PATHOGENIC GENE DISCOVERY IN THE DEVASTATING RICE BLAST FUNGUS MAGNAPORTHE ORYZAE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0223733
• Project Start Date: Sep 1, 2010
• Project End Date: Aug 31, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
Plant Pathology

Non Technical Summary
Rice contributes 23 percent of the calories consumed worldwide, but each year 10 to 30 percent of the global rice harvest enough to feed 60 million people - is lost to infection by the rice blast fungus Magnaporthe oryzae. Because traditional plant breeding strategies have been unable to produce durable blast-resistant rice cultivars, containment of this severe threat to global food security is likely to come from a better understanding of the molecular and cellular processes underlying the plant-fungal interaction. M. oryzae is ideally suited to such study because: 1) it can be cultured away from its host plant, 2) it is amenable to rapid gene functional analysis, and 3) both M. oryzae and its rice host plant have sequenced genomes. M. oryzae plant infection is mediated by a specialized structure, called the appressorium, which develops from an asexual spore of M. oryzae adhered to the leaf surface. The mature appressorium generates enormous turgor through the accumulation of glycerol, and this pressure is translated into mechanical force acting on a penetration peg emerging at the base of the cell, causing it to breach the leaf cuticle and enter the plant. However, the physical nature of M. oryzae host entry belies the fact that once within the plant, the fungus forms an intimate association with the plant tissue and proliferates from cell to cell in a symptomless, biotrophic manner for the first 72 hours of infection. Therefore, there is a critical need to determine how M. oryzae is able to evade or suppress the host response, how it controls the development of infectious hyphae during the biotrophic growth phase, and what triggers the switch to symptom development following the first 72 hours.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	4020	1000	25%
212	4020	1040	25%
212	4020	1102	25%
212	4020	1080	25%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1040 - Molecular biology; 1102 - Mycology; 1000 - Biochemistry and biophysics; 1080 - Genetics;

Keywords

magnaporthe oryzae

genetic regulation

rice blast

nadph

redox

Goals / Objectives
The overall objective of the proposal is two-fold: 1) to identify Tps1-dependent transcription factors necessary for pathogenicity, and the genes they control; and 2) to fully characterize this novel NADPH signaling pathway to enhance our basic understanding of how NADPH production is regulated in the cell and provide important knowledge on the connections between nutrient availability, NADPH metabolism, and gene regulation. Together, this study will enhance our basic understanding of the M. oryzae-rice interaction and provide a critical first step toward finding a solution to the intractable problem of rice blast disease. Our goals include: a) Identification and characterization of transcription factors controlling pathogenicity in M. oryzae. b) Functional characterization of the dynamics of Nmr function and the role of Nmrs in pathogenicity. c) Identification of Tps1-dependent genes necessary for pathogenicity. d) Characterization of the connection between NADPH metabolism and gene expression. e) Determining how the subcellular compartmentalization of NADPH biosynthesis influences cell signaling. Objective 1: Identification and functional characterization of TFs necessary for virulence, their interaction with the Nmr proteins, and the pathogenicity genes they control. Objective 2 Determine the connection between NADPH metabolism and gene expression. Outcomes: Through the comparison of gene expression in pathogenic and non-pathogenic strains, we will identify currently unknown pathogenic determinants, and their likely cellular functions, which will significantly advance current knowledge of the M. oryzae-rice interaction. In addition, novel mechanisms of regulation that integrate metabolism with gene expression in M. oryzae will likely shed light on fundamental genetic regulation in other fungi. These advances will allow us to move forward in our search for durable control strategies. Moreover, strategies developed here that target general fungal processes necessary for virulence, such as blocking trehalose metabolism in other plant pathogenic fungi, or determining how the integration of carbon and nitrogen metabolism can be altered to reduce toxic fungal secondary metabolite production, will be applicable to other severe crop diseases such as those of wheat and corn. Therefore, one measurable outcome of our work will be the enhancement of plant health in a range of agricultural systems. Progress will also be evaluated based on our success in obtaining competitive extramural grant support and on our ability to publish research results in refereed scientific journals that will impact scientific colleagues in related fields of study.

Project Methods
Growth of fungi on minimal media containing NO3- as sole nitrogen source results in increased NADPH levels, compared to growth on other nitrogen sources, to supply reducing power to nitrate reductase. The Δtps1 mutant is unable to elevate NADPH levels, through an inability to activate G6PDH in response to G6P, and cannot grow on NO3- media. In addition, some genes encoding NADPH-dependent enzymes, including nitrate reductase, are expressed in Guy11 under these NADPH elevated conditions, but are much reduced in expression in Δtps1 strains. In order to perturb NADPH metabolism, we will delete from the M. oryzae genome genes involved in NADPH metabolic pathways and determining the affect on NADPH production and gene expression. We will use the split marker approach to delete genes from Guy11, and assess the effect on NADPH production and gene expression. Following identification of deletion mutants we will grow the strains on NO3 -media and analyze NADPH levels in the mutants compared to Guy11, using the enzymatic approach described in Wilson et al. (2007).

Progress 09/01/10 to 08/31/15

Outputs
Target Audience: The proposed project will yield new insights into the M. oryzae- rice interaction that will be of specific interest to other researchers in the rice blast field. In addition, this project will yield important discoveries about fundamental mechanisms that integrate metabolism with gene expression that will be applicable to other plant pathogenic fungi. Breeders and the agrochemical industry are also likely to benefit from the work undertaken here. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project involves undergraduates researchers and thus provided considerable scope for training the next generation of scientists. This project provided postdoctoral training in molecular biology. How have the results been disseminated to communities of interest? Yes, via publication and talks given by the PI and graduate students at international, national and local meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Rice blast caused by the fungus Magnaporthe oryzae is a global food security threat that annually results in a 10-30% reduction in rice yield, yet little is known about how the fungus grows and survives in the host. For the first days of infection before causing disease, M. oryzae acquires nutrients and colonizes living plant tissue without activating the robust plant defenses that normally work to keep the host plant disease-free.This project seeks to determine the molecular mechanisms that sustain M. oryzae growth in the host cell. Because M. oryzae devastates up to 30% of global rice crops each year, the identification of significant weaknesses in M. oryzae physiology could potentially lead to the development of strategies to protect rice plants from blast disease. Such strategies would also be applicable to a wide range of fungi with lifestyles similar to M. oryzae. Thus, by addressing the fundamental question "What molecular mechanisms coordinate sustained fungal growth in the host cell?" this project aims to resolve substantial issues in plant pathology regarding the molecular mechanisms by which fungal pathogens thrive in plant cells. Achieving the stated objectives will define key exploitable differences in the metabolism of M. oryzae compared to host rice cells. This could lead to the development of novel crop protection strategies targeting molecular pathways that are critical for the biotrophic growth of the fungus but are not required for the normal function of the host cell. This work will also shed new light on the basic principles of cell growth. In the current reporting period, progress has been made in understanding the genes regulating how the fungus senses and responds to sources of glucose, and the molecular mechanisms by which these signals result in colonizaton of the host cell. We have used a combination of forward and reverse genetics to identify novel components of the glucose signaling pathway which are currently being functioanlly charaxcterized, and described a novel role for a fungal sirtuin in regulating early rice cell infection. This work thus not only contributes to our understanding of plant pathogenic processes but, due to the new knowledge generated about sirtuin, has broad implications for understanding molecular mechanisms of gene regulation in eukaryotes. Objective 1: Understand the role of glucose-6-phosphate signaling in disease. 1) Major activities completed / experiments conducted; We have undertaken a screen of Tps1 suppressor mutants that are revealing how glucose metabolism is functionally connected with downstream outputs necessary for viurlence. 2) Data collected: Five mutants obtained and two suppressor mutations identified. Full characterization is underway. 3) Summary statistics and discussion of results and 4) Key outcomes or other accomplishments realized: on going. Objective 2: Characterize carbon-derepressed mutants to elucidate how glucose signalling is transmitted to the nucleus to control fungal nutrient adaptability in the plant. 1) Major activities completed / experiments conducted; We have used forward genetics to identify undertaken a screen of suppressor mutants that are revealing novel comonents of the glucose signaling pathway in eukaryotes. 2) Data collected: Six mutants obtained and two suppressor genes identified. Full characterization is underway. 3) Summary statistics and discussion of results and 4) Key outcomes or other accomplishments realized: on going. Objective 3: Characterize a newly discovered M. oryzae gene required for glucose tolerance and essential for rice infection. 1) Major activities completed / experiments conducted; Previously, we performed the first functional characterization of a sirtuin from a fungal plant pathgen. We showed it was necessary for antioxidation, suppressing host ROS burst and thus suppressing the host defenses. Forward genetic screens coupled with genome wide acetylomic and proteomic studies (the first of their kind performed using a fungal plant pathogen) revealed a JmjC histone demethylase as a downstream target regulating the expression of superoxide dismutase. Together, this work revelaed the sirtuin and JmJc as novel mediators of early rice cell infection. 2) Data collected: Live cell-imaging, genetic, genomic, proteomic, acetylomic. Going forward, we have used ChIP to identify interacting proteins of this sirtuin, including an E3 ubiquitin ligase that likely regulates sirtuiin function. We are currently characterizing this regulator of a regulator. 3) Summary statistics and discussion of results and 4) Key outcomes or other accomplishments realized: This work identified sirtuins as major mediators of plant. Inhibiting fungal sirtuins would thus boost plant defenses and abolish fungal infection of host cells. This work has thus significantly enhanced our understanding of how fungal cells thrive in host plant cells.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, G., Marroquin-Guzman, M. and Wilson, R. A. (2015). Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein  DNA Interactions in Magnaporthe oryzae. Bio-protocol 5(21): e1643. http://www.bio- protocol.org/e1643

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: This work will contribute to our understanding of the metabolic strategies, and their regulation, that facilitate the interactions between pathnogeic fungi and their hosts. For society-wide problems such as crop devastation resulting from rice blast, this could reveal how disruptive interventions might be developed. This work is thus targeted at the plant pathogen/ plant sciences research communites. Moreover, this project will continue to provide a rich and rewarding platform for engaging underrepresented graduate, undergraduate, and high school students in the research challenges associated with rice blast disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? I have trained 1 post doc, 4 gradstudents and 12 undergraduates through this project. How have the results been disseminated to communities of interest? Through publication in international, peer-reviewed journals, conference presentations, and using online platforms such as ResearchGate and Twitter. What do you plan to do during the next reporting period to accomplish the goals? Continue identifying and characterizing genes important for mediating the rice-magnaporthe interaction.

Impacts
What was accomplished under these goals? Rice blast caused by the fungus Magnaporthe oryzae is a global food security threat that annually results in a 10-30% reduction in rice yield, yet little is known about how the fungus grows and survives in the host. For the first days of infection before causing disease, M. oryzae acquires nutrients and colonizes living plant tissue without activating the robust plant defenses that normally work to keep the host plant disease-free.This project seeks to determine the molecular mechanisms that sustain M. oryzae growth in the host cell. Because M. oryzae devastates up to 30% of global rice crops each year, the identification of significant weaknesses in M. oryzae physiology could potentially lead to the development of strategies to protect rice plants from blast disease. Such strategies would also be applicable to a wide range of fungi with lifestyles similar to M. oryzae. Thus, by addressing the fundamental question "What molecular mechanisms coordinate sustained fungal growth in the host cell?" this project aims to resolve substantial issues in plant pathology regarding the molecular mechanisms by which fungal pathogens thrive in plant cells. Achieving the stated objectives will define key exploitable differences in the metabolism of M. oryzae compared to host rice cells. This could lead to the development of novel crop protection strategies targeting molecular pathways that are critical for the biotrophic growth of the fungus but are not required for the normal function of the host cell. This work will also shed new light on the basic principles of cell growth. In the current reporting period, progress has been made in understanding the genes regulating how the fungus senses and responds to sources of glucose, and the molecular mechanisms by which these signals result in colonization of the host cell. We have used a combination of forward and reverse genetics to identify novel components of the glucose signaling pathway which are currently being functionally characterized, and described a novel role for a fungal sirtuin in regulating early rice cell infection. This work thus not only contributes to our understanding of plant pathogenic processes but, due to the new knowledge generated about sirtuin, has broad implications for understanding molecular mechanisms of gene regulation in eukaryotes. Objective 1: Identification and functional characterization of transcription factors necessary for virulence, their interaction with the Nmr proteins, and the pathogenicity genes they control. 1) Major activities completed / experiments conducted: We have fully characterized two GATA factors required for appressorium development and the growth of M. oryzae in planta, thus revealing how these important regulators are functionally connected to Tps1 and essential for virulence (publication in revision). We 2) Data collected: Gene deletion mutants were identified. Full characterization is underway. 3) Summary statistics and discussion of results and 4) Key outcomes or other accomplishments realized: on going. Objective 2 Determine the connection between NADPH metabolism and gene expression. 1) Major activities completed / experiments conducted: We coupled plate tests with forward genetic analyses to characterize how NADPH production results in the expression of antioxidation genes critical for biotrophic growth. Further analysis is revealing novel components of the glucose signaling pathway in eukaryotes. 2) Data collected: Six antioxidation mutants obtained and two suppressor genes identified. Full characterization is underway. 3) Summary statistics and discussion of results and 4) Key outcomes or other accomplishments realized: on going.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Fernandez J, Wilson RA (2014) Characterizing Roles for the Glutathione Reductase, Thioredoxin Reductase and Thioredoxin Peroxidase-Encoding Genes of Magnaporthe oryzae during Rice Blast Disease. PLoS ONE 9: e87300.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Donofrio NM, Wilson RA (2014) Redox and rice blast: new tools for dissecting molecular fungalplant interactions. New Phytol 201: 367369.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Fernandez J, Marroquin-Guzman M, Nandakumar R, Shijo* S, Cornwell* KM, Li G, Wilson RA (2014) Plant defence suppression is mediated by a fungal sirtuin during rice infection by Magnaporthe oryzae. Mol Microbiol 94:70-88.
• Type: Book Chapters Status: Published Year Published: 2014 Citation: Donofrio NM, Hu J, Mitchell TK, Wilson RA (2014) Facilitating the Fungus: Insights from the Genome of the Rice Blast Fungus, Magnaporthe oryzae. In: Genomics of Plant-Associated Fungi: Monocot Pathogens. pp.141-160. Ed: Dean RA, Lichens-Park A, Kole C. Published by Springer Berlin Heidelberg.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Fernandez J, Marroquin-Guzman M, Wilson RA (2014) Mechanisms of nutrient acquisition and utilization during fungal infections of leaves. Annu Rev Phytopathol 52:155-74.
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Fernandez J, Marroquin-Guzman M, Wilson RA (2014) Evidence for a Transketolase-Mediated Metabolic Checkpoint Governing Biotrophic Growth in Rice Cells by the Blast Fungus Magnaporthe oryzae. PLoS Pathog 10: e1004354.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Target audiences include professionals in the area of plant pathology reached by publications and presentations at international conferences, and students trained through working on this project, including those from underrepressented groups. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has trained 15 undergraduates, resulting in two included as authors on our recent Sci Rep publication. How have the results been disseminated to communities of interest? Via publications and presentations at international meetings. What do you plan to do during the next reporting period to accomplish the goals? Continue our functional investigations of this important system and remain at the fore-front of our field.

Impacts
What was accomplished under these goals? We made significant progress towards addressing these goals, as evidenced by our publications in high-impact journals.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Glycogen metabolic genes are involved in trehalose-6-phosphate synthase-mediated regulation of pathogenicity by the rice blast fungus Magnaporthe oryzae. Badaruddin M, Holcombe LJ, Wilson RA, Wang ZY, Kershaw MJ, Talbot NJ.PLoS Pathog. 2013;9(10):e1003604. doi: 10.1371/journal.ppat.1003604. Epub 2013 Oct 3.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Cells in cells: morphogenetic and metabolic strategies conditioning rice infection by the blast fungus Magnaporthe oryzae.Fernandez J, Wilson RA.Protoplasma. 2013 Aug 29. [Epub ahead of print]
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Growth in rice cells requires de novo purine biosynthesis by the blast fungus Magnaporthe oryzae. Fernandez J, Yang KT, Cornwell KM, Wright JD, Wilson RA.Sci Rep. 2013;3:2398. doi: 10.1038/srep02398.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: We have identified metabolic capabilities that are over- or underrepresented in M. oryzae compared to the soil saprophyte A. nidulans and are critical to the M. oryzae lifestyle. We identified metabolic processes present in wild type and suppressor strains, but not non-pathogenic mutants, that are required for virulence. We identified novel genes expressed in wild type and suppressor strains, but not non-pathogenic mutants, that are required for virulence. We continued to functionally characterize regulatory transcription factors and determine their epistatic relationship to each other, and to other signaling pathways, via high through-put gene deletion strategies. We have functionally characterized genes involved in maintaining the redox balance. We have successfuly developed our forward genetic screen for identifying pathways and regulators that are essential for fungal virulence, and have determined for the first time the mechanism by which plant pathogenic fungi acquire nutrients from the host. I have taught the following courses: Semester 20101-2012 Spring Course: AGRO370: Biology of Fungi. Semester:2012-2013 Fall Course:PLPT110 Molds & Man Semester:2010-2013 Fall Course:PLPT801 Plant Pathology I am currently advising the following students: Undergrad Advisees: 2 UCARE, 2 workstudy, 3 research assistants: 2 Graduate Students that I alone advise 2 PhD Residents: 1 I was invited to speak at the following conferences: Purdue Plant Pathology seminar series: Mechanisms of in planta nutroent acquisition by the rice blast fungus. I am organizing a session for the 27th Fungal Genetics Conference, Asilomar, CA.I hosted 30 highschool students for three days as part of the Nebraska EPSCoR summercamp program, where my goal was to use the rice-Magnaporthe interaction as an introduction to the importance of crop diseases and inspire them to become the next generation of scientists. PARTICIPANTS: PARTICIPANTS: Individuals who work on this project include Janet wright, technologist, Hayden Hopkins (undergraduate), Jessie Fernandez (graduate student) and Margarita Marroquin (graduate student). TARGET AUDIENCES: TARGET AUDIENCES: Target audiences include undergraduates and graduates, where my lab provides practical training and my courses involve instructional training in plant pathology related matters. I also reach highschool students through my participation in the Nebraska EPSCoR summercamp program. Finally, by disseminating my work through publications and presentations, I target other fungal researchers with my work. PROJECT MODIFICATIONS: PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Results from this work formed the basis for a successful NSF submission. Forward genetic analysis of the mutants described in this objective has generated enough novel data that we have submitted this work for publication. This will impact our ability to secure Federal funds in the future. Our preliminary data on the regulation of pathogenicity has been presented at several international meetings, has been published in high impact journals, and thus will impact the wider research community. This work is novel, high impact, and is not being studied in any other system.

Publications

• Wilson RA, Fernandez J, Quispe CF, Gradnigo J, Seng A, et al. (2012) Towards Defining Nutrient Conditions Encountered by the Rice Blast Fungus during Host Infection. PLoS ONE 7(10): e47392. doi:10.1371/journal.pone.0047392.
• Fernandez, J, and Wilson, RA (2012) Why no feeding frenzy Mechanisms of nutrient acquisition and utilization during infection by the rice blast fungus Magnaporthe oryzae. Mol Plant-Microbe Interact. 25:1286-1293.
• Fernandez J, Wright JD, Hartline D, Quispe CF, Madayiputhiya N, and Wilson, RA (2012) Principles of Carbon Catabolite Repression in the Rice Blast Fungus: Tps1, Nmr1-3, and a MATE Family Pump Regulate Glucose Metabolism during Infection. PLoS Genet 8(5): e1002673. doi:10.1371/journal.pgen.1002673.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: We have identified metabolic capabilities that are over- or underrepresented in M. oryzae compared to the soil saprophyte A. nidulans and are critical to the M. oryzae lifestyle. We identified metabolic processes present in wild type and suppressor strains, but not non-pathogenic mutants, that are required for virulence. We identified novel genes expressed in wild type and suppressor strains, but not non-pathogenic mutants, that are required for virulence. We continued to functionally characterize regulatory transcription factors and determine their epistatic relationship to each other, and to other signaling pathways, via high through-put gene deletion strategies. We have functionally characterized five nitroalkane dioxygenases (ndos), and revealed an essential role for at least two in virulence. We are functionally characterized genes involved in maintaining the redox balance. We have successfuly developed our forward genetic screen for identifying pathways and regulators that are essential for fungal virulence, and have determined for the first time the mechanism by which plant pathogenic fungi acquire nutrients from the host.. I have taught the following courses: Semester 2010-2011 Spring Course: AGRO370: Biology of Fungi. Semester:2011-2012 Fall Course:PLPT110 Molds & Man Semester:2011-2012 Fall Course:PLPT801 Plant Pathology I am currently advising the following students: Undergrad Advisees: 2 UCARE, 2 workstudy, 3 research assistants: 2 Graduate Students that I alone advise MS Resident: 1 PhD Resident: 1 I was invited to speak at the following conferences: A novel NADPH-dependent genetic switch regulates infection by the rice blast fungus. 25th Fungal Gentics Conference, Asilomar, USA. I was an invited plenary speaker at the IX National Meeting on Fungal Molecular and Cellular Biology, San Luis Potosi, Mexico: Feeding Frenzy: Mechanisms of in planta nutroent acquisition by the rice blast fungus. I hosted 30 highschool students for three days as part of the Nebraska EPSCoR summercamp program, where my goal was to use the rice-Magnaporthe interaction as an introduction to the importance of crop diseases and inspire them to become the next generation of scientists PARTICIPANTS: Individuals who work on this prect include Janet wright, technologist, Jonathan Hinz (undergraduate), David Hartline (undergraduate), Anya Seng (undergraduate), Jessie Fernandez (graduate student) and Cristian Quispe (graduate student). TARGET AUDIENCES: Target audiences include undergraduates and graduates, where my lab provides practical training and my courses involve instructional training in plant pathology related matters. I also reach highschool students through my participation in the Nebraska EPSCoR summercamp program. Finally, by disseminating my work through publications and presentations, I target other fungal researchers with my work. PARTICIPANTS: Individuals who work on this prect include Janet wright, technologist, Jonathan Hinz (undergraduate), David Hartline (undergraduate), Anya Seng (undergraduate), Jessie Fernandez (graduate student) and Cristian Quispe (graduate student). TARGET AUDIENCES: Target audiences include undergraduates and graduates, where my lab provides practical training and my courses involve instructional training in plant pathology related matters. I also reach highschool students through my participation in the Nebraska EPSCoR summercamp program. Finally, by disseminating my work through publications and presentations, I target other fungal researchers with my work. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Results from this work formed the basis for two NSF submissions, and one USDA submission. Forward genetic analysis of the mutants described in this objective has generated enough novel data that we have submitted this work for publication. This will impact our ability to secure Federal funds in the future. Our preliminary data on the regulation of pathogenicity has been presented at several international meetings, has been published in high impact journals, and thus will impact the wider research community. The significance of the ndo results to the field is such that we are preparing a manuscript of this work for publication. The significance of the NADPH work to the field is such that we are preparing a manuscript for publication.

Publications

• Franceschetti, M., Bueno, E., Wilson, R. A. ,Tucker, S. L., Gomez-Mena, C., Calder, G. and Sesma, A. RBP35 is an RNA 3 End Processing Factor that Interacts with Cleavage Factor I 25kDa to Control Fungal Development and Virulence. Plos Pathogens (2011).
• Fernandez, J. and Wilson, R. A. The sugar sensor, trehalose-6-phosphate synthase (Tps1), regulates primary and secondary metabolism during infection by the rice blast fungus: Will Magnaporthe oryzaes sweet tooth become its Achilles heel Mycology. 2, 46- 53 (2011).
• Wilson, R. A., Gibson, R. P., Quispe, C. F. Littlechild, J. A. and Talbot, N. J. 2010. An NADPH dependent genetic switch regulates plant infection by the rice blast fungus. Proc. Natl. Acad. Sci. U. S. A. 107, 21902-21907 (2010).

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: We have identified metabolic capabilities that are over- or underrepresented in M. oryzae compared to the soil saprophyte A. nidulans and are critical to the M. oryzae lifestyle. We identified metabolic processes present in wild type and suppressor strains, but not non-pathogenic mutants, that are required for virulence. We identified novel genes expressed in wild type and suppressor strains, but not non-pathogenic mutants, that are required for virulence. We continued to functionally characterize regulatory transcription factors and determine their epistatic relationship to each other, and to other signaling pathways, via high through-put gene deletion strategies. We have functionally characterized four out of five nitroalkane dioxygenases (ndos), and revealed an essential role for at least two in virulence. We are functionally characterized genes involved in maintaining the redox balance. We have successfuly developed our forward genetic screen for identifying pathways and regulators that are essential for fungal virulence. I have taught the following courses: Semester:2009-2010 Fall Course:PLPT369 Intro Plant Path Lab. Semester:2009-2010 Fall Course:PLPT110 Molds & Man Semester:2009-2010 Fall Course:PLPT864 Plant Pathology I am currently advising the following students: Undergrad Advisees: 1 UCARE: 2 Graduate Students that I alone advise MS Resident: 1 PhD Resident: 1 I was invited to speak at the following conferences: Trehalose-6-phosphate synthase integrates metabolism and virulence in Magnaporthe oryzae via a novel NADP(H)-dependent genetic switch. X International Fungal Biology Conference, Ensenada, Mexico. 12/07/2009 New solutions to old problems: using scalable "-omics" technologies to understand the fungal cell as an integrated dynamic living system. La Universidad Autonoma de San Luis Potosi. Mexico. 02/25/2010 Biochemistry at full blast: A new role for NADPH metabolism in the pathogenicity of the rice blast fungus Magnaporthe oryzae. Instituto Potosino de investigacion Cientifica y Tecnologica (IPICYT), Mexico. 02/26/2010 A novel NADPH-dependent genetic switch regulates infection by the rice blast fungus. 5th International Rice Blast Conference, Little Rock, Arkansas. 08/12/2010 I attended the American Phytopathology Society meeting in Charlotte, NC, in August 2010. I hosted 30 highschool students for three days as part of the Nebraska EPSCoR summercamp program, where my goal was to use the rice-Magnaporthe interaction as an introduction to the importance of crop diseases and inspire them to become the next generation of scientists PARTICIPANTS: Individuals who work on this prect include Janet wright, technologist, Jonathan Hinz (undergraduate), David Hartline (undergraduate), Anya Seng (undergraduate), Jessie Fernandez (graduate student) and Cristian Quispe (graduate student). TARGET AUDIENCES: Target audiences include undergraduates and graduates, where my lab provides practical training and my courses involve instructional training in plant pathology related matters. I also reach highschool students through my participation in the Nebraska EPSCoR summercamp program. Finally, by disseminating my work through publications and presentations, I target other fungal researchers with my work. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Results from this work has allowed us to secure an EPSCoR First Award, formed the basis for one NSF submission, and was included in a DoE grant. Forward genetic analysis of the mutants described in this objective has generated enough novel data that we are currently writing up this work for publication. This will impact our ability to secure Federal funds in the future. Our preliminary data on the regulation of pathogenicity has been presented at several international meetings, has been included in a research paper in PNAS (in press), and thus will impact the wider research community. The significance of the ndo results to the field is such that we are preparing a manuscript of this work for publication. The significance of the NADPH work to the field is such that we are preparing a manuscript for publication.

Publications

• Wilson, R. A. and Talbot, N. J. 2009. Fungal Physiology- A Future Perspective. Microbiology. 155: 3810-3815.