics Conference | 267