Progress 10/01/02 to 09/30/07
Outputs
OUTPUTS: Completed phenotypic characterization of mutants carrying a gene deletion of the adenylate cyclase-encoding gene (sac1). Mutants exhibit defects in growth, development and pathogenicity. Finished characterization of Pac1 activating mutants. These mutants exhibit altered gene expression relative to ambient pH, accumulate high levels of oxalic acid under acidic growth conditions, and exhibit a small attenuation of virulence. Created and characterized a mutant in the ssp1 gene of S. sclerotiorum. This gene plays roles in sclerotium function and has provided insights into the mechanisms fungi potentially use to persist in the environment. A collection of approximately 1,045 Agrobacterium-mediated insertional transformants was established and screened for defects in apothecial development. Fifty-eight strains with defects in apothecial development phenotypes were identified. Insertion-site flanking DNA recovery, phenotypic characterization and genetic complementation studies were established. The Sclerotinia sclerotiorum genome was sequenced and a joint community annotation project with the European Botrytis cinerea community has been completed. PARTICIPANTS: Dov Prusky, international collaborator. Kathy Dobinson, international collaborator. Wayne Jurick, PhD student training. Moyi Li, PhD student training. Young Tae Kim, , postdoctoral research training. Selva Veluchamy, postdoctoral research training. John Reid,undergraduate student training. Loumarie Colon, undergraduate student training. Ron Wilson, undergraduate student training. Fressia Torres,undergraduate student training. Chris Roeser, undergraduate student training. Kelly Chacon, high school student training. TARGET AUDIENCES: Graduate students, university faculty, agriculture industry in the United States and globally. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The functional analysis of genes has been developed as a routine procedure for evaluating Sclerotinia pathogenicity and development. Our analyses have revealed essential molecular factors for these processes. Targeted functional gene analysis as well as the genome sequencing and insertional mutagenesis projects have provided fundamental knowledge concerning necrotrophic pathogenicity and development. This research has facilitated progress in identifying and characterizing critical factors that can be targeted for controlling diseases caused by this devastating plant pathogen.
Publications
- Drori, N., Kramer-Haimovich, H., Rollins, J., Dinoor, A., Okon, Y., Pines, O., and Prusky, D. 2003. External pH and nitrogen source affect secretion of pectate lyase by Colletotrichum gloeosporioides. Appl. Environ. Microbiol. 69:3258-3262.
- Veluchamy S., and J. A. Rollins. 2008. A CRY-DASH-type photolyase/cryptochrome from Sclerotinia sclerotiorum mediates minor UV-A-specific effects on development. Fungal Genetics and Biology 45:1265-1276.
- Miyara, I., Shafran, H., Kramer-Haimovich, H. Rollins, J., Sherman, A., and Prusky, D. 2008. Multifactor regulation of pectate lyase secretion by Colletotrichum gloeosporioides pathogenic on avocado fruits. Molecular Plant Pathology 9:281-291.
- Liberti, D., Grant, S.J., Benny, U., Rollins, J.A. and Dobinson, K.F.. 2007. Development of an Agrobacterium tumefaciens-mediated gene disruption method for Sclerotinia sclerotiorum. Canadian Journal of Plant Pathology 29:394-400.
- Jurick, W. M. & Rollins, J. A. 2007. Deletion of the adenylate cyclase (sac1) gene affects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorum. Fungal genetics and Biology. 44:521-530.
- Kim, Y., Prusky, D. & Rollins, J. A. 2007. An activating mutation of the Sclerotinia sclerotiorum pac1 gene increases oxalic acid production at low pH but decreases virulence. Molecular Plant Pathology. 8:611-622.
- Kramer-Haimovich, H., Servi, E., Katan, T., Rollins, J., Okon, Y. & Prusky. D. 2006. Effect of ammonia production by Colletotrichum gloeosporioides on pelB activation, pectate lyase secretion, and fruit pathogenicity. Appl. Environ. Microbiol. 72:1034-1039.
- Jurick II, W., Dickman , M.B., and Rollins, J. A. 2004. Characterization and functional analysis of a cAMP-dependent protein kinase A catalytic subunit gene (pka1) in Sclerotinia sclerotiorum. Physiol. Mol. Plant Pathol. 64:155-163.
- Chen, H., Dekkers, K., Cao, L., Rollins, J. A., Ko, N., Timmer, L. W. & Chung, K. R. 2005. A gene with domains related to transcription regulation is required for pathogenicity in Colletotrichum acutatum causing Key lime Anthracnose. Mol. Plant
- Rodrigues, F., Jurick II, W. M.*, Datnoff, L. E., Jones, J. B. & Rollins, J. A. 2005. Silicon influences cytological and molecular events in compatible and incompatible rice-Magnaporthe grisea interactions. Physiol. Mol. Plant Pathol. 66:144-159.
- Rollins J. A. 2003. The Sclerotinia sclerotiorum pac1 gene is required for sclerotial development and virulence. Mol. Plant-Microbe Interact. 16:785-795.
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Progress 10/01/05 to 09/30/06
Outputs
Completed phenotypic characterization of mutants carrying a gene deletion of the adenylate cyclase-encoding gene (sac1). Mutants exhibit defects in growth, development and pathogenicity. Manuscript was submitted and accepted for publication. Finished characterization of Pac1 activating mutants. These mutants exhibit altered gene expression relative to ambient pH, accumulate high levels of oxalic acid under acidic growth conditions, and exhibit a small attenuation of virulence. Manuscript was submitted for publication and is currently being revised. A collection of approximately 1,045 Agrobacterium-mediated insertional transformants has been established. Greater than 600 of these have been screened for apothecial disc development defects. Three strains with disc development defect phenotypes have been identified. Insertion-site flanking DNA recovery, phenotypic characterization. And genetic complementation studies are continuing. As a continuing component of the
Sclerotinia sclerotiorum genome sequencing project, a joint community annotation project with the European Botrytis cinerea community has been established. I serve as an annotation coordinator for growth and development genes.
Impacts
The functional analysis of genes has been developed as a routine procedure for evaluating Sclerotinia pathogenicity and development. Our analyses have revealed essential molecular factors for these processes. The genome sequencing and insertional mutagenesis projects will increase the pace at which we are able to identify and characterize critical factors that can be targeted for controlling diseases caused by this devastating plant pathogen.
Publications
- Kramer-Haimovich, H., Servi, E., Katan, T., Rollins, J., Okon, Y. & Prusky. D. 2006. Effect of ammonia production by Colletotrichum gloeosporioides on pelB activation, pectate lyase secretion, and fruit pathogenicity. Appl. Environ. Microbiol. 72:1034-1039.
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Progress 10/01/04 to 09/30/05
Outputs
For this period, progress is reported on the functional characterization of five genes hypothesized to play a role in the pathogenesis or multicellular development of Sclerotinia sclerotiorum. To further analyze genes with roles in pathogenesis and multicellular development, methods were developed and put to use for creating a tagged mutant collection of S. sclerotiorum. Additionally the initiation and completion of a whole genome sequencing project and the development of an Expressed Sequence Tag (EST) project for S. sclerotiorum is reported. Signal transduction pathways involving cAMP have been implicated in the regulation of oxalic acid production sclerotial development. The targeted functional characterization of two genes involved in cAMP-dependent signaling, pka1 and sac1, was initiated. The pka1 characterization is complete and the results have been published. The sac1 gene has been successfully knocked out and the loss-of-function mutant is being
phenotypically characterized. EST data has allowed us to identify a gene hypothesized to encode enzymatic activity required for oxalic acid biosynthesis. This gene, oah1, was targeted for disruption and two independent mutants with a deletion in this gene have been established. Complementation of the mutation is underway and phenotypic characterization will follow. The relationship between pH-dependent signaling and carbon nutrition was explored from two perspectives. The first was through functional characterization of the snf1 gene, a gene hypothesized to encode a protein kinase that functions in the carbon catabolite repression pathway. A gene deletion mutant was established for this gene and awaits complementation and full phenotypic characterization. The second approach was through transcriptional kinetic profiling of a number of transcripts identified through the EST project. The accumulation kinetics of these transcripts have been characterized in response to pH-and carbon
nutrition through quantitative Northern hybridization analysis. A manuscript detailing the results of this study is in preparation. Work has continued on the characterization of the pH-responsive transcriptional regulator Pac1. An activating muation allele was created and reintroduced into wild-type and pac1 loss-of-function backgrounds. Phenotypic characterization of these mutants has revealed an attenuated virulence phenotype, but the affect is not as severe as the loss-of-function mutation. These results have revealed that the ability to respond to the dynamic ambient pH environment rather than low or high pH responsiveness per se is important for Sclerotinia virulence. With funding from the USDA microbial genome sequencing program and in collaboration with colleagues at the University of Nebraska, the University of Toronto, and MIT's BROAD institute a project to sequence the genome of S. sclerotiorum was initiated and completed. The sequence was assembled into 36 non-overlapping
contigs representing 38 million base pairs. An automated gene calling annotation has identified 14,522 genes. This genome data represents a genetic resource that will facilitate molecular genetic analysis of this fungus.
Impacts
The functional analysis of genes has been developed as a routine procedure for evaluating Sclerotinia pathogenicity and development. Our analyses have revealed essential molecular factors for these processes. The genome sequencing and insertional mutagenesis projects will increase the pace at which we are able to identify and characterize critical factors that can be targeted for controlling diseases caused by this devastating plant pathogen.
Publications
- Chen, H.-Q., Cao, L., Dekkers, K.L., Rollins, J.A., Ko, N.J., Timmer, L.W., and Chung, K.-R. 2005. A gene with domains related to transcription regulation is required for pathogenicity in Colletotrichum acutatum causing Key lime Anthracnose. Mol. Plant Pathol. 6:513-525.
- Rodrigues, F. A., Datnoff, L.E., Jurick, W. M., Jones, J. B., and Rollins, J. A. 2004. Silicon influences cytological and molecular events in compatible and incompatible rice-Magnaporthe grisea interactions. Physiol. Mol. Plant Pathol. 66:144-159.
- Jurick , W., Dickman , M.B., and Rollins, J. A. 2004. Characterization and functional analysis of a cAMP-dependent protein kinase A catalytic subunit gene (pka1) in Sclerotinia sclerotiorum. Physiological and Molecular Plant Pathology 64:155-163
- Drori N, Kramer-Haimovich H, Rollins J, Dinoor A, Okon Y, Pines O, Prusky D. 2003. External pH and nitrogen source affect secretion of pectate lyase by Colletotrichum gloeosporioides. Applied and Environmental Microbiology 69:3258-3262.
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Progress 10/01/02 to 10/01/03
Outputs
Sclerotinia sclerotiorum is a broad host range necrotrophic phytopathogen. The aggressive and nondiscriminate nature of its pathogenicity has stymied efforts to control Sclerotinia disease through host resistance breeding. Progress is reported on the identification and evaluation of molecular factors important for Sclerotinia pathogenesis. S. sclerotiorum requires the production of oxalic acid, a dicarboxyillic acid that chelates divalent cations, for pathogenicity. In culture oxalic acid production is regulated by pH. Alkaline pH promotes oxalic acid biosynthesis and acid pH inhibits production and facilitates its enzymatic breakdown. Long-term survival and propagation of S. sclerotiorum is dependent on its production of sclerotia, highly compact and melanized aggregates of fungal mycelia. Sclerotial development is also dependent on ambient pH conditions. A gene encoding a pH responsive transcription factor (pac1) has been isolated and characterized from S.
sclerotiorum. Functional characterization of this gene was carried out through the creation of loss-of-function pac1 alleles. Under the most favorable cultural conditions, pH 7, for oxalic acid production, pac1 loss-of-function mutants produced approximately one fifth of wild type oxalic acid levels. Although greatly attenuated, oxalic acid production remained alkaline pH inducible suggesting a secondary molecular mechanisms for the pH regulation of oxalic acid production independent of Pac1. Pac1 mutants, but not wild type, were unable to grow in culture above pH 6.2 and developed aberrant sclerotia that coalesced and failed to elaborate outer melanized rind layers independent of culture pH. Pathogenicity assays with Arabidopsis plants and detached tomato leaflets indicated an approximately 75% reduction in virulence with pac1 mutants. Expression of pH-regulated endopolygalacturonase encoding genes was altered in a manner consistent with the function of Pac1 as a positive regulator
of alkaline-expressed genes and a repressor of acid-expressed genes. These studies have indicated a role for pH-mediated gene expression via Pac1 in oxalic acid production, endopolygalacturonase gene expression, sclerotial development, alkaline adaptation, and pathogenicity. To identify genes that are specific to the processes of oxalic acid biosynthesis or sclerotial development we have established an EST collection including more than 1200 sequences from a developing sclerotia cDNA library. Individual clones with putative roles in sclerotial development and oxalic acid synthesis are currently being evaluated. We have established a collection of approximately 350 REMI mutants. Twelve mutants from this collection exhibit defects in sclerotial development. Genes responsible for these phenotypes are being characterized by gene rescue and functional analysis.
Impacts
The first defined mutation resulting in a loss of S. sclerotiorum virulence has been created and characterized. This research contributes to our basic understanding of Sclerotinia diseases and the underlying molecular regulators of pathogenesis. This information contributes to our efforts to define critical factors in pathogenesis that can be targeted for controlling disease development in agriculture.
Publications
- Rollins, J.A. 2003. The Sclerotinia sclerotiorum pac1 gene is required for sclerotial development and virulence. Mol. Plant-Microbe Ineract. 16:785-795.
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