Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSof Ave1-like genes in pathogenicity. Ave1-like genes from non-Venturia fungi (and the bacterial gene) do not contain predicted introns, however, several(not all) V. inaequalis Ave1-like genes are predicted to contain introns. Codon usage bias among fungal, plant, and bacterial Ave1 orthologs, are beingcompared with the aim of inferring the kingdom of origin of the Venturia Ave1 orthologs. At least two ViAve1 orthologs are up-regulated during infectionof apple. To determine whether the Venturia Ave1 proteins also activate a Ve1-mediated hypersensitive response, each has been co-expressed withtomato Ve1 in Nicotiana tabacum, using an Agrobacterium tumefaciens-mediated transient transformation assay.527. Identification of targets of mycorrhizal effector proteins in planta. Natalia Requena, Carolin Heck, Ruben Betz. Molecular Phytopathology, KarlsruheInstitute of Technology, Karlsruhe, Germany.Plant roots are constantly approached by a myriad of microorganisms and are thus challenged to recognize friends from foes. Most plant roots engage ina mutualistic association with fungi from the Glomeromycota Phylum forming the arbuscular mycorrhiza (AM) symbiosis. The establishment of thisbeneficial association requires an intensive signal exchange including the down-regulation of PAMP triggered responses. We have shown that secretionand delivery of the effector proteins contribute to the manipulation of the plant defense response. In a previous work we showed that one of this proteinstravels to the plant nucleus and interacts with a plant transcription factor to down-regulate plant defenses. In order to identify further mechanisms of howsymbiotic effectors function we are investigating new plant targets of mycorrhizal effector proteins and how do they differ from targets from pathogenicfungi. Progress in this area will be presented.528. Structural basis for interactions of the Phytophthora sojae RXLR effector Avh5 with phosphatidylinositol 3-phosphate and for host cell entry.Furong Sun 2,3 , Shiv Kale 4 , Hugo Azurmendi 3 , Dan Li 2 , Brett M. Tyler 1,4 , Daniel Capelluto 2 . 1) Center for Genome Research and Biocomputing, Oregon StateUniversity, Corvallis, OR; 2) Dept of Biological Sciences, Virginia Tech, Blacksburg, VA 24061; 3) Dept of Chemistry, Virginia Tech, Blacksburg, VA 24061; 4)Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA 24061.Oomycetes, such as Phytophthora sojae, employ protein effectors that enter host cells to facilitate infection. Entry of some effector proteins into plantcells is mediated by conserved RxLR motifs in the effectors and phosphoinositides (PIPs) resident in the host plasma membrane such asphosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effector proteins involved in PIP recognition. To clarifythese differences, we have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5). Using NMR spectroscopy, wedemonstrate that Avh5 is helical in nature with a long N-terminal disordered region. Heteronuclear single quantum coherence titrations of Avh5 with thePtdIns(3)P head group, inositol 1,3-bisphosphate (Ins(1,3)P2), allowed us to identify a C-terminal lysine-rich helical region (helix 2) as the principal lipidbindingsite in the protein, with the N-terminal RxLR (RFLR) motif playing a more minor role. Mutations in the RFLR motif slightly affected PtdIns(3)Pbinding, while mutations in the basic helix almost abolished it. Avh5 exhibited moderate affinity for PtdIns(3)P, which increased the thermal stability of theprotein. Mutations in the RFLR motif or in the basic region of Avh5 both significantly reduced protein entry into plant and human cells. Both regionsindependently mediated cell entry via a PtdIns(3)P-dependent mechanism. Our findings support a model in which Avh5 transiently interacts withPtdIns(3)P by specific electrostatic interactions mainly through its positively charged helix 2 region, enabling the RFLR domain to promote PI3P-mediatedhost entry. This study, including the identification of the PtdIns(3)P-binding site, provides an improved and updated model for how RxLR effector proteinsrecognize phosphoinositides and for the contributions of the RxLR motif and basic-rich C-terminal regions to the internalization process.529. Dose-dependent induction of plant immunity by application of the Fusarium mycotoxin deoxynivalenol. Antje Blümke, Christian A. Voigt. MolecularPhytopathology, Biocenter Klein Flottbek, Hamburg, Germany.The mycotoxin deoxynivalenol (DON) is associated with Fusarium head blight (FHB). This disease causes vast losses by reducing grain quantity andquality. Our study was aimed at analyzing cell wall changes in the host model grass Brachypodium distachyon due to infection with different F.graminearum mutants. The mutants vary in their ability to infect spikelets and to produce DON. As a reference, we used a F. graminearum wild-type strainwith the full capacity to produce DON and to cause FHB. The results were compared to the infection with the Dtri5, the Dfgl1, and the Dgpmk1 mutant.The Dtri5 mutant cannot produce DON due to disruption of the DON biosynthetic pathway. This mutant infects the directly inoculated spikelet withoutfurther propagation into the head tissue. A similar disease phenotype is described for the lipase-disruption mutant Dfgl1, which is able to produce DON.The MAP kinase disruption mutant Dgpmk1 is apathogenic but still able to produce DON. We observed similar disease phenotypes and amounts of DONfor all F. graminearum mutants during B. distachyon infection as described for wheat. 7 days post-inoculation (dpi), we analyzed the non-cellulosicmonocarbohydrate cell wall composition of B. distachyon spikelets by high-performance anion exchange chromatography with pulsed amperometricdetection (HPAEC-PAD). Only the infection with F. graminearum mutants that showed reduced virulence but still produce DON, namely the Dfgl1 andDgpmk1 mutants, resulted in compositional changes of the cell wall, an increase in the amount of glucose. Next, we wanted to know to what extend themycotoxin DON itself can induce cell wall changes. We applied DON solutions at different concentrations to B. distachyon spikelets. 7 dpi, the HPAEC-PADanalysis revealed an increase in the glucose amount only at relatively low DON concentrations of 1, 10, 100, and 1000 ppb whereas higher DONconcentrations of 50 and 100 ppm did not change the cell wall composition. However, only these high DON concentrations caused necrosis of florets.Interestingly, F. graminearum wild-type infection was significantly reduced on spikelets sprayed with a DON solution at a concentration of 1000 ppb 7 daysprior fungal inoculation. This suggests that the mycotoxin DON can induce an effector-triggered-like immunity in a dose-dependent manner.530. How Oomycete Pathogens Exploit PI3P to Target Secreted RxLR Effectors into Host Cells. Q. Wang 1 , S. Ferrer 1 , J. Carlough 1 , F. Arredondo 1 , S. Kale 2 ,B. Tyler 1 . 1) Botany and Plant Pathology, OREGON STATE UNIVERSITY, Corvallis, OR. 97330; 2) Virginia Bioinformatics Institute, Virginia Tech, Blacksburg,VA 24060.Effector proteins from diverse oomycetes and fungi can enter plant cells to facilitate infection. Recent research suggests that phosphoinositides (PIPs)resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PI3P) mediate the entry of some oomycete RxLR effectors. The PIPrecognition domain of these effectors is still controversial. Current evidence shows that either the RxLR domain or positive residues in the C-terminaldomains (Ct) of some effectors such as the P. sojae effectors Avr1b and Avh5 can bind PI3P, it has been unresolved which of these domains, if either, orboth, are involved in cell entry during natural pathogen infection. Here we have used heterologous PI3P-binding proteins, such as the yeast VAM7p PXdomain to replace the RxLR or Ct domains of Avr1b in P.sojae transformants. Our results reveal that the VAM7p PX domain can replace the RxLR domain ofAvr1b in carrying the C-terminal domain of Avr1b into soybean cells, conferring an avirulent phenotype on the transformants. Mutations that abolish thebinding of VAM7p to PI3P substantially reduce but do abolish avirulence conferred by the construct. Mutations in the PI3P-binding residues of the Avr1b Ctalso substantially reduce avirulence, while the double mutant cannot confer avirulence at all. These results strongly support the hypothesis that PI3Pbindingis essential for Avr1b cell entry during natural infection, and further suggest that efficient entry by Avr1b may require two PI3P binding sites.27th Fungal Genetics Conference | 251