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The Pennsylvania State University Introduction Objectives

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The Pennsylvania State University Introduction Objectives Generating Secretomes to Investigate Unique Pathogen-Host Interactions Matthew R. Sheltra, Christopher W. Benson, and David R. Huff The Pennsylvania State University Introduction Methodology Results The secretome of Salmacisia constitutes 4.2% of the The consequences of plant-fungal interactions can range Using the genomes of Salmacisia, four related Tilletia from mutually beneficial to detrimental for the host plant. proteome, while the secretomes of other evaluated fungi range species, and the model organism Ustialgo maydis, a Infection of buffalograss with the fungus Salmacisia from 6.9-7.8% of the proteome (Figure 3). Of the 16 Salmacisia combination of web based tools were utilized to develop a buchloëana results in altered physiology of the host. Upon effectors, ten have unknown functions and three are ribosomal list of secreted proteins. The automated pipeline Secretools1 infection of male and female plants, hermaphroditism is proteins. There is also a thioredoxin domain containing protein, was combined with a modified series of tools discussed in induced in the otherwise dioecious plants, resulting in flowers a proteasome regulatory subunit, and a chitin deacetylase. Wang et al., 20152 as both pathways yielded different having both pistils and stamens. Rather than developing seed, classically secreted proteins. The resulting combination of the ovary of infected flowers becomes filled with fungal Organism Total Protein Proteins in Effector Proteins classically and non-classically secreted proteins is Isoforms Secretome in Secretome teliospores. Although infected flowers do not produce seed, considered the secretome. Each secretome was screened for Salmacisia buchloëana 6167 260 16 infected plants have greater reallocation of resources towards effector proteins with EffectorP. Tilletia caries 10204 804 214 reproductive structures than uninfected plants1. The underlying Tilletia walkeri 7999 614 114 mechanisms for this drastic change in reproductive biology is Tilletia controversa 9873 761 190 not yet known. Genomes Tilletia indica 9548 662 135 Ustilago maydis 6783 512 124 Derived from Wang Secretools Table 1) Secretome size for each organism and the number of predicted et al., 2015 effectors in each secretome dataset. Signal P + SignalP TargetP PredGPI TMHMM Secretome Relative to Proteome ListMerge 100% 98% 4.21% Signal Peptide Signal Classically secreted Classically 6.93% 96% 7.88% 7.68% 7.71% 7.55% No TM helix SeqRet 94% TMHMM 92% PROTEOME 90% SeqRet PERCENTAGE OF OF PERCENTAGE 88% SecretomeP Salmacisia T. caries T. walkeri T. controversa T. indica U. maydis Chandra and huff 2010 Chandra and huff 2010 Chandra and huff 2010 2.0 WolfPSort SPECIES Anther D E Non-secreted proteins Secretome SeqRet Figure 3) Percentage of proteomes represented by each secretome. BLAST HMMPfam Style TargetP Conclusions This has shown Salmacisia has a reduced secretome and far fewer effectors compared to related fungi. The generated secretome has also provided a list of candidate proteins which Teliospores Ovary Stigma may be responsible for the induction of hermaphroditism in Salmacisia. Figure 1a) Uninfected male inflorescence with orange anthers visible. 1b) Uninfected female inflorescence with purple stigmas visible. 1c) Infected PredGPI male with purple stigmas visible. 1d) Teliospores within the ovary in an anchor No GPI Future Work infected male plant. 1e) Salmacisia in culture on charcoal and agar. The proteins in the secretome will be tested through a - proteomic screen of infected plants at different developmental stages as well as uninfected plants and Salmacisia in culture. Objectives Secretome EffectorP Effectors Transformation of the fungus will allow knockouts to validate • Develop a comprehensive secretome for Salmacisia the role of these proteins. buchloëana and related fungi to identify proteins that may Figure 2) Pipeline used to generate secretomes. Secretools (red box) generated classically secreted proteins. The Wang et al., 2015 derived be responsible for the induction of hermaphroditism in References buffalograss. pathway (black box) generated classically and non-classically secreted 1) Chandra, A., and D.R. Huff. 2010. A Fungal Parasite Regulates a Putative Female-Suppressor Gene Homologous to MaizeTasselseed2and Causes Induced Hermaphroditism in Male Buffalograss. Molecular Plant-Microbe Interactions 23: 239–250. proteins. Individual tools are represented in blue. Beginning and end 2) Cortázar AR, Aransay AM, Alfaro M, Oguiza JA, Lavín JL (2014) SECRETOOL: integrated secretome analysis tool for fungi. Amino Acids. 2014 • Compare the Salmacisia secretome to those of well Feb;46(2):471-3. products are displayed in purple. Arrows show the workflow and selection 3) Wang, S., W. Wei, and X. Cai. 2015. Genome-wide analysis of excretory/secretory proteins in Echinococcus multilocularis: insights into characterized and related fungi. functional characteristics of the tapeworm secretome. Parasites & Vectors 8: . Available at: criteria applied. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696181/pdf/13071_2015_Article_1282.pdf..
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