Progress 01/01/99 to 12/31/04
Outputs
The project identified and characterized a transcription factor in the fungal plant pathogen Nectria haematococca (Anamorph: Fusarium solani). This transcription factor is associated with the induction of PDA1, a gene encoding the cytochrome P450 gene that detoxifies pisatin, the host plant's isolfavonoid defense compound. The transcription factor was cloned based upon its ability to bind a pisatin-responsive element identified in the PDA1 promoter and is referred to as the pisatin-responsive factor (PRF). The sequence of the PRF gene indicates that it belongs to the C6-Zn2 family of transcription factors unique to fungi. RNAi suppression of PRF expression was found to eliminate pisatin-induction of the PDA1 gene, supporting a regulatory role of PDA1. Further manipulation in N. haematococca was hampered by problems with transformation; therefore heterologous systems were used to characterize PRF. In the yeast Saccharomyces cerevisiae, expression of PRF was able to
modulate transcription of a reporter gene fused to the pisatin-responsive element. However, pisatin was found to reduce reporter expression rather than induce expression as in the homologous system. Deletion of portions of the PRF gene identified a transcriptional activator region and another inhibitory region that suppressed the activator activity. Removal of the inhibitory region removed the pisatin-response, indicating that this C-terminal portion controls its regulatory activity. A second heterologous system used to characterize PRF was the genetic model filamentous fungus Neurospora crassa. A reporter gene fused to the pisatin-response element displayed strong induction when it was transformed into N. crassa along with the PRF gene. However, a weaker pisatin-responsive expression of the reporter gene was also observed even without PRF co-transformation. This indicated that a N. crassa protein was able to both recognize the pisatin-responsive DNA element and modulate activity in
response to pisatin. Two predicted genes in the N. crassa genome, NCU00217 and NCU02752, were the closest in sequence similarity to PRF. Repeat-induced polymorphisms (RIP) was used to separately mutate each of these genes in N. crassa to null alleles. Only mutation of NCU02752 resulted in a lack of pisatin induction of the reporter gene. Further analysis of the N. crassa transformants indicated that the range of compounds that could induce the response was broader than pisatin alone. Compounds that disrupt the membrane potential, either through acting as an ionophore or by changing membrane fluidity, were strong inducers of this pathway. These results indicate that pisatin recognition in N. haematococca is likely through a novel PRF/ NCU02752 signal pathway that recognizes the membrane stress that pisatin causes, rather than through direct recognition of the pisatin molecule itself. Further, it appears that the pathway is not specific to N. haematococca and may be common to other
fungi beyond N. crassa as well.
Impacts
The interaction of plants and pathogens is regulated by recognition of the other partner in the interaction. Understanding the signals that are exchanged will allow the control of the interaction and thus the diseases caused by these fungi. At the onset of this project, it was believed that N. haematococca was able to specifically recognize its plant host through the physical recognition of its host-specific isoflavonoid phytoalexin. This would be consistent with the mode of regulation of animal detoxification-related cytochrome P450 genes by nuclear receptors that bind inducing molecules. However, this study has indicated a different form of regulation that is novel to fungi through recognition of the physiological stress that the compound causes. This signaling pathway can respond to other membrane active plant compounds and synthetic antifungal agents. Since the pathway is not restricted to N. haematococca, it may play a role in the regulation of other
detoxification or transport genes conferring resistance to chemical defenses in other plant-fungal interactions. It may also play a role in the native resistance to synthetic fungicides observed in many fungi. This is particularly relevant since recent genome sequencing indicates a large number of cytochrome P450 genes exist in fungi, particularly the plant pathogens. Further research will indicate whether it is unique or if there is common regulation of other cytochrome P450 genes, potentially responding to other plant or synthetic anti-fungal compounds.
Publications
- R. Khan, R. Tan, A. Galvez Mariscal, and D. Straney. 2003 A binuclear zinc transcription factor binds a host isoflavonoid-responsive element in a fungal cytochrome p450 gene responsible for detoxification. Molecular Microbiology. 49: 117-130.
- X. Wei, F. Yang, D.C. Straney 2005. Multiple Non-ribosomal Peptide Synthetase Genes Determine Peptaibol Synthesis in Trichoderma virens. Canadian Journal of Microbiology.51: 423-29
- U. Gunawardena, M. Rodriguez, D. Straney, J.T. Romeo, H.D. VanEtten, M.C. Hawes. 2005 Tissue-specific localization of pea root infection by Nectria haematococca. Mechanisms and consequences Plant Physiol. 137: 1363-74
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Progress 01/01/02 to 12/31/02
Outputs
Our progress has continued in characterizing the pisatin-responsive regulation of the PDA1 gene. In particular, we have been determining the function of the transcription factor (PRF) that binds the pisatin-responsive element of the PDA1 gene. Transformation of Fusarium solani with constructs that produce an RNAi-suppression of the gene encoding PRF caused a loss of PRF expression as well as a loss of pisatin-inducibility of the PDA gene. Further, transformation of the PRF gene into a heterologous fungus, along with GUS reporter gene linked to the pisatin-responsive element, produces a pisatin-responsive expression of the GUS gene in this fungus. These two tests provide evidence that not only does PRF mediate pisatin response in Fusarium, but that it may be the sole component required for conferring pisatin response. A function of PRF as a pisatin receptor is currently being tested.
Impacts
The characterization of the pisatin-responsive transcription factor may show it to be a central element in the ability of this host-specific fungus to specifically recognize its host through a chemical signal. The identification of this pathway and its relation to that used by other fungi should indicate whether we can interfere with host recognition and so prevent pathogenesis.
Publications
- S.E. Wilhite , R.D. Lumsden and D.C. Straney 2001. Peptide synthetase gene in Trichoderma virens. Applied and Environmental Microbiology 67: 5055-5062.
- R. Khan, R. Tan, A. Galvez Mariscal, and D. Straney.2003 A binuclear zinc transcription factor binds a host isoflavonoid-responsive element in a fungal cytochrome p450 gene responsible for detoxification. Molecular Microbiology. Revised manuscript submitted.
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Progress 11/01/00 to 10/31/01
Outputs
Our work has progress mainly in the first area of our project in characterizing the pisatin-response of the PDA1 gene. Over this past year we have identified and sequenced a 4Kb genomic clone of the putative pisatin-responsive transcription factor. With this sequence and further characterization of the cDNA clone isolated from the screen, we have identified a short ORF directly upstream of that encoding the DNA binding protein, which was previously thought to be part of the latter's coding region. We have been able to express the main ORF in E. coli so as to recover expressed protein. We are now using this protein to study DNA binding specificity. We have utilized out new 4Kb genomic clone to construct a better gene-disruption vector. In order to produce a gene disruption, a transformation procedure was developed using electroporation of germinating spores. A set of putative gene-disruptants are now being analyzed. The second area of the project deals with the
developmental response to pisatin and flavonoids. From our previous results, detailed in the previous progress report, it was obvious that pisatin plays a central role in triggering germination in pea root exudates. We intend to use our gene-disruption constructs to test if the pisatin-responsive transcription factor is also controlling the pisatin-responsive development as well.
Impacts
The characterization of the pisatin-responsive transcription factor may show it to be a central element in the ability of this host-specific fungus to specifically recognize its host through a chemical signal. The identification of this pathway and its relation to that used by other fungi should indicate whether we can interfere with host recognition and so prevent pathogenesis.
Publications
- H.D. VanEtten, D. Straney, S. Covert, H.C. Kistler. 2001. Update on Selected Topics in the Genetics of Nectria haematococca Mating Population VI with Special Emphasis on its Conditionally Dispensable Chromosomes: A Source of Habitat Specific Genes. In: Fusarium: Paul Nelson Memorial Symposium. J.F. Leslie, ed. American Phytopathological Society Press, St. Paul MN. In press.
- David Straney, Rana Khan, Reynold Tan and Savita Bagga 2001 Host Recognition By Pathogenic Fungi Through Plant Flavonoids.. In:Flavonoids in Cell Function. Volume in the Advances in Experimental Medicine and Biology Series. Eds. J.A. Manthey and B.S. Buslig. Plenum Press
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Progress 01/01/99 to 12/31/99
Outputs
The project focuses upon two areas of Nectria haematococca response to host flavonoids: 1) characterization of the regulatory components of the PDA1 gene and 2) the role of signals which stimulate spore germination. 1) PDA regulation. We have defined a 40 bp pisatin-responsive site in the PDA1 promoter. Critical evidence for its function is provided by the ability of 5-copies of this 40 bp site to enhance trancription of a minimal promoter in response to pisatin. We have cloned a DNA-binding protein that binds this 40 bp element using a yeast one-hybrid system screen. The sequence indicates that it is a DNA binding protein of the binuclear zinc- 6 cystein class common to fungi. Gene disruption and heterologous expression is currently being applied to this gene to test the exclusivity of its function in pisatin regulation. 2) Germination stimuli. We have pursed the detection and identification of germination stimuli present in pea root exudates. Pisatin is present in
all exudates except for under aseptic conditions and provides at least 50% of the germination stimulus activity, increasing with greater pisatin production. Another fraction of the exudate also induces germination, but this fraction also induced nod-gene expression in the pea-specific rhizobium. The germination stimulus in pea roots appears to be a balance between pisatin and this (or these) nod inducers.
Impacts
This project has identified a gene which appears to encode a regulatory protein which mediates fungal recognition of a host-specific compound. Cloning this gene allow us to perform specific tests upon the function of this gene in regulationg a number of traits associated with pea pathogenesis. Our biochemical analysis shows that pea roots exude this compound, pisatin, into the soil around the plant and so indicates a broader role in the host-recognition process.
Publications
- KHAN, R., STRANEY, D.C. 1999. Regulatory Signals Influencing Expression of the PDA1 Gene of Nectria haematococca MP VI in Culture and During Pathogenesis of Pea. Molecular Plant-Microbe Interactions. 12: 733-742
- KHAN, R. 1999. Molecular analysis of a fungal virulence gene regulation by host signals during pathogenesis. Ph.D. Thesis. Department of Cell Biology & Molecular Genetics, University of Maryland, College Park.
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