@article{oh_franck_dean_2018, title={Sequential Phosphopeptide Enrichment for Phosphoproteome Analysis of Filamentous Fungi: A Test Case Using Magnaporthe oryzae}, volume={1848}, ISBN={["978-1-4939-8723-8"]}, ISSN={["1940-6029"]}, DOI={10.1007/978-1-4939-8724-5_7}, abstractNote={A number of challenges have to be overcome to identify a complete complement of phosphorylated proteins, the phosphoproteome, from cells and tissues. Phosphorylated proteins are typically of low abundance and moreover, the proportion of phosphorylated sites on a given protein is generally low. The challenge is further compounded when the tissue from which protein can be recovered is limited. Global phosphoproteomics primarily relies on efficient enrichment methods for phosphopeptides involving affinity binding coupled with analysis by fast high-resolution mass spectrometry (MS) and subsequent identification using various software packages. Here, we describe an effective protocol for phosphopeptide enrichment using an Iron-IMAC resin in combination with titanium dioxide (TiO2) beads from trypsin digested protein samples of the filamentous fungus Magnaporthe oryzae. Representative protocols for LC-MS/MS analysis and phosphopeptide identification are also described.}, journal={PLANT PATHOGENIC FUNGI AND OOMYCETES: METHODS AND PROTOCOLS}, author={Oh, Yeonyee and Franck, William L. and Dean, Ralph A.}, year={2018}, pages={81–91} }
 @article{sharpee_oh_yi_franck_eyre_okagaki_valent_dean_2017, title={Identification and characterization of suppressors of plant cell death (SPD) effectors from Magnaporthe oryzae}, volume={18}, ISSN={["1364-3703"]}, DOI={10.1111/mpp.12449}, abstractNote={Phytopathogenic microorganisms, including the fungal pathogen Magnaporthe oryzae, secrete a myriad of effector proteins to facilitate infection. Utilizing the transient expression of candidate effectors in the leaves of the model plant Nicotiana benthamiana, we identified 11 suppressors of plant cell death (SPD) effectors from M. oryzae that were able to block the host cell death reaction induced by Nep1. Ten of these 11 were also able to suppress BAX-mediated plant cell death. Five of the 11 SPD genes have been identified previously as either essential for the pathogenicity of M. oryzae, secreted into the plant during disease development, or as suppressors or homologues of other characterized suppressors. In addition, of the remaining six, we showed that SPD8 (previously identified as BAS162) was localized to the rice cytoplasm in invaded and surrounding uninvaded cells during biotrophic invasion. Sequence analysis of the 11 SPD genes across 43 re-sequenced M. oryzae genomes revealed that SPD2, SPD4 and SPD7 have nucleotide polymorphisms amongst the isolates. SPD4 exhibited the highest level of nucleotide diversity of any currently known effector from M. oryzae in addition to the presence/absence polymorphisms, suggesting that this gene is potentially undergoing selection to avoid recognition by the host. Taken together, we have identified a series of effectors, some of which were previously unknown or whose function was unknown, that probably act at different stages of the infection process and contribute to the virulence of M. oryzae.}, number={6}, journal={MOLECULAR PLANT PATHOLOGY}, author={Sharpee, William and Oh, Yeonyee and Yi, Mihwa and Franck, William and Eyre, Alex and Okagaki, Laura H. and Valent, Barbara and Dean, Ralph A.}, year={2017}, month={Aug}, pages={850–863} }
 @article{franck_franck_birke_chang_sangurdekar_cytryn_joshi_sadowsky_stacey_xu_et al._2014, title={Comparative transcriptomic analysis of symbiotic Bradyrhizobium japonicum}, volume={63}, number={3}, journal={Symbiosis}, author={Franck, S. and Franck, W. L. and Birke, S. R. and Chang, W. S. and Sangurdekar, D. P. and Cytryn, E. and Joshi, T. and Sadowsky, M. and Stacey, G. and Xu, D. and et al.}, year={2014}, pages={123–135} }
 @article{medeiros-silva_franck_borba_pizzato_strodtman_emerich_stacey_poacco_carlini_2014, title={Soybean ureases, but not that of Bradyrhizobium japonicum, are involved in the process of soybean root nodulation}, volume={62}, number={16}, journal={Journal of Agricultural and Food Chemistry}, author={Medeiros-Silva, M. and Franck, W. L. and Borba, M. P. and Pizzato, S. B. and Strodtman, K. N. and Emerich, D. W. and Stacey, G. and Poacco, J. C. and Carlini, C. R.}, year={2014}, pages={3517–3524} }
 @article{franck_gokce_oh_muddiman_dean_2013, title={Temporal Analysis of theMagnaporthe OryzaeProteome During Conidial Germination and Cyclic AMP (cAMP)-mediated Appressorium Formation}, volume={12}, ISSN={1535-9476 1535-9484}, url={http://dx.doi.org/10.1074/MCP.M112.025874}, DOI={10.1074/mcp.m112.025874}, abstractNote={Rice blast disease caused by Magnaporthe oryzae is one of the most serious threats to global rice production. During the earliest stages of rice infection, M. oryzae conidia germinate on the leaf surface and form a specialized infection structure termed the appressorium. The development of the appressorium represents the first critical stage of infectious development. A total of 3200 unique proteins were identified by nanoLC-MS/MS in a temporal study of conidial germination and cAMP-induced appressorium formation in M. oryzae. Using spectral counting based label free quantification, observed changes in relative protein abundance during the developmental process revealed changes in the cell wall biosynthetic machinery, transport functions, and production of extracellular proteins in developing appressoria. One hundred and sixty-six up-regulated and 208 down-regulated proteins were identified in response to cAMP treatment. Proteomic analysis of a cAMP-dependent protein kinase A mutant that is compromised in the ability to form appressoria identified proteins whose developmental regulation is dependent on cAMP signaling. Selected reaction monitoring was used for absolute quantification of four regulated proteins to validate the global proteomics data and confirmed the germination or appressorium specific regulation of these proteins. Finally, a comparison of the proteome and transcriptome was performed and revealed little correlation between transcript and protein regulation. A subset of regulated proteins were identified whose transcripts show similar regulation patterns and include many of the most strongly regulated proteins indicating a central role in appressorium formation. A temporal quantitative RT-PCR analysis confirmed a strong correlation between transcript and protein abundance for some but not all genes. Collectively, the data presented here provide the first comprehensive view of the M. oryzae proteome during early infection-related development and highlight biological processes important for pathogenicity.}, number={8}, journal={Molecular & Cellular Proteomics}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Franck, William L. and Gokce, Emine and Oh, Yeonyee and Muddiman, David C. and Dean, Ralph A.}, year={2013}, month={May}, pages={2249–2265} }
 @article{gokce_franck_oh_dean_muddiman_2012, title={In-Depth Analysis of the Magnaporthe oryzae Conidial Proteome}, volume={11}, ISSN={["1535-3907"]}, DOI={10.1021/pr300604s}, abstractNote={The filamentous fungus Magnaporthe oryzae (M. oryzae) is the causative agent of rice blast disease and presents a significant threat to worldwide rice production. To establish the groundwork for future research on the pathogenic development of M. oryzae, a global proteomic study of conidia was performed. The filter aided sample preparation method (FASP) and anion StageTip fractionation combined with long, optimized shallow 210 min nanoLC gradients prior to mass spectrometry analysis on an Orbitrap XL was applied, which resulted in a doubling of protein identifications in comparison to our previous GeLC analysis. Herein, we report the identification of 2912 conidial proteins at a 1% protein false discovery rate (FDR) and we present the most extensive study performed on M. oryzae conidia to date. A similar distribution between identified proteins and the predicted proteome was observed when subcellular localization analysis was performed, suggesting the detected proteins build a representative portion of the predicted proteome. A higher percentage of cytoplasmic proteins (associated with translation, energy, and metabolism) were observed in the conidial proteome relative to the whole predicted proteome. Conversely, nuclear and extracellular proteins were less well represented in the conidial proteome. Further analysis by gene ontology revealed biological insights into identified proteins important for central metabolic processes and the physiology of conidia.}, number={12}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Gokce, Emine and Franck, William L. and Oh, Yeonyee and Dean, Ralph A. and Muddiman, David C.}, year={2012}, month={Dec}, pages={5827–5835} }
 @article{oh_franck_han_shows_gokce_muddiman_dean_2012, title={Polyubiquitin Is Required for Growth, Development and Pathogenicity in the Rice Blast Fungus Magnaporthe oryzae}, volume={7}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0042868}, DOI={10.1371/journal.pone.0042868}, abstractNote={Protein ubiquitination, which is highly selective, regulates many important biological processes including cellular differentiation and pathogenesis in eukaryotic cells. Here, we integrated pharmacological, molecular and proteomic approaches to explore the role of ubiquitination in Magnaporthe oryzae, the leading fungal disease of rice world-wide. Inhibition of ubiquitin-mediated proteolysis using the 26S proteasome inhibitor, Bortezomib, significantly attenuated conidia germination, appressorium formation and pathogenicity in M. oryzae. Gene expression analysis revealed that many genes associated with protein ubiquitination were developmentally regulated during conidia germination. Only a few, including a polyubiquitin encoding gene, MGG_01282, were more abundantly expressed during appressorium formation and under nitrogen starvation. Targeted gene deletion of MGG_01282, in addition to a significant reduction in protein ubiquitination as determined by immuno blot assays, resulted in pleiotropic effects on M. oryzae including reduced growth and sporulation, abnormal conidia morphology, reduced germination and appressorium formation, and the inability to cause disease. Mutants were also defective in sexual development and were female sterile. Using mass spectrometry, we identified 63 candidate polyubiquitinated proteins under nitrogen starvation, which included overrepresentation of proteins involved in translation, transport and protein modification. Our study suggests that ubiquitination of target proteins plays an important role in nutrient assimilation, development and pathogenicity of M. oryzae.}, number={8}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Oh, Yeonyee and Franck, William L. and Han, Sang-Oh and Shows, Angela and Gokce, Emine and Muddiman, David C. and Dean, Ralph A.}, editor={Nielsen, KirstenEditor}, year={2012}, month={Aug}, pages={e42868} }
 @article{gokce_shuford_franck_dean_muddiman_2011, title={Evaluation of Normalization Methods on GeLC-MS/MS Label-Free Spectral Counting Data to Correct for Variation during Proteomic Workflows}, volume={22}, ISSN={["1879-1123"]}, DOI={10.1007/s13361-011-0237-2}, abstractNote={Normalization of spectral counts (SpCs) in label-free shotgun proteomic approaches is important to achieve reliable relative quantification. Three different SpC normalization methods, total spectral count (TSpC) normalization, normalized spectral abundance factor (NSAF) normalization, and normalization to selected proteins (NSP) were evaluated based on their ability to correct for day-to-day variation between gel-based sample preparation and chromatographic performance. Three spectral counting data sets obtained from the same biological conidia sample of the rice blast fungus Magnaporthe oryzae were analyzed by 1D gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Equine myoglobin and chicken ovalbumin were spiked into the protein extracts prior to 1D-SDS- PAGE as internal protein standards for NSP. The correlation between SpCs of the same proteins across the different data sets was investigated. We report that TSpC normalization and NSAF normalization yielded almost ideal slopes of unity for normalized SpC versus average normalized SpC plots, while NSP did not afford effective corrections of the unnormalized data. Furthermore, when utilizing TSpC normalization prior to relative protein quantification, t-testing and fold-change revealed the cutoff limits for determining real biological change to be a function of the absolute number of SpCs. For instance, we observed the variance decreased as the number of SpCs increased, which resulted in a higher propensity for detecting statistically significant, yet artificial, change for highly abundant proteins. Thus, we suggest applying higher confidence level and lower fold-change cutoffs for proteins with higher SpCs, rather than using a single criterion for the entire data set. By choosing appropriate cutoff values to maintain a constant false positive rate across different protein levels (i.e., SpC levels), it is expected this will reduce the overall false negative rate, particularly for proteins with higher SpCs.}, number={12}, journal={JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY}, author={Gokce, Emine and Shuford, Christopher M. and Franck, William L. and Dean, Ralph A. and Muddiman, David C.}, year={2011}, month={Dec}, pages={2199–2208} }
 @article{collier_sarkar_franck_rao_dean_muddiman_2010, title={Direct Comparison of Stable Isotope Labeling by Amino Acids in Cell Culture and Spectral Counting for Quantitative Proteomics}, volume={82}, ISSN={["1520-6882"]}, DOI={10.1021/ac101978b}, abstractNote={Numerous experimental strategies exist for relative protein quantification, one of the primary objectives of mass spectrometry based proteomics analysis. These strategies mostly involve the incorporation of a stable isotope label via either metabolic incorporation in cell or tissue culture (¹⁵N/¹⁴N metabolic labeling, stable isotope labeling by amino acids in cell culture (SILAC)), chemical derivatization (ICAT, iTRAQ, TMT), or enzymatically catalyzed incorporation (¹⁸O labeling). Also, these techniques can be cost or time prohibitive or not amenable to the biological system of interest (i.e., metabolic labeling of clinical samples, most animals, or fungi). This is the case with the quantification of fungal proteomes, which often require auxotroph mutants to fully metabolically label. Alternatively, label-free strategies for protein quantification such as using integrated ion abundance and spectral counting have been demonstrated for quantification affording over 2 orders of magnitude of dynamic range which is comparable to metabolic labeling strategies. Direct comparisons of these quantitative techniques are largely lacking in the literature but are highly warranted in order to evaluate the capabilities, limitations, and analytical variability of available quantitative strategies. Here, we present the direct comparison of SILAC to label-free quantification by spectral counting of an identical set of data from the bottom-up proteomic analysis of human embryonic stem cells, which are readily able to be quantified using both strategies, finding that both strategies result in a similar number of protein identifications. We also discuss necessary constraints for accurate quantification using spectral counting and assess the potential of this label-free strategy as a viable alternative for quantitative proteomics.}, number={20}, journal={ANALYTICAL CHEMISTRY}, author={Collier, Timothy S. and Sarkar, Prasenjit and Franck, William L. and Rao, Balaji M. and Dean, Ralph A. and Muddiman, David C.}, year={2010}, month={Oct}, pages={8696–8702} }