Progress 10/01/07 to 09/30/13

Outputs
Target Audience: -Delivered research presentations to scientists in the Departments of Plant Pathology at the University of Kentucky and the University of Wisconsin -Provided classroom instruction to graduate students in the Department of Plant Pathology at the University of Kentucky -Delivered a Next Generation Sequencing and Bioinformatics workshop to particpants both from within Kentucky and from other states Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Over 30 scientists (undergraduate and graduate students, postdocs and faculty) from Kentucky and other states received training through the Next Generation Sequencing Workshop How have the results been disseminated to communities of interest? Through scientific presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Using live cell imaging, we gained evidence that Magnaporthe oryzae penetrates host cell membranes during host invasion and grows directly in the host cytoplasm. This is counter to all current views of how the fungus grows inside host cells and may establish a new paradigm for the infection strategies of certain biotrophic fungal pathogens. It also throws into question current models of fungal effector protein translocation.

Publications

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: 1. developed a web-based tool for facilitating development of fungal gene knockout/protein fusion constructs; 2. created a suite of Gateway-based, plasmid vectors for high throughput localization of fungal proteins; 3. performed localization studies for over 400 secreted proteins from the fungus Magnaporthe oryzae; 4. performed a gene knockout study for a fungal protein that accumulates in the nuclei of host cells; 5. made protein localization data available in a publicly available online database; 6. gave several poster and oral presentations at various conferences, and invited talks at various universities; 7. provided plasmid vectors to a large number of researchers worldwide. PARTICIPANTS: Eleanor Johnson, undergraduate, characterized a number of Magnaporthe strains expressing secreted protein fusion construct that produced differential expression patterns among individuals supposed to be genetically identical. This provided Eleanor with an independent research opportunity in the summer of 2012. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. demonstrated that protein:autofluorescent protein (AFP) fusions generated using Gateway technology yield localization patterns equivalent to direct protein:AFP fusions; 2) gained insight into the localization patterns of over 200 secreted proteins; 3) identified a comprehensive set of protein fusion constructs that allow marking of various subcellular compartments; 4) discovered novel subcellular structures based on protein localization patterns; 5) found that appressoria continue to express proteins long after penetration of the host has occurred; 6) showed that two nuclei remain in the appressoria even after penetration has occurred; 7) identified a fungal secreted protein that accumulates in the nuclei of host cells; 8) demonstrated that the nucleus-targeted protein is non-essential for pathogenesis; 9) identified a number of secreted proteins that are expressed only in certain infection sites; 10) gained insight into temporal changes in protein localization patterns.

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Activities: We continued studies on the localization of Magnaporthe secreted proteins during host penetration and invasive growth. We expanded the number of secreted proteins studied to more than 400. We developed plasmid constructs which allow for simultaneous labeling of two proteins with different colors. We used confocal microscopy to study the expression/localization of certain proteins in more detail. We generated gene knockouts for a fungal protein that accumulates in the nuclei of the invaded host cells and tested the effect of pathogenicity. Events: All project participants attended the Fungal Genetics Conference at the Asilomar Conference Grounds, Pacific Grove, CA. Services: The Farman lab provided plasmid constructs to numerous researchers from around the globe. A website and database allowing gene knockout/gene fusion primer design was generated for the Colletotrichum graminicola research community. Products. Five plasmid vectors with broad utility for the fungal research community. Dissemination (outreach): Results from the project were presented in poster sessions at the Fungal Genetics Conference. PARTICIPANTS: Individuals: Mark Farman (PI) directed the project, generated the gene knockout (KO) construct, screened transformants for KOs and analyzed the KO strains; Xiaoyan Gong (postdoc) and Baohua Wang (postdoc) worked on the production and analysis of secreted protein fusions. Melanie Heist (research Analyst) generated knockout transformants and performed general technical support. Collaborators/Partner Organizations: Michael Goodin, Chris Schardl and Lisa Vaillancourt, University of Kentucky; Barbara Valent, Kansas State University; Cari Soderlund, University of Arizona. Training: Postdocs 2; graduate student 1. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We discovered that Magnaporthe appressoria retain two nuclei throughout the biotrophic stages of infection. Coupled with our earlier discovery that appressoria accumulate certain proteins long after the fungus has gained entry into the host tissues, this leads us to propose that appressoria continue to serve a function post-penetration. Deletion of a gene for a secreted protein that accumulates in the nuclei of invaded host cells had no effect on pathogenicity. We found that wild-type Magnaporthe produces fluorescent compounds in the appressorial walls; and on the walls of hyphae where they emerge from the spore. We identified a novel appressorial structure that is labeled by a histone 2B:GFP fusion, as well as two different secreted protein:RFP fusions. change in actions: We adopted a simplified Agrobacterium-based method of introducing protein:fusion constructs into Magnaporthe spores.

Publications

• No publications reported this period

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: During the past year we have focused our efforts on studying the role of a G-protein gamma subunit in phytopathogenesis. We previously identified a T-DNA tagged mutant with an insertion in Magnaporthe gene MGG_10193 which is predicted to code for a G-protein gamma subunit. The original mutant was identified as one that did not form appressoria and which germinated to form aerial hyphae. The mutant is also unable to infect rice. Previously, we had confirmed that the T-DNA insertion is responsible for the mutant phenotype, we recreated the insertion in the wild-type strain and performed a clean knock-out of the entire open reading frame. As a final confirmation of the gene's role in pathogenesis, we tried to complement the original T-DNA insertion mutant with an intact copy of the MGG_10193 gene. This had been unsuccessful due to an inability to re-transform the mutant. In the current review period, we sought to circumvent this problem by trying to recreate the original T-DNA insertion in a different (more easily transformed) Magnaporthe strain. To develop a sensitive reporter system to detect delivery of fungal proteins into the cytoplasm of a plant cell. Due to difficulties in transforming rice plants with the reporter construct, we are using an alternative and more direct approach to detect translocation of fungal proteins. We have fused a large number of fungal secreted proteins to a red fluorescent protein tag. These constructs were introduced into Magnaporthe via Agrobacterium-mediated transformation. The transformants were inoculated onto rice plants and the fusion proteins were then visualized using epifluorescence microscopy of infection sites. Due to problems with the generation of transgenic rice plants, we have abandoned Objective 3. Events and Dissemination: Farman and other lab members presented data from this project at several conferences/events: Plant and Animal Genome Conference, San Diego, CA; KY Innovation and Entrepreneurship conference, Lexington, KY; Mycological Society of America-International Symposium on Fungal Endophytes of Grasses Conference, Lexington, KY; International Rice Blast Meeting, Little Rock, AR; USDA Functional Genomic of Microbes awardees' workshop. Plant Pathology seminar series, Ohio State University; Plant Pathology seminar series, Kansas State University. Services: Farman taught a graduate level class on Critical Methods in Plant Microbe Interactions and mentored 4 undergraduate students who were conducting independent research projects in the lab. PARTICIPANTS: Individuals: Jihan Ahmed (postbaccalaureate intern) performed the experiments on the G-protein gamma subunit (Objective 1); Xiaoyan Gong (postdoc), Junhuan Xu (postdoc), Angela Tronzo (undergraduate)and Baohua Wang (visiting scientist) worked on the production and analysis of secreted protein fusions (Objective 2); David Thornbury (Scientist II) and Melanie Heist (research Analyst) performed technical support roles. Collaborators/Partner Organizations: Michael Goodin, Chris Schardl and Lisa Vaillancourt, University of Kentucky; Tom Mitchell and Guo-Liang Wang, Ohio State University; Yinong Yang, Penn State University; Barbara Valent, Kansas State University; Cari Soderlund, University of Arizona. Training: Postdocs 2; visiting scientist 1; undergraduates 1; graduate student 1. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Approximately 20 candidate transformants were generated. Unfortunately, however, none of these transformants had integrated the disruption cassette in the MGG_10193 locus. Over three hundred secreted proteins were analyzed. Approximately fifty percent of the proteins were expressed at one or more points during pathogenic development. More than twenty distinct localization patterns were identified. These included the following: Spore patterns: cytoplasm, cell wall and septa, tip cell, tip and middle cell, middle cell, spore tip mucilage, endoplasmic reticulum, vesicles and vacuoles, vacuolar membrane. Appressorium patterns: cytoplasm, cell wall, vesicles and vacuole, appressorial "glue," a novel appressorial structure Biotrophic hyphae: bitrophic interfacial complex, primary invasive hypha and biotrophic interfacial complex, cytoplasm, endoplasmic reticulum, plasma membrane, trapped in the space between the fungal cell wall and the extra invasive hyphal membrane, host cell cytoplasm, host cell nucleus, entire lumen of invaded cell. Two of the proteins studies were translocated into the host cytoplasm. One of these translocated proteins was found to accumulate in the nucleus of the host cell. In addition to identifying the expression and localization patterns for over 150 secreted proteins, we made a number of new and important findings: i) fungal spores contain large quantities of secreted proteins; ii) several secreted proteins require plant-derived signal for their expression; iii) a novel appressorial structure may be required for penetration of the host leaf epidermis; iv) cell wall degrading enzymes are down-regulated during biotrophic growth but massively expressed during lesion development; v) we identified the first fungal protein that is translocated into the host cell nucleus. Further studies of these proteins will help us understand how fungal pathogens grow inside plant cells without triggering plant defenses. Such knowledge will help us develop new strategies for controlling plant disease that specifically target the mechanisms that fungi use to subvert host resistance.

Publications

• No publications reported this period

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Activities: Objective 1. To characterize a collection of Magnaporthe oryzae non-pathogenic mutants to identify strains deficient in biotrophic growth. a. During the past year we have focused our efforts on studying the role of a G-protein gamma subunit in phytopathogenesis. We previously identified a T-DNA tagged mutant with an insertion in Magnaporthe gene MGG_10193 which is predicted to code for a G-protein gamma subunit. The original mutant was identified as one that did not form appressoria and which germinated to form aerial hyphae. The mutant is also unable to infect rice. To confirm that the T-DNA insertion is responsible for the mutant phenotype, we recreated the insertion in the wild-type strain and also performed a clean knock-out of the entire open reading frame. This year we have performed a quantitative phenotypic analysis as well as molecular characterization of the knock-out strains, as well as a number of control transformants with ectopic insertions (non knock-outs). This has identified a potential new phenotype associated with the MGG_10193 null phenotype, that being a dramatic reduction in sporulation. b. As a final confirmation of the gene's role in pathogenesis, we attempted to complement the original T-DNA insertion mutant with an intact copy of the MGG_10193 gene. This was unsuccessful due to an inability to re-transform the mutant. Therefore, we are currently attempting to re-create the T-DNA insertion in a new strain that is highly transformable. Large numbers of transformants have been generated and we are currently characterizing the transformants to identify ones that carry the desired mutation. Objective 2. To develop a sensitive reporter system to detect delivery of fungal proteins into the cytoplasm of a plant cell. Initial attempts to express a translocation reporter protein in rice plants were unsuccessful due to an inability to obtain stable transformants. Objective 3. To develop rice plants with fluorescently tagged cell compartments for cytological studies of fungus:plant interactions. a. Several vectors for that have been used for protein localization in maize were obtained from the Jackson laboratory (Cold Spring Harbor). Plasmid DNAs were prepared form these constructs and sequenced to confirm their veracity. Preliminary attempts to express the constructs in rice leaves following particle bombardment were unsuccessful. Services: Farman taught a graduate level class on Critical Methods in Plant Microbe Interactions and gave a total of four guest lectures in other classes. Thornbury had a leading role in a Building Emergency Action Plan within the College of Agriculture. Dissemination: Farman presented work on fungal protein localization during plant infection at the USDA Functional Genomic of Microbes awardees' workshop. PARTICIPANTS: Individuals: Mark Farman (PI): directed the research of a part-time employee and a graduate student who performed all of the lab work. David Thornbury (lab manager): was responsible for the day-to-day running of the lab (maintenance, ordering supplies, training of the part-time employee). Training: The project provided training to two individuals: Jihan Ahmed who is learning molecular biology techniques in preparation for applying to graduate school; and Melanie Heist, a PhD student. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: We are currently finding ourselves spread too thin over the three objectives outlined in the original proposal. Therefore, we will likely abandon our attempts to develop a reporter for fungal protein translocation into plant cells (Objective 2) in order to improve our chances of success with Objectives 1 and 3.

Impacts
G-protein signaling is known to be involved in developmental processes required for plant pathogenesis - in particular it is important for appressorium formation and function. Genes for G-protein alpha and beta subunits have been extensively characterized in a number of plant pathogens. However, there has been no research on the function(s) of the gamma subunit, at least in pathogenic filamentous fungi. Research on this particular gene promises to provide new insights into the G-protein signaling cascade and, in particular, how the gamma subunit modulates the inputs and outputs of the cascade to affect different processes required for pathogenic success (i.e. surface sensing, appressorium formation/function, invasive growth and sporulation). This new information on the cellular mechanisms that allow fungi to infect plants ultimately will facilitate the development of rational, knowledge-based approaches to controlling disease.

Publications

• No publications reported this period

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The overall goals of these experiments are to examine the functions of genes related to pathogenesis (with a specific focus on secretion), and to investigate the regulation of genes and transposons found in telomere regions. Specific activities were as follows: 1) We used gene replacement to generate a number of Magnaporthe oryzae mutants. The genes that were targeted for mutagenesis were: i) MgAPT2 - codes for an aminophosphlipid translocase that is predicetd to be responsible for secretion of avirulence proteins; ii) MGG00786 - codes for a protein that is predicted involved in the secretion of proteins lacking a canonical secretion signal; iii) MGG02848 - codes for a secreted protein; iv) MGG06515 codes for a putative chromodomain protein; v) MgSir2 - codes for a histone deacetylase that is involved in telomeric silencing; vi) MgHP1 - codes for heterochromatin protein 1, a factor that is required for DNA methylation and, hence, gene repression in the related fungus Neurospora crassa; and vii) MgTERT - codes for telomerase. Gene disruption constructs were generated for each gene and a collection of transformants was generated for each target gene. To date, we have confirmed the deletion of four genes: MgAPT2, MgHP1, MgSir2 and MgTERT. 2) We developed web-based programs to facilitate the design of oligonucleotide primers for gene knockout and protein fusion constructs. The programs are available to the broader research community through the Farman lab website. 3) The PI attended the USDA Functional Genomics of Microbes workshop in Washington DC. PARTICIPANTS: 1) Mark Farman, PI: Developed all of the gene disruption constructs and performed the fungal transformation experiments. Analyzed candidate gene knockout strains. Wrote the computer programs and developed the CGI interfaces for the primer design web applications. 2) David Thornbury, staff scientist: Assisted in the preparation of fungal DNA, and in other general laboratory procedures. TARGET AUDIENCES: Target audiences: Basic science researchers; The "Magnaporthe" research community. Efforts: Provided internships to four undergraduate students with the goal of providing research experience. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Most of the work that was performed in 2008 entailed development of resources for future studies. The strains that are deleted for the MgAPT2 and MGG00786 genes will be used in future experiments to study the effect of these deletions on the secretion of fungal proteins required for establishing infections inside plant cells. The MgHP1, MgSir2 and MgTERT deletion strains will be used to study the effects of these genetic modifications on the expression of genes and transposons near telomeres. This studies are important because we predict that some of the telomere-proximal genes have roles in pathogenesis. Ultimately, these studies will provide inportant insights into mechanisms that fungi use to colonize plants. This in turn, will allow for rationale development of strategies and/or plant germplasm to minimize the diseases caused by fungi.

Publications

• Wu C, Kim, Y-S, Smith K, Li W, Hood H, Staben C, Selker E, Sachs M and Farman M (2008) Characterization of chromosome ends in the filamentous fungus Neurospora crassa. Genetics [Epub ahead of print].
• Vincelli P, Dixon E, and Farman M (2008) Gray leaf spot of annual ryegrass: susceptibility of selected cultivars of forage grasses and relatedness of the pathogen to strains from other grasses. Forage and Grazinglands, doi:10.1094/FG-2008-0226-01-RS.