Source: UNIVERSITY OF GEORGIA submitted to
GENETIC DETERMINANTS OF DIMORPHIC AND PATHOGENICITY IN USTILAGO MAYDIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0187510
Grant No.
(N/A)
Project No.
GEO00917
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2001
Project End Date
Jun 30, 2007
Grant Year
(N/A)
Project Director
Gold, S. E.
Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
PLANT PATHOLOGY
Non Technical Summary
Corn smut is a plant disease of cultivated maize. The fungus and its relatives grow as yeast or filaments. The yeast form cannot attack the plant but the filaments can. We have discovered genes that control the ability of the fungus to grow in these two forms that are critical for pathogenicity. This information will have wide significance for understanding fungal growth and disease in general. It may provide new insights into novel targets for plant disease control.
Animal Health Component
(N/A)
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121510104050%
2121510110250%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1510 - Corn;

Field Of Science
1102 - Mycology; 1040 - Molecular biology;
Goals / Objectives
Objective 1. Utilize differential screening to identify genes involved in dimorphism. 1A) Identify clones for transcripts specific to filamentous or budding growth, and characterize the temporal expression of these genes. 1B) Disrupt genes expressed early and specifically in the bi-directional transitions between budding and filamentous growth. Objective 2. Determine the roles of genes specifically upregulated during gall formation. 2A) Isolate genes from either the plant or the fungus that are specifically upregulated upon gall induction. 2B) Determine the sequence and source organism of the differential clones. 2C) Obtain and characterize specific mutants in either organism. Objective 3. Determine the roles in dimorphism of genes encoding catalytic subunits of serine/threonine protein phosphatase. 3A) Initially we will focus our attention for characterization, disruption and mutant phenotype characterization on the calcineurin (PP2B) gene to determine its importance in dimorphism and/or role in cAMP signaling. Objective 4: Ubc2 Protein-Protein Interactions. 4A: Targeted two-hybrid assays. 4B: Targeted in vitro protein-protein interaction assays. 4C: Analysis of important functional domains of Ubc2 by site directed mutagenesis. Objective 5: Expression Analysis of Filament Specific Genes. 5A: In vitro and in planta assays for filament specific gene expression. 5B: Promoter analysis of two filament specific genes. Objective 6: Continuation of Mutant Analysis. 6A: Complementation of additional ubc mutants.
Project Methods
Corn smut is a plant disease of cultivated maize. The fungus that causes this disease is a close relative of other pathogens of many grain crops. The corn smut fungus and its relatives can grow in two forms. The first form is a yeast with small dividing cells. The second form is typical of most fungi and is made up of long tubular cells or filaments. In corn smut the yeast form cannot attack the plant but the filaments can. These two forms are also common in animal and human pathogens and their form is often correlated with the ability to cause disease. We have discovered genes that control the ability of the fungus to grow in these two forms that are critical for pathogenicity. In this grant we will characterize the interactions of some of these genes with particular focus on a gene (ubc2) that encodes a novel adaptor protein. Protein phosphorylation plays a critical role in regulation of morphogenesis and pathogenicity. Consequently we are identifying and mutating genes encoding several protein phosphatases. We will identify transcripts that are upregulated in host-pathogen interactions and in morphogenesis. We will disrupt several of these genes and possibly identify mutant maize plants defective in identified genes. We also plan to analyze the timing of expression patterns of particular filament specific genes during the infection process to better understand the order of events. Additionally, we will attempt to identify other genes required for the maintenance of the budding growth form of the fungus. This information will have wide significance for understanding fungal growth and disease in general. It may provide new insights into novel targets for plant disease control. The results will likely have important implications for animal and human fungal diseases as well.

Progress 07/01/01 to 06/30/07

Outputs
OUTPUTS: The objectives of this project are shown below. Objective 1. Utilize differential screening to identify genes involved in dimorphism. Objective 2. Determine the roles of genes specifically upregulated during gall formation. Objective 3. Determine the roles in dimorphism of genes encoding catalytic subunits of serine/threonine protein phosphatase. Objective 4: Ubc2 Protein-Protein Interactions. Objective 5: Expression Analysis of Filament Specific Genes. Objective 6: Continuation of Mutant Analysis. Progress on objective 1: We have analyzed 60 genes by northern analysis for their level of differential expression. We have disrupted 6 filament specific genes to date. We have deleted 7 genes from the budding form thus far including a putative xylanase and ammonium transporter gene. We have published one paper on the ammonium transporter and have two submitted manuscripts in review on this objective. Progress on objective 2: We have constructed two libraries one from infected galled tissue and one from infected non-galled tissue which we have been confirmed to contain mostly differentially expressed genes. We screened for differential expression by northern blots and determined species of origin by genome searches. One interesting gene is highly upregulated in gall tissue and absent in non-galled tissue. A new graduate student is pursuing full analysis of this gene that appears to be involved in regulating cell cycle. Most genes identified are from maize. Progress on objective 3: We have isolated small fragments of several protein phosphatase genes. We have cloned, sequenced and disrupted the U. maydis calcineurin gene and find it important in morphogenesis, mating and pathogenicity. We have also cloned a PP2A homolog and showed that it is likely essential. A manuscript on this work was submitted and is in revision for resubmission. With its publication this objective will have been accomplished. Progress on objective 4: Progress to date: We have made constructs for the analysis of these interactions. Site directed mutants have been characterized. They identify important function for the SAM and RA domains but none detected for the SH3 or P-loop domains. Two hybrid assays continue. We have clear evidence for interaction between the SAM domain of Ubc2 and the Ubc4 protein. A manuscript has been submitted to MPMI. Progress on objective 5: We have observed differential expression by northern blot of a large number of these genes. We are still in early stages in the other approaches. We have identified the genes that we plan to manipulate. We identified a major regulator (ust1) of filament down regulated gene transcription. When deleted the ust1 mutant appears to produce spore-like structures in vitro, a phenomenon normally requiring plant infection. A manuscript is under revision regarding the ust1 gene. Progress on objective 6: We have thoroughly analyzed the ubc mutant collection and have found that we have identified most genes. Some strains are difficult to manipulate, but it appears that we have more or less exhausted this screen. Thus this objective is completed. PARTICIPANTS: Dr. Maria Garcia-Pedrajas PDF Dr. Steven Klosterman PDF Dr. David Andrews, Research Coordinator Ms Marina Nadal PhD student Ms Johanna Takach PhD student Ms Nadia Chacko PhD student Ms. Laura Kappa (undergraduate researcher) Mr. David Smith PhD student (U. Louisville) Dr. Michael Perlin collaborator (U. Louisville) TARGET AUDIENCES: Fungal biology researchers Plant Pathologists PROJECT MODIFICATIONS: None

Impacts
Impact will be accomplished in the long-term through an increased understanding of the genetic basis of fungal plant diseases. This should eventually lead to novel disease control methods. The DelsGate procedure is universal for fungi and will be widely disseminated and we plan to develop high throughput procedures.

Publications

  • Garcia-Pedrajas, M.D., Nadal, M., Kapa, L.B., Perlin, M.H., Gold, S.E. (2008) DelsGate, a robust and rapid gene deletion construction method. Fungal Genetics and Biology. (2008 in press).
  • Klosterman, S., Martinez-Espinoza, A., Andrews, D., Seay, J. and Gold, S. (2008) Ubc2, an Ortholog of the Yeast Ste50p Adaptor, Possesses a Basidiomycete-Specific Carboxy Terminal Extension Essential for Pathogenicity Independent of Pheromone Response. Molecular Plant-Microbe Interactions 21:110-121


Progress 01/01/06 to 12/31/06

Outputs
The objectives of this project are shown below. Objective 1. Utilize differential screening to identify genes involved in dimorphism. Objective 2. Determine the roles of genes specifically upregulated during gall formation. Objective 3. Determine the roles in dimorphism of genes encoding catalytic subunits of serine/threonine protein phosphatase. Objective 4: Ubc2 Protein-Protein Interactions. Objective 5: Expression Analysis of Filament Specific Genes. Objective 6: Continuation of Mutant Analysis. Progress on objective 1: We have analyzed 60 genes by northern analysis for their level of differential expression. We have disrupted 6 filament specific genes to date. We have deleted 7 genes from the budding form thus far including a putative xylanase and ammonium transporter gene. We have published one paper on the ammonium transporter and have two submitted manuscripts in review on this objective. Progress on objective 2: We have constructed two libraries one from infected galled tissue and one from infected non-galled tissue which we have been confirmed to contain mostly differentially expressed genes. We screened for differential expression by northern blots and determined species of origin by genome searches. One interesting gene is highly upregulated in gall tissue and absent in non-galled tissue. A new graduate student is pursuing full analysis of this gene that appears to be involved in regulating cell cycle. Most genes identified are from maize. Progress on objective 3: We have isolated small fragments of several protein phosphatase genes. We have cloned, sequenced and disrupted the U. maydis calcineurin gene and find it important in morphogenesis, mating and pathogenicity. We have also cloned a PP2A homolog and showed that it is likely essential. A manuscript on this work was submitted and is in revision for resubmission. With its publication this objective will have been accomplished. Progress on objective 4: Progress to date: We have made constructs for the analysis of these interactions. Site directed mutants have been characterized. They identify important function for the SAM and RA domains but none detected for the SH3 or P-loop domains. Two hybrid assays continue. We have clear evidence for interaction between the SAM domain of Ubc2 and the Ubc4 protein. A manuscript has been submitted to MPMI. Progress on objective 5: We have observed differential expression by northern blot of a large number of these genes. We are still in early stages in the other approaches. We have identified the genes that we plan to manipulate. We identified a major regulator (ust1) of filament down regulated gene transcription. When deleted the ust1 mutant appears to produce spore-like structures in vitro, a phenomenon normally requiring plant infection. A manuscript is under revision regarding the ust1 gene. Progress on objective 6: We have thoroughly analyzed the ubc mutant collection and have found that we have identified most genes. Some strains are difficult to manipulate, but it appears that we have more or less exhausted this screen. Thus this objective is completed.

Impacts
Impact will be accomplished in the long-term through an increased understanding of the genetic basis of fungal plant diseases. This may eventually lead to novel disease control methods.

Publications

  • Kamper, J., Kahmann, R., Bolker, M., Ma, L.J., Brefort, T., Saville, B.J., Banuett, F., Kronstad, J.W., Gold, S.E., Muller, O. et al. (2006) Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature, 444, 97-101.
  • Fletcher, J., Bender, C., Budowle, B., Cobb, W. T., Gold, S. E., Ishimaru, C. A., Luster, D., Melcher, U., Murch, R. Scherm, H., Seem, R. C., Sherwood, J. L., Sobral, B. W. and Tolin, S.A. 2006. Plant Pathogen Forensics: Capabilities, Needs and Recommendations. Microbiol Mol Biol Rev. 70: 450-471


Progress 01/01/05 to 12/31/05

Outputs
The objectives of this project are shown below. Objective 1. Utilize differential screening to identify genes involved in dimorphism. Objective 2. Determine the roles of genes specifically upregulated during gall formation. Objective 3. Determine the roles in dimorphism of genes encoding catalytic subunits of serine/threonine protein phosphatase. Objective 4: Ubc2 Protein-Protein Interactions. Objective 5: Expression Analysis of Filament Specific Genes. Objective 6: Continuation of Mutant Analysis. Progress on objective 1: We have analyzed 60 genes by northern analysis for their level of differential expression. We have disrupted 5 filament specific genes to date. We have deleted 4 genes from the budding form thus far including a putative xylanase and ammonium transporter gene. We have published one paper on the ammonium transporter and have two submitted manuscripts in review on this objective. Progress on objective 2: We have constructed two libraries one from infected galled tissue and one from infected non-galled tissue which we have been confirmed to contain mostly differentially expressed genes. We have begun screening for differential expression by northern blots and determining species of origin by genome searches. We will continue to sequencing a large number of clones to get a global sense of these libraries. One interesting gene is highly upregulated in gall tissue and absent in non-galled tissue. A new graduate student is pursuing full analysis of this gene that appears to be involved in regulating cell cycle. Most genes identified are from maize. Progress on objective 3: We have isolated small fragments of several protein phosphatase genes. We have cloned, sequenced and disrupted the U. maydis calcineurin gene and find it important in morphogenesis, mating and pathogenicity. We have also cloned a PP2A homolog and showed that it is likely essential. A manuscript on this work has been submitted and is under review. With its publication this objective will have been accomplished. Progress on objective 4: Progress to date: We have made constructs for the analysis of these interactions. Site directed mutants have been characterized. They identify important function for the SAM and RA domains but none detected for the SH3 or P-loop domains. Two hybrid assays continue. We have clear evidence for interaction between the SAM domain of Ubc2 and the Ubc4 protein. A manuscript is under preparation. Progress on objective 5: We have observed differential expression by northern blot of a large number of these genes. We are still in early stages in the other approaches. We have identified the genes that we plan to manipulate. We identified a major regulator (ust1) of filament down regulated gene transcription. Progress on objective 6: We have thoroughly analyzed the ubc mutant collection and have found that we have identified most genes. Some strains are difficult to manipulate, but it appears that we have more or less exhausted this screen. Thus this objective is completed.

Impacts
Impact will be accomplished in the long-term through an increased understanding of the genetic basis of fungal plant diseases. This may eventually lead to novel disease control methods.

Publications

  • Horton, J.S., Bakkeren, G., Klosternam, S.J., Garcia-Pedrajas M. and Gold, S.E. 2005. Genetics of morphogenesis in Basidiomycetes. In: Appled Mycology and Biotechnology Vol 5. pp 353-422. Elsevier. Arora,D.K. and Berka, R. eds.
  • Bakkeren, G. and S. Gold (2004). "The path in fungal plant pathogenicity: many opportunities to outwit the intruders?" Genet Eng (N Y). 26: 175-223.
  • Martinez-Espinoza, A.D., Ruiz-Herrera, J., Leon-Ramirez C.G. and Gold, S.E. 2004. MAP kinase and cAMP signaling pathways modulate the pH-induced yeast to mycelium dimorphic transition in the corn smut fungus Ustilago maydis Curr. Microbiol. 49:274-281


Progress 01/01/04 to 12/31/04

Outputs
The objectives of this project are shown below. Objective 1. Utilize differential screening to identify genes involved in dimorphism. Objective 2. Determine the roles of genes specifically upregulated during gall formation. Objective 3. Determine the roles in dimorphism of genes encoding catalytic subunits of serine/threonine protein phosphatase. Objective 4: Ubc2 Protein-Protein Interactions. Objective 5: Expression Analysis of Filament Specific Genes. Objective 6: Continuation of Mutant Analysis. Progress on objective 1: To date we have isolated unique PCR products upregulated in filaments (40 different genes) or in budding cells (about 37 genes). We have analyzed 60 genes by northern analysis for their level of differential expression. We have disrupted 5 filament specific genes to date. We have deleted 4 genes from the budding form thus far including a putative xylanase and ammonium transporter gene. We have published one paper on the ammonium transporter and have two submitted manuscripts in review on this objective. Progress on objective 2: We have constructed two libraries one from infected galled tissue and one from infected non-galled tissue which we have been confirmed to contain mostly differentially expressed genes. We have begun screening for differential expression by northern blots and determining species of origin by genome searches. We will continue to sequencinga large number of clones to get a global sense of these libraries. One interesting gene is highly upregulated in gall tissue and absent in non-galled tissue. A new graduate student is pursuing full analysis of this gene that appears to be involved in regulating cell cycle.Most genes identified are from maize. Only a few genes have been identified from the fungus thus far. Progress on objective 3: We have isolated small fragments of several protein phosphatase genes. We have cloned, sequenced and disrupted the U. maydis calcineurin gene and find it important in morphogenesis, mating and pathogenicity. We have also cloned a PP2A homolog and showed that it is likely essential. A manuscript on this work has been submitted and is under review. With its publication this objective will have been accomplished. Progress on objective 4: Progress to date: We have made constructs for the analysis of these interactions. Site directed mutants have been characterized . They identify important function for the SAM and RA domains but none detected for the SH3 or P-loop domains. Two hybrid assays continue. We have clear evidence for interaction between the SAM domain of Ubc2 and the Ubc4 protein. We have identified numerous interactors with Ubc2. Progress on objective 5: We have observed differential expression by northern blot of a large number of these genes. We are still in early stages in the other approaches. We have deleted several genes to date and published on paper on this. Progress on objective 6: We have thoroughly analyzed the ubc mutant collection and have found that we have identified most genes. Some strains are difficult to manipulate, but it appears that we have more or less exhausted this screen. Thus this objective is completed.

Impacts
Impact will be accomplished in the long-term through an increased understanding of the genetic basis of fungal plant diseases. This may eventually lead to novel disease control methods.

Publications

  • Garcia-Pedrajas M.D. and Gold, S.E. 2004. Kernel Knowledge: Smut of Corn. In: Advances in Applied Microbiology V 56: pp263-289. Elsevier Academic Press. Laskin, Bennett and Gadd eds.
  • Gold S.E., Garcia-Pedrajas, M.D., Andrews, D.L., Klosterman, S.J., Mayorga, M.E., Egan J.D., Martinez-Espinoza, A.D., Smith, D. and Perlin, M.H. 2004. Molecular Basis of Virulence in Ustilago maydis. In: Genomic and Genetic Analysis of Plant Parasitism and Defense. pp 30-49. APS Press. Tsuyumu, Leach, Shiraishi and Wolpert eds.
  • Garcia-Pedrajas M.D., Klosterman, S.J., Andrews, D.A. and Gold, S.E. 2004. The Ustilago maydis maize interaction. In: Plant-Pathogen Interactions, Annual Plant Reviews. Blackwell Publishing. Talbot, N. ed. 11:166-201.
  • Martinez-Espinoza, A.D., Ruiz-Herrera, J., Leon-Ramirez C.G. and Gold, S.E. 2004. MAP kinase and cAMP signaling pathways modulate the pH-induced yeast to mycelium dimorphic transition in the corn smut fungus Ustilago maydis. Current Microbiology 49:274-281.
  • Andrews, D.L., Garcia-Pedrajas, M.D. and Gold, S.E. 2004. Fungal dimorphism regulated gene expression in Ustilago maydis: I. Filament upregulated genes. Molecular Plant Pathology 5:281-293.
  • Garcia-Pedrajas, M.D. and Gold, S.E. 2004. Fungal dimorphism regulated gene expression in Ustilago maydis: II. Bud upregulated genes. Molecular Plant Pathology 5:295-307.


Progress 01/01/03 to 12/31/03

Outputs
The objectives of this project are shown below. Objective 1. Utilize differential screening to identify genes involved in dimorphism. Objective 2. Determine the roles of genes specifically upregulated during gall formation. Objective 3. Determine the roles in dimorphism of genes encoding catalytic subunits of serine/threonine protein phosphatase. Objective 4: Ubc2 Protein-Protein Interactions. Objective 5: Expression Analysis of Filament Specific Genes. Objective 6: Continuation of Mutant Analysis. Progress on objective 1: To date we have isolated unique PCR products upregulated in filaments (40 different genes) or in budding cells (about 50 genes). We have analyzed 60 genes by northern analysis for their level of differential expression. We have disrupted 5 filament specific genes to date. We have deleted 4 genes from the budding form thus far including a putative xylanase and ammonium transporter gene. We have published one paper on the ammonium transporter and have two submitted manuscripts in review on this objective. Progress on objective 2: We have constructed two libraries one from infected galled tissue and one from infected non-galled tissue which we have been confirmed to contain mostly differentially expressed genes. We have begun screening for differential expression by northern blots and determining species of origin by genome searches. We will continue to sequencinga large number of clones to get a global sense of these libraries. One interesting gene is highly upregulated in gall tissue and absent in non-galled tissue. A new graduate student is pursuing full analysis of this gene that appears to be involved in regulating cell cycle.Most genes identified are from maize. Only a few genes have been identified from the fungus thus far. Progress on objective 3: We have isolated small fragments of several protein phosphatase genes. We have cloned, sequenced and disrupted the U. maydis calcineurin gene and find it important in morphogenesis, mating and pathogenicity. We have also cloned a PP2A homolog and showed that it is likely essential. A manuscript on this work has been submitted and is under review. With its publication this objective will have been accomplished. Progress on objective 4: Progress to date: We have made constructs for the analysis of these interactions. Site directed mutants have been characterized . They identify important function for the SAM and RA domains but none detected for the SH3 or P-loop domains. Two hybrid assays continue. We have clear evidence for interaction between the SAM domain of Ubc2 and the Ubc4 protein. Progress on objective 5: We have observed differential expression by northern blot of a large number of these genes. We are still in early stages in the other approaches. We have identified the genes that we plan to manipulate. One manuscript with the northern analysis is submitted. Progress on objective 6: We have thoroughly analyzed the ubc mutant collection and have found that we have identified most genes. Some strains are difficult to manipulate, but it appears that we have more or less exhausted this screen. Thus this objective is completed.

Impacts
Impact will be accomplished in the long-term through an increased understanding of the genetic basis of fungal plant diseases. This may eventually lead to novel disease control methods.

Publications

  • A ste20 homologue in Ustilago maydis plays a role in mating and pathogenicity. 2004. Smith, D.G., Garcia-Pedrajas M.D., Hong, W., Yu, Z. Gold, S.E. and Perlin, M.H. Eukaryotic Cell, 3: 180-189.
  • Fungal dimorphism regulated gene expression in Ustilago maydis: I. Filament upregulated genes. Andrews, D.L., Garcia-Pedrajas, M.D. and Gold, S.E. 2004 (Submitted to Molecular Plant Pathology).
  • Fungal dimorphism regulated gene expression in Ustilago maydis: II. Bud upregulated genes. Garcia-Pedrajas, M.D. and Gold, S.E. 2004 (Submitted to Molecular Plant Pathology).


Progress 01/01/02 to 12/31/02

Outputs
The objectives of this project are shown below. Objective 1. Utilize differential screening to identify genes involved in dimorphism. Objective 2. Determine the roles of genes specifically upregulated during gall formation. Objective 3. Determine the roles in dimorphism of genes encoding catalytic subunits of serine/threonine protein phosphatase. Objective 4: Ubc2 Protein-Protein Interactions. Objective 5: Expression Analysis of Filament Specific Genes. Objective 6: Continuation of Mutant Analysis. Progress on objective 1: To date we have isolated unique PCR products upregulated in filaments (40 different genes) or in budding cells (about 65 genes). We have analysed 60 genes by northern analysis as to their level of differential expression. We have isolated full length genomic clones of 3 filament upregulated transcripts and sequenced them. We have disrupted 3 filament specific genes to date. We have deleted 2 genes from the budding form thus far including a putative xylanase and ammonium transporter gene. Progress on objective 2: We have constructed two libraries one from infected galled tissue and one from infected non-galled tissue which we have been confirmed to contain mostly differentially expressed genes. We have begun screening for differential expression by northern blots and determining species of origin by genome searches. We will be sequencing a large number of clones in the near future to get a global sense of these libraries. One interesting gene is highly upregulated in gall tissue and absent in non-galled tissue. Interestingly this transcript was identified in a maize root EST library. This suggests possible ectopic induction of the gene. Progress on objective 3: We have isolated small fragments of several protein phosphatase genes. We have cloned, sequenced and disrupted the U. maydis calcineurin gene and find it important in morphogenesis, mating and pathogenicity. We have also cloned a PP2A homolog and showed that it is likely essential. A manuscript is reaching the final stages of preparation for the publication of this work. Progress on objective 4: Progress to date: We have made constructs for the analysis of these interactions. The Postdoctoral fellow working on this took a faculty position in January 2002. A new Postdoc started in July and this caused a slow down in progress. The interaction work is progressing nicely and we have identified a number of putative interacters including a ras gene that we predicted we might identify. Most of the site directed mutants have been identified that inhibit the function of ubc2 and final experiments are being set up to characterize the entire set. Progress on objective 5: We have observed differential expression by northern blot of a large number of these genes. We are still in early stages in the other approaches in this aim although we expect to make good progress on this calendar year. We have identified the genes that we plan to manipulate. Progress on objective 6: We have thoroughly analyzed the ubc mutant collection and have found that we have identified most genes. Some strains are difficult to manipulate, but it appears that we have more or less exhausted this screen.

Impacts
Impact will be accomplished in the long-term through an increased understanding of the genetic basis of fungal plant diseases. This should eventually lead to novel disease control methods.

Publications

  • Gold, S.E. Ustilago Pathogenicity. 2003. Plant-Microbe Interactions Volume 6, Stacey and Keen eds. APS Press.
  • Martinez-Espinoza, A.D., Garcia-Pedrajas, M.D. and Gold, S.E. 2002. The Ustilaginales as plant pests and model systems. Fungal Genetics and Biology 35:1-20


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

Outputs
The objectives of this project are shown below. Objective 1. Utilize differential screening to identify genes involved in dimorphism. Objective 2. Determine the roles of genes specifically upregulated during gall formation. Objective 3. Determine the roles in dimorphism of genes encoding catalytic subunits of serine/threonine protein phosphatase. Objective 4: Ubc2 Protein-Protein Interactions. Objective 5: Expression Analysis of Filament Specific Genes. Objective 6: Continuation of Mutant Analysis. Progress on objective 1: To date we have isolated unique PCR products representing genes likely upregulated in filaments (about 40 different genes) or in budding cells (about 15 genes). We are still at an early stage with the budding form. We have analysed 20 genes by northern analysis as to their level of differential expression. We have isolated full length genomic clones of 3 filament upregulated transcripts and sequenced them. We have isolated about 60 upregulated genes in the budding form and we are more fully characterizing these. We have disrupted 3 filament specific genes to date. We plan to disrupt at least 2 of these, including a putative xylanase gene. Progress on objective 2: We have constructed the library and we are screening for observed expression by northern blots and determining species of origin by genome searches. Many of the genes appear to be of maize origin but do not identify maize ESTs. Progress on objective 3: We have isolated small fragments of several protein phosphatase genes. We have cloned, sequenced and disrupted the U. maydis calcineurin gene and find it important in morphogenesis, mating and pathogenicity. We have also cloned a PP2A homolog and are currently characterizing it. Progress on objective 4: Progress to date: We have made constructs for the analysis of these interactions. The Postdoctoral fellow working on this took a faculty position in January and I am in the process of refilling the position. Therefore this is currently on hold. Several site directed mutants have been identified that inhibit the function of ubc2. Again this is on hold due to the departure of the postdoctoral fellow. Progress on objective 5: We are still in early stages although we expect to make good progress on this this calendar year. We have identified the genes that we plan to manipulate. Progress on objective 6: We have thoroughly analyzed the ubc mutant collection and have found that we have identified most genes. Some strains are difficult to manipulate, but it appears that we have more or less exhausted this screen.

Impacts
Impact will be accomplished in the longterm through an increased understanding of the genetic basis of fungal plant diseases. This should eventually lead to novel disease control methods.

Publications

  • Topp, C.N., Ruiz-Herrera, J., Martinez-Espinoza, A.D. and Gold S.E. 2002. Integration of the gene for carboxin resistance does not impact the Ustilago maydis - maize interaction. Current Microbiology. 44:67-70.
  • Gold, S.E., Marinez-Espinoza, A.D. and Garcia-Pedrajas M.D. 2001. New and Used Approaches to the Study of Fungal Pathogenicity. Requested Review. Annual Review of Phytopathology, 39:337-65.
  • Mayorga, M.E. and Gold, S.E. 2001. The ubc2 gene of Ustilago maydis encodes a putative novel adaptor protein required for filamentous growth, pheromone response and virulence. Molecular Microbiology. 41:1365-1380.
  • Scott E. Gold, Maria D. Garcia-Pedrajas, David L. Andrews, Alfredo D. Martinez-Espinoza, Maria E. Mayorga, John D. Egan, and Karen E. Snyder. 2000. The genetics of the Ustilago maydis - maize interaction: Messages from the fungus and from the host. Research Trends; Current Topics in Plant Biology 2:21-33.