Progress 09/15/02 to 03/14/06
Outputs
This research was focused on understanding the genetic basis for the biotrophic to necrotrophic transition in the hemibiotrophic fungus Colletotrichum graminicola, which causes anthracnose disease of corn. A nonpathogenic mutant was identified during a large-scale screening experiment. The mutant initiates the biotrophic phase of the disease normally, but does not switch to necrotrophic growth in leaves. The mutant appears to be deficient in one component of the signal peptidase enzyme responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum system of the cell. The gene encoding this protein was named CPR1. A CPR1 cDNA was cloned and transformed into two different yeast signal peptidase mutants. However, CPR1 did not complement either mutant yeast strain, perhaps because CPR1 is too divergent from the yeast homologue of this protein. To obtain indirect evidence for the function of CPR1, a gene encoding a putative
pSEC11 homologue of C. graminicola (Cgs11), was cloned and characterized. pSEC11 is one of four subunits of the signal peptidase in yeast, and has been shown to physically interact with pSPC3. The predicted protein encoded by the Cgs11 cDNA was 67 % similar to pSEC11. The Cgs11 cDNA complemented two different yeast sec11 mutants. Additional research was focused on the potential role of CPR1 in quantitative and qualitative regulation of secretion. Measurements of protein secreted by the mutant versus the wild-type into media containing various carbon and nitrogen sources suggested that CPR1 is a negative regulator of secretion under conditions of carbon starvation, and a positive regulator under nutrient-rich conditions. Other investigations suggested that CPR1 plays a role in the specific secretion of enzymes important for pathogenic growth. Work was also undertaken to characterize the growth and development of the mutant in host tissues: these studies revealed that the mutant
pathogen probably dies in the host tissues at or before the transition to necrotrophy.
Impacts
This work will help us to understand more about how pathogenic fungi cause harmful symptoms in their hosts, and how those symptoms relate to fungal colonization of the host tissues.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
This work is focused on understanding the genetic basis for the lifestyle transition in the hemibiotrophic fungus Colletotrichum graminicola, which causes anthracnose disease of corn. A nonpathogenic mutant was identified during a large-scale screening experiment. The mutant initiates the biotrophic phase of the disease normally, but does not switch to necrotrophic growth in leaves. The mutant appears to be deficient in one component of the signal peptidase enzyme responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum system of the cell. The CPR1 protein cDNA was cloned and transformed into two different yeast signal peptidase mutants. However, CPR1 did not complement either mutant yeast strain, perhaps because CPR1 is too divergent from the yeast protein pSPC3. To obtain indirect evidence for the function of CPR1, a gene encoding a putative pSEC11 homologue of C. graminicola (Cgs11) was cloned and
characterized. pSEC11 is one of four subunits of the signal peptidase in yeast, and has been shown to physically interact with pSPC3. The predicted protein encoded by the Cgs11 cDNA was 67 % similar to pSEC11. The Cgs11 cDNA complemented two different yeast sec11 mutants. Co-immunoprecipitation and co-immunolocalization experiments are in progress to test for an interaction between CGS11 and CPR1. Additional research was focused on the potential role of CPR1 in quantitative and qualitative regulation of secretion. Measurements of protein secreted by the mutant versus the wild-type into media containing various carbon and nitrogen sources suggested that CPR1 is a negative regulator of secretion under conditions of carbon starvation. Other investigations suggested that CPR1 plays a role in the specific secretion of enzymes important for pathogenic growth.
Impacts
This work will help us to understand more about how pathogenic fungi cause harmful symptoms in their hosts, and how those symptoms relate to fungal colonization of the host tissues.
Publications
- Flowers, J.L., and Vaillancourt, L.J. 2005. Parameters affecting the efficiency of Agrobacterium tumefaciens-mediated transformation of Colletotrichum graminicola. Current Genetics 48: 380-388
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Progress 01/01/04 to 12/31/04
Outputs
Hemibiotrophic pathogenic microbes switch during their life cycles from a relatively non-damaging mode of parasitic growth on plants (biotrophy) to a relatively damaging one (necrotrophy). This work is focused on understanding the genetic basis for this transition in the hemibiotrophic fungus Colletotrichum graminicola, which causes anthracnose disease of corn. A nonpathogenic mutant was identified during a large-scale screening experiment. The mutant initiates the biotrophic phase of the disease normally, but appears to be unable to switch to necrotrophic growth in leaves. The mutant is evidently deficient in one component of the signal peptidase enzyme responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum system of the cell. Our most recent goal has been to test the hypothesis that the mutant protein, CPR1, is indeed a component of the C. graminicola signal peptidase. The CPR1 cDNA was cloned and
transformed into two different yeast signal peptidase mutants. However, CPR1 did not complement either mutant yeast strain, perhaps because CPR1 is too divergent from the yeast homologue, pSPC3. To obtain indirect evidence for the function of CPR1, a gene encoding a putative pSEC11 homologue of C. graminicola (Cgs11) was cloned and characterized. pSEC11 is one of four subunits of the signal peptidase in yeast, and has been shown to physically interact with pSPC3. The predicted protein encoded by the Cgs11 cDNA was 67 % similar to pSEC11. The Cgs11 cDNA complemented two different yeast sec11 mutants. Co-immunoprecipitation and co-immunolocalization experiments are now in progress to test for an interaction between CGS11 and CPR1.
Impacts
This work will help us to understand more about how pathogenic fungi cause harmful symptoms in their hosts, and how those symptoms relate to fungal colonization of the host tissues.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Hemibiotrophic pathogenic microbes switch during their life cycles from a relatively non-damaging mode of parasitic growth on plants (biotrophy) to a relatively damaging one (necrotrophy). This work is focused on understanding the genetic basis for this transition in the hemibiotrophic fungus Colletotrichum graminicola, which causes anthracnose disease of corn. A nonpathogenic mutant was identified during a large-scale screening experiment. The mutant initiates the biotrophic phase of the disease normally, but appears to be unable to switch to necrotrophic growth. The mutant is evidently deficient in one component of the signal peptidase enzyme responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum system of the cell. The hypothesis that is currently being tested is that the mutant is unable to secrete sufficient quantities of one or more proteins that are necessary for the switch between biotrophy and
necrotrophy. Current experiments are focused on labeling the relevant protein with an epitope tag so that it can be localized using immunological techniques. In other experiments, we are attempting to use the two-hybrid system to identify proteins that interact with the putative signal peptidase protein.
Impacts
This work will help us to understand more about how pathogenic fungi cause harmful symptoms in their hosts, and how those symptoms relate to fungal colonization of the host tissues.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Hemibiotrophic pathogenic microbes switch during their life cycles from a relatively non-damaging mode of parasitic growth on plants (biotrophy) to a relatively damaging one (necrotrophy). This work is focused on understanding the genetic basis for this transition in the hemibiotrophic fungus Colletotrichum graminicola, which causes anthracnose disease of corn. Mutagenesis resulted in identification of nine mutants that are deficient in the ability to infect, colonize, and cause symptoms on corn. One of these mutants is being studied in detail. This mutant causes no symptoms on corn leaves or stalks. It initiates the biotrophic phase of the disease normally, but it appears to be unable to switch to necrotrophic growth. The mutant appears to be deficient in one component of the signal peptidase enzyme responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum system of the cell. The hypothesis that is currently
being tested is that the mutant is unable to secrete sufficient quantities of one or more proteins that are necessary for the switch between biotrophy and necrotrophy.
Impacts
The goal of this project is to identify and characterize fungal genes that are essential for pathogenicity of a representative stalk rot fungus to corn stalks. Little is presently known about the process of colonization and rotting of corn stalks by stalk rot fungi. This project will provide potential targets for therapies that are designed to control stalk rot diseases of corn.
Publications
- Mims, C.W., Vaillancourt, L.J. 2002. Ultrastructural characterization of infection and colonization of maize leaves by Colletotrichum graminicola, and by a C. graminicola pathogenicity mutant. Phytopathology 92: 803-812
- Thon, M.R., Nuckles, E.M., Takach, J.E., and Vaillancourt, L.J. 2002. CPR1: A gene encoding a putative signal peptidase that functions in pathogenicity of Colletotrichum graminicola to maize. Molecular Plant-Microbe Interactions 15: 120-128
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