Progress 09/15/05 to 09/14/09
Outputs
OUTPUTS: The goal of the project is to study mechanisms involved in the development and germination of asexual spores in P. INFESTANS. Experiments were used to help identify genes expressed at the earliest stages of sporulation. This included performing detailed timecourses and analyzing expression in a mutant strain that is blocked at an early stage of sporulation. Initial data were obtained using an Affymetrix GeneChip representing about 15,000 genes from P. INFESTANS. During sporulation, about 12% of genes were found to be up-regulated compared to vegetative hyphae and 5% were down-regulated. The most prevalent induced genes had functions in signal transduction, flagella assembly, cellular organization, metabolism, and molecular or vesicular transport. Distinct patterns of expression were discerned based on the kinetics of mRNA induction and their persistence in sporangia. For example, most flagella-associated transcripts were induced very early in sporulation and maintained in sporangia, while many participants in metabolism or small molecule transport were also induced early but had low levels in sporangia. Several genes identified by this study were used as sources of promoters for detailed analysis, with a longer-term goal of identifying the transcription factors that activate gene expression during development. Promoters from several were fused to GUS and transformed into P. INFESTANS to identify good candidates for detailed analysis. For the CDC14 gene, analyses of promoters containing 5' and 3' deletions, individual base mutations, and chimeras between different promoter fragments showed that at least two independent mechanisms are responsible for stimulating transcription. While both pathways are activated early in spore development, the activation pathways appear to involve distinct transcription factors that have somewhat different spatial and temporal patterns of expression. Biochemical chromatography techniques were used to enrich nuclear protein extracts for the relevant DNA-binding proteins, which appear to be present in both hyphae and sporulating cultures. Progress was also made in characterizing transcription factor binding sites in other spore-specific promoters, and a zoosporogenesis-inducible promoter, by combining mutagenesis experiments, bioinformatics approaches, and gel-shift assays. In addition, studies targeted at specific transcription factor families were initiated, such as the bZIP and Myb families. Findings from the research were disseminated through talks presented at the annual Oomycete Molecular Genetics Network meeting, the American Phytopathology Society annual meeting, and the Fungal Genetics Conference. Another output from the research involved hosting summer interns in the laboratory chosen from local community colleges that have an interest in teaching. PARTICIPANTS: PI H. Judelson participated in management and conducted experiments related to gene silencing and expression systems in PHYTOPHTHORA. Other participants were A. Ah Fong, X. Niu, S. Roy, and Q. Xiang who studied promoter structures; and K. Madishetty who was involved in gene silencing studies. Undergraduates included J. Manson, C. Thurber, and Q. Tea. Professional development: All but the PI were trainees, either graduate students and postdoctorals, or undergraduates. Collaborators: these included the Broad Institute of MIT and Harvard, Ohio State University, University of Aberdeen, Scottish Crop Research Institute, and Wageningen University. TARGET AUDIENCES: Internships were provided to undergraduates, usually in summer research projects. Workshops were run for community college students interested in careers in teaching. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. The expression pattern of the majority of the P. INFESTANS transcriptome during asexual development was characterized. This yielded information about when proteins needed for certain life-stages are made, and identified candidate targets for future fungicides, which should lead to a safer and more economical food supply for U.S. citizens. 2. Transcription factor binding sites in stage-specific genes were identified, which in the future will enable the factors regulating the steps in the spore cycle to be revealed. This will identify additional targets for fungicide design, and may reveal cultural methods that could be used to reduce pathogen disseminiation. 3. To develop alternative methods for testing the function of genes, we adapted the Tet-on system to P. INFESTANS, which should also accelerate studies of gene function in oomycetes.
Publications
- Judelson, H. S., Narayan, R., Fong, A. M., Tani, S. & Kim, K. S. (2007). Performance of a tetracycline-responsive transactivator system for regulating transgenes in the oomycete Phytophthora infestans. Curr Genet 51, 297-307.
- Judelson, H. S. & Tani, S. (2007). Transgene-induced silencing of the zoosporogenesis-specific PiNIFC gene cluster of Phytophthora infestans involves chromatin alterations. Eukaryot Cell 6, 1200-1209.
- Judelson, H. S., Narayan, R. D., Ah-Fong, A. M. & Kim, K. S. (2008b). Gene expression changes during asexual sporulation by the late blight agent Phytophthora infestans occur in discrete temporal stages. Mol Genet Genomics 281, 193-206.
- Prakob, W. & Judelson, H. S. (2007). Gene expression during oosporogenesis in heterothallic and homothallic Phytophthora. Fungal Genet Biol 44, 726-739.
- Ah-Fong, A. M., Bormann-Chung, C. A. & Judelson, H. S. (2008). Optimization of transgene-mediated silencing in Phytophthora infestans and its association with small-interfering RNAs. Fungal Genet Biol 45, 1197-1205.
- Judelson, H. S. (2007). Genomics of the plant pathogenic oomycete Phytophthora: insights into biology and evolution. Adv Genet 57, 97-141.
- Judelson, H. S., Ah-Fong, A. M., Aux, G., Avrova, A. O., Bruce, C., Cakir, C., da Cunha, L., Grenville-Briggs, L., Latijnhouwers, M. & other authors (2008a). Gene expression profiling during asexual development of the late blight pathogen Phytophthora infestans reveals a highly dynamic transcriptome. Mol Plant Microbe Interact 21, 433-447.
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Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: The goal of the project is to study mechanisms involved in the development and germination of asexual spores in P. INFESTANS. To add to our earlier data comparing mRNA levels in hyphae with sporangia, a series of experiments were used to help identify genes expressed at the earliest stages of sporulation. This included performing detailed timecourses and analyzing expression in a mutant strain that is blocked at an early stage of sporulation. Initial data was obtained using an Affymetrix GeneChip representing about 15,000 genes from P. INFESTANS. Results were confirmed by reverse transcription-polymerase chain reaction analyses of sporulation on artificial media and infected tomato. During sporulation, about 12% of genes were up-regulated compared to vegetative hyphae and 5% were down-regulated. The most prevalent induced genes had functions in signal transduction, flagella assembly, cellular organization, metabolism, and molecular or vesicular transport. Distinct patterns of expression were discerned based on the kinetics of mRNA induction and their persistence in sporangia. For example, most flagella-associated transcripts were induced very early in sporulation and maintained in sporangia, while many participants in metabolism or small molecule transport were also induced early but had low levels in sporangia. Several genes identified by this study were used as sources of promoters for detailed analysis, with a longer-term goal of identifying the transcription factors that activate gene expression during development. Promoters from several were fused to GUS and transformed into P. INFESTANS to identify good candidates for detailed analysis. For the CDC14 gene, analyses of promoters containing 5' and 3' deletions, individual base mutations, and chimeras between different promoter fragments showed that at least two independent mechanisms are responsible for stimulating transcription. While both pathways are activated early in spore development, the activation pathways appear to involve distinct transcription factors that have somewhat different spatial and temporal patterns of expression. Biochemical chromatography techniques were used to enrich nuclear protein extracts for the relevant DNA-binding proteins, which appear to be present in both hyphae and sporulating cultures. Progress was also made in characterizing transcription factor binding sites in other spore-specific promoters, and a zoosporogenesis-inducible promoter, by combining mutagenesis experiments, bioinformatics approaches, and gel-shift assays. Findings from the research were disseminated through talks presented at the annual Oomycete Molecular Genetics Network meeting and the American Phytopathology Society annual meeting. Another output from the research involved hosting summer interns in the laboratory chosen from local community colleges that have an interest in teaching. PARTICIPANTS: PI H. Judelson participated in managment and conducted experiments related to gene silencing and expression systems in PHYTOPHTHORA. Other participants were A. Ah Fong. S. Roy, and Q. Xiang who studied promoter structures, and K. Madishetty who was involved in gene discovery and functional testing studies. Professional development: All but the PI were trainees, either graduate students and postdoctorals. In addition, 7 interns participated in laboratory activities during the summer. These were community college students interested in pursuing careers in science education. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Data from this study are a resource for understanding sporogenesis, which is critical to the pathogenic success of P. INFESTANS and other oomycetes. By characterizing the expression patterns of genes expressed during spore formation and germination, candidates for potential targets for fungicide development were identified. This will lead to a safer and more economical food supply for U.S. citizens. The project also contributed to the training of graduate students, postdoctorals, and future high school and elementary school science teachers. In addition, PHYTOPHTHORA cloning vectors were developed and disseminated to researchers in other laboratories, that will accelerate research progress by others.
Publications
- Howard S. Judelson and Audrey Ah-Fong. 2008. Molecular Strategies for Identifying Determinants of Oomycete Pathogenicity. C.S. Nautiyal, P. Dion, (eds.) Molecular Mechanisms of Plant and Microbe Coexistence. Soil Biology 15: 383-410.
- Howard S. Judelson, Audrey M. V. Ah-Fong, George Aux, Anna O. Avrova, Catherine Bruce, Cahid Cakir, Luis da Cunha, Laura Grenville-Briggs, Maita Latijnhouwers, Wilco Ligterink, Harold J. G. Meijer, Samuel Roberts, Carrie S. Thurber, Stephen C. Whisson, Paul R. J. Birch, Francine Govers, Sophien Kamoun, Pieter van West, and John Windass. 2008. Gene Expression Profiling During Asexual Development of the Late Blight Pathogen PHYTOPHTHORA INFESTANS Reveals a Highly Dynamic Transcriptome. Molecular Plant Microbe Interactions 21: 433-447
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Progress 09/15/06 to 09/14/07
Outputs
OUTPUTS: The goal of the project is to study mechanisms involved in the development and germination of asexual spores in P. INFESTANS. To identify genes induced during these stages, Affymetrix GeneChip studies characterized mRNA levels of 18K genes in hyphae, sporangia, sporangia undergoing zoosporogenesis, motile zoospores, and germinated cysts forming appressoria. Many genes were regulated during the spore cycle, having roles in metabolism, structure, regulation, colonization of plants, etc. The data were validated by qRT-PCR using RNA from tissue grown on lab media and in detached tomato leaflets. Several experiments, such as detailed timecourses, helped to identify genes induced at the earliest stage of sporulation; such results were confirmed by pRT-PCR using tissue from lab media and infected plants, which helped distinguish authentic "early genes" from laboratory artifacts. The expression-profiling of strains silenced for the CDC14 gene, which are blocked in sporulation, also
helped distinguish genes activated at early and late stages. Interestingly, genes involved in zoosporogenesis were found to be induced very early during sporulation. Such stage-specific genes were used as sources of promoters for detailed analysis, with a longer-term goal of identifying the transcription factors that activate gene expression during development. Promoters from several genes were fused to GUS and transformed into P. INFESTANS to identify good candidates for such studies. CDC14 transcription was expressed at a very early stage, so deleted and mutagenized promoters from its gene were tested in transformants to define the site binding its cognate transcription factors. This was proved to be a repeated CTYAAC motif based on the functional tests and gel-shift assays. A second and potentially functionally redundant site inducing CDC14 during sporulation was also identified, as were motifs required for the accurate initiation of transcription. Biochemical chromatography
techniques were used to enrich nuclear protein extracts for the CTYAAC-binding proteins, and candidate proteins were sequenced by tandem mass spectrometry. Progress was also made in characterizing transcription factor binding sites in other spore-specific promoters, by combining mutagenesis experiments, bioinformatics approaches, and gel-shift assays. Several motifs were identified, and proteins binding the motifs partially purified. Other work relevant to spore biology included the silencing of various genes to test their function, and the testing of different methods for silencing genes (or forcing their expression in transformants). Several genes induced during spore stages were silenced (mostly genes induced during zoospore formation and appressoria formation), however the resulting phenotypes were unexciting or intermediate. Findings were disseminated through publications and talks presented at national and international meetings in plant pathology and in fungal or oomycete
biology.
PARTICIPANTS: PI H. Judelson participated in managment and conducted experiments related to gene silencing and expression systems in PHYTOPHTHORA. Other participants were A. Ah Fong. S. Roy, and Q. Xang who studied promoter structures; R. Narayan who characterized gene expression patterns; and K. Madishetty who was involved in gene silencing studies. Professional development: All but the PI were trainees, either graduate students and postdoctorals. Collaborators included Ohio State University, University of Aberdeen, Scottish Crop Research Institute, Syngenta Corporation, and Wageningen University.
Impacts
1. The expression pattern of the majority of the P. INFESTANS transcriptome was characterized. This yielded information about when proteins needed for certain life-stages are made; for example proteins required for zoospore function are made early during sporulation. Also, such findings identified candidates for future studies and potential targets for fungicide development, leading to a safer and more economical food supply for U.S. citizens. 2. Transcription factor binding sites in stage-specific genes were identified, which in the future will enable the factors regulating the steps in the spore cycle to be revealed. 3. The P. INFESTANS gene silencing method was optimized (hairpin constructs gave the best results), which will accelerate studies of genes in this species and other important oomycete pathogens. 4. To develop alternative methods for testing the function of genes, we adapted the Tet-on system to P. INFESTANS, which should also accelerate studies of gene
function in oomycetes.
Publications
- Ah Fong, A., Q. XIang, and H. S. Judelson. 2007. Architecture of the sporulation-specific Cdc14 promoter from the oomycete PHYTOPHTHORA INFESTANS. Eukaryotic Cell 6:2222-2230.
- Judelson, H. S., R. Narayan, A. M. Fong, S. Tani, and K. S. Kim. 2007. Performance of a tetracycline-responsive transactivator system for regulating transgenes in the oomycete PHYTOPHTHORA INFESTANS. Current Genetics 51:297-307.
- Judelson, H. S., and S. Tani. 2007. Transgene-induced silencing of the zoosporogenesis-specific PiNIFC gene cluster of PHYTOPHTHORA INFESTANS involves chromatin alterations. Eukaryotic Cell 6:1200-1209.
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Progress 09/15/05 to 09/15/06
Outputs
Genes relevant to the biology of asexual spores are being studied in PHYTOPHTHORA INFESTANS, the fungus-like oomycete that causes the economically important late blight diseases of potato and tomato. Using cDNA macroarray and oligonucleotide microarray techniques, genes activated during the formation of sporangia and their germination (via zoospores) have been identified. Since such data came from tissues grown in artificial media (rye-sucrose agar), experiments were performed in planta to validate the relevance of the described expression patterns. Based on quantitative reverse transcription-polymerase chain reaction (PCR) assays, about 98% of the genes were confirmed to be spore-induced. The rest were recalcitrant to amplification by PCR. Moreover, the timing of sporulation in planta was measured and found to be stimulated in the middle of the darkness period. Temporal differences in gene induction during the dark were demonstrated, possibly pointing towards the
earliest gene expression changes in the sporulation process. In related work, promoter analyses have been performed of sporulation and zoosporogenesis-induced genes, in particular of genes PiCDC14 and PiPKS1. This has involved increasingly detailed directed mutagenesis experiments (involving tests of promoter activity in transformants using the GUS reporter gene) and electrophoretic shift mobility assays (EMSA) using wild-type and mutant competitor and probes. For example, with PiCdc14 a triplet-repeat motif was identified as the likely binding site for a major inducer of sporulation. However, there appears to be functional redundancy within the promoter as an independent site driving sporulation-specific expression also exists. Experiments to fractionate nuclear proteins to aid purification studies of the cognate transcription factors are underway. EMSA studies of the zoosporogenesis-induced gene PiNIFC1 were also pursued with a goal of purifying the relevant transcription factors.
However, this area is proceeding slowly due to apparent instability of the binding activity in protein extracts. Studies of other zoosporogenesis-induced genes (regulated by factors different from PiNIFC1) have therefore been initiated. Nevertheless, studies of the PiNIFC genes themselves have been completed. This indicated that the genes play a role in cyst germination, however gene silencing studies indicate only a partial phenotype results from knock-downs. Interesting results were obtained from characterization of the silenced strains, in which an entire cluster of PiNIFC genes was simultaneously silenced. The basis of silencing was shown to be transcriptional based on run-on assays, and involves a shift towards tighter chromatin packing based on nuclease accessibility studies. Moreover, in some cases these changes spread along chromosomes, moving several hundred bases beyond the limits of the transcription unit.
Impacts
Spores are central to the life cycle of plant pathogens such as PHYTOPHTHORA, as they are the agents of dissemination and infection. By understanding the processes used by PHYTOPHTHORA to produce the spores, it should be possible to identify genes and their corresponding proteins that are essential for spore development. Such proteins are potential targets for environmentally benign and effective crop protection chemicals, leading to a safer and more economical food supply for citizens.
Publications
- Tani, S., and Judelson, H. S. 2006. Activation of zoosporogenesis-specific genes in PHYTOPHTHORA INFESTANS involves a seven-nucleotide promoter motif and cold-induced membrane rigidity. Eukaryotic Cell 42:745-752.
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