Progress 09/01/05 to 08/31/09
Outputs
OUTPUTS: The goal of this proposal was to identify, characterize, and confirm genes involved in resistance of plant pathogenic Cercospora fungi to the active-oxygen generating toxin cercosporin which they produce for parasitism of plants. This goal was approached by characterizing genes regulated by a regulatory protein (CRG1), which was identified based on its requirement for normal expression of cercosporin resistance as well as for normal production of the toxin. Research performed in this project resulted in the recovery of a genomic library containing 185 expressed sequence tags that were differentially regulated between the wild type cercosporin-resistant strain, and a cercosporin-sensitive null mutant for the CRG1 transcription factor. All library genes were sequenced, and classified based on putative function. Genes that encoded proteins known to be involved in cercosporin resistance were targeted for further analysis. These were genes encoding membrane transporters as well as those with oxidation/reduction activity as both toxin export and toxin reduction are known to be involved in resistance. Evidence for a role of the library genes was determined by expressing genes constitutively in the crg1 mutant as well as by creating mutants in the wild type strain. Mutant analysis was done using established techniques for targeted gene disruption in the wild type strain. In addition, protocols for gene silencing were developed for use in Cercospora nicotianae. Methods were developed for cloning inverted repeats into two different plasmids, and transformed strains of wild type C. nicotianae were recovered that were silenced for selected library genes. Transformed strains (overexpression in crg1, disrupted strains, silenced strains) were screened for changes in cercosporin resistance and cercosporin production, changes in developmental stages such as sporulation, and for changes in pathogenicity. In addition to characterizing specific genes, the role of the CRG1 transcription factor in regulating an interaction between pH and cercosporin resistance was documented. Research funded by this project was disseminated through publications and presentations at the Fungal Genetics Conference in 2007, the American Phytopathological Society Annual meeting in 2006, at USDA National Research Initiative competitive grants program principle investigator meetings in 2007 and 2008, and through invited seminars in various academic departments. Sequences of all library genes were made publicly available through the National Center for Biotechnology Information. This project contributed to the mentoring and research training of four postdoctoral researchers, one graduate student, and two undergraduate students. Knowledge gained through this project contributed to the teaching of an advanced graduate level Fungal Genetics course as well as lectures in an undergraduate Plant Biology course at North Carolina State University. PARTICIPANTS: The principle investigators for this grant are Drs. Margaret Daub and Sonia Herrero. Dr. Daub was responsible for oversight of the project, planning, and analysis of the work. Dr. Herrero conducted the subtractive hybridization studies and developed the silencing technology. Dr.Alongkorn Amnuaykanjanasin was a postdoctoral associate supported from this grant who characterized the transporter genes and their role in cercosporin biosynthesis and resistance. Dr. Amnuaykanjanasin is now employed at the National Center for Genetic Engineering and Biotechnology in Thailand, and is continuing to collaborate with us on this project. Dr. Nafisa Ibrahim was another postdoctoral associate supported from this grant who cloned and transformed silencing constructs, and screened silencing strains for cercosporin resistance, sporulation, and pathogenicity. Dr. Ibrahim has returned to Egypt, and is collaborating with us to continue studies on pathogenicity of silenced strains. The grant supported training for a short-term postdoctoral researcher (Eddy Velez) who cloned genes into silencing vectors. Dr. Velez is now a postdoctoral associate at Upsala University, Sweden. The grant also supported training of a graduate student (Aydin Beseli) and two undergraduate students (Andrew Mealin, Zhenghua Zhang) who worked with Dr. Herrero on assay of library gene induction by cercosporin, cloning of silencing vectors, screening of silenced and disrupted strains, and initial efforts to transform plants with ATR1. TARGET AUDIENCES: This grant provided a research opportunity for six undergraduate, graduate, and postdoctoral scientists who received training in molecular biology, plant-microbe interactions, mycology, biotechnology, and agriculture. Four were international, one was female, and one was a US citizen from an underrepresented group (hispanic). During the duration of this grant, Dr. Herrero developed and taught a joint graduate/undergraduate course in use of eukaryotic microbes in biotechnology. This course provides training to students from multiple disciplines (biology, engineering, agriculture) in use of fungi as model systems for research and biotechnology applications. Dr. Daub co-teaches an advanced course in fungal genetics and physiology for graduate students, which is enhanced by the work supported by this grant. She also teaches a professionalism course for graduate students from diverse curricula that conduct research in biotechnology, and provides lectures to in-coming botany majors, exposing them to research, biotechnology, and agriculture. She participates in training and outreach activities supported by the Center for Integrated Fungal Research. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Subtractive hybridization led to the recovery of 338 sequences that were differentially regulated between the cercosporin-resistant wild type strain of Cercospora nicotianae and a cercosporin sensitive null mutant for the CRG1 transcription factor. Sequence analysis identified 185 unique sequences that were classified by putative function as well as by KEGG functional classification. Genes recovered included ones encoding proteins involved in detoxification of noxious compounds, multidrug membrane transporters, antioxidant enzymes, polyketide biosynthesis enzymes, ATPases and ATP synthases, and enzymes involved in oxidation/reduction reactions. Both toxin export and toxin reduction have been implicated in cercosporin resistance, and genes encoding transporters and reductases were prioritized for characterization. Of the six genes encoding major facilitator superfamily (MFS) and ATB binding cassette (ABC) transporters, three were found to contribute to resistance: CnCFP, ATR1, and ATR2. ATR1 in particular was shown to be a major efflux protein for transport of cercosporin out of hyphae, and to play a significant role in resistance, demonstrated both by targeted gene disruption in the wild type strain as well as constitutive expression in the crg1 mutant. Disruption of ATR1 did not affect growth or sporulation. Efforts to transform ATR1 into plants are currently in progress. Silencing protocols were developed using two different fungal silencing vectors. Genes encoding homologues to an ABC transporter, an alcohol dehydrogenase, an alcohol monooxygenase, a monophenol monooxygenase, a succinate seminalde dehydrogenase, a quinine oxidoreductase, and a hypothetical protein were successfully silenced. Silencing of the ABC transporter and quinine oxidoreductase decreased cercosporin resistance, although changes were small. Gene expression studies showed that silencing is affected by medium composition and temperature, confounding analysis of gene function. The subtractive library genes were also characterized for induction by cercosporin using a cercosporin-deficient mutant generated by targeted gene disruption of the polyketide synthase (CTB1) responsible for the initial steps in biosynthesis. Both the ABC transporter and quinine oxidoreductase were induced by cercosporin, consistent with the silencing data suggesting a role in cercosporin resistance. The research conducted through this proposal is advancing our understanding of the mechanisms used by Cercospora fungi to parasitize their hosts, and confirms earlier studies suggesting a central role for transporters and reductases. The project has led to the identification of the ATR1 gene currently being investigated as a resistance gene in plants. Engineering resistance will enhance breeding efforts to reduce the current dependence on use of fungicides to protect against Cercospora diseases.
Publications
- Herrero, S., Amnuaykanjanasin, A., and Daub, M. E. 2007. Identification of genes differentially expressed in the phytopathogenic fungus Cercospora nicotianae between cercosporin toxin-resistant and -susceptible strains. FEMS Microbiol. Lett. 275:326-337.
- Daub, M. E., and Chung, K. R. 2009. Photoactivated perylenequinone toxins in plant pathogenesis. Chapt. 11 in: The Mycota V, Plant Relationships, 2nd Edition. H. Deising, Ed. Springer-Verlag, Berlin Heidelberg.
- Daub, M. E., Herrero, S., and Taylor, T. V. 2010. Strategies for the development of resistance to cercosporin, a toxin produced by Cercospora species. Chapter 14 in: Cercospora Leaf Spot of Sugar Beet, APS Press, Minneapolis (In Press).
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: The goal of this proposal is to identify genes involved in resistance of plant pathogenic Cercospora fungi to the active-oxygen generating toxin cercosporin which they produce for parasitism of plants. This goal was approached by characterizing genes regulated by a regulatory protein (CRG1) which was identified based on its requirement for normal expression of cercosporin resistance as well as for normal production of the toxin. In previous work, we identified genes that were regulated by CRG1. Recovered genes included ones that code for mechanisms that have been shown to be important in cercosporin resistance, including genes encoding membrane transporters and processes associated with reducing reactions in cells. Microscopy analysis suggested that crg1 mutants were deficient in exporting cercosporin out of the cell, thus we initially focused on genes encoding major facilitator superfamily (MFS) and ATP-binding cassette (ABC) transporters whose expression was decreased in the crg1 mutant. The role of transporters in cercosporin resistance was assayed by both overexpression in the crg1 mutant and by either targeted gene disruption or by gene silencing in the wild type strain. Using these approaches, we showed that one MFS transporter (MFS2) plays no role in cercosporin resistance, but another MFS transporter (CFP) and two ABC (ATR1, ATR2) transporters all contribute to resistance. All three provided moderately enhanced cercosporin resistance when constitutively expressed in the crg1 mutant, however, none were able to restore resistance to wild type levels indicating that other gene(s) are required to restore full resistance to the crg1 mutant. Disruption and silencing experiments with ATR1 and ATR2, respectively, in the wild type strain also confirmed that these transporters play a moderate role in cercosporin resistance. These experiments also identified ATR1 as a major transporter involved in export of cercosporin out of cells, as constitutive expression of ATR1 alone was able to restore full cercosporin production to the crg1 mutant, whereas constitutive expression of CFP did not. Other transporters with redundant activities likely exist, however, as atr1 disruption mutants export a small, but measurable amount of cercosporin out of cells. Current efforts are directed at using gene silencing technology to screen for the role of genes that encode proteins involved in reduction reactions to identify additional genes involved in cercosporin resistance. The results of this work have been disseminated through presentations at scientific meetings and through publications. PARTICIPANTS: The principle investigators for this grant are Dr. Margaret Daub and Dr. Sonia Herrero. Dr. Daub is responsible for oversight of the project, planning, and analysis of the work. Dr. Herrero conducted the subtractive hybridization studies and is taking the lead to use gene silencing to investigate the role of reduction proteins in resistance. Dr.Alongkorn Amnuaykanjanasin is a postdoctoral associate supported from this grant who characterized the transporter genes and their role in cercosporin biosynthesis and resistance. Dr. Amnuaykanjanasin is now employed at the National Center for Genetic Engineering and Biotechnology in Thailand, and is continuing to collaborate with us on this project. This grant provided training for Dr. Amnuaykanjanasin and also supports an undergraduate student who works with Dr. Herrero on screening silenced and disrupted strains for cercosporin resistance. TARGET AUDIENCES: Dr. Daub co-teaches an advanced course in Fungal Genetics and Physiology for graduate students at NC State University, which is enhanced by the work supported by this grant. She also teaches a professionalism course for graduate students from diverse curricula that conduct research in biotechnology. She participates in training and outreach activities supported by the Center for Integrated Fungal Research at NC State University. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The research conducted through this proposal is advancing our understanding of the mechanisms used by Cercospora fungi to parasitize their hosts, and has the potential to lead to the development of novel methods of disease control. This work also has the potential to define novel mechanisms used by cells to tolerate reactive oxygen species, general toxicants that play a role in damaging processes in all organisms. The cercosporin toxin has almost universal toxicity to cells of all organisms (plants, animals, fungi, bacteria) due to its production of toxic reactive oxygen species, but the Cercospora fungi are immune to the toxic effects. Genes encoding essential resistance functions may have utility in engineering plants to resist the toxic effects of cercosporin, and thus resist disease. Engineering resistance will enhance breeding efforts to reduce the current dependence on use of fungicides to protect against Cercospora diseases. The putative cercosporin resistance genes that were isolated in this study confirmed earlier reports that suggested that membrane transporters and reduction proteins are central to cercosporin resistance, and allows us to focus on these classes of proteins for further characterization. Also, previous studies had identified two transporters involved in cercosporin biosynthesis, with one of these playing a dual role in resistance. Our current work identified an additional two transporters that share a dual role in cercosporin biosynthesis and resistance. We are beginning to characterize reduction-related proteins for roles in resistance. All of these genes have the potential to be used in genetic engineering efforts to enhance resistance to these important diseases.
Publications
- Amnuaykanjanasin, A., and Daub, M. E. 2009. The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae. Fung. Genet. Biol. (In Press).
- Daub, M. E., and Chung, K. R. 2009. Photoactivated perylenequinone toxins in plant pathogenesis. Chapt. 11 in: The Mycota V, Plant Relationships, 2nd Edition. H. Deising, Ed. Springer-Verlag, Berlin Heidelberg
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Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: The goal of this proposal is to identify genes involved in resistance of Cercospora fungi to the active-oxygen generating toxin cercosporin which they produce for parasitism of plants, by characterizing genes regulated by a transcription factor (CRG1) required for normal expression of cercosporin resistance and for biosynthesis. In our previous report, we reported on the isolation and sequence identification of genes recovered by subtractive hybridization that were differentially regulated between the wild type and a mutant deficient in CRG1. Because the phenotype of the crg1 mutant suggests that it is deficient in transport of cercosporin out of the cell, we have focused on genes encoding major facilitator superfamily (MFS) and ATP-binding cassette (ABC) transporters down-regulated in the crg1 mutant. One of two MFS transporter genes (MFS2) was shown to have no role in cercosporin resistance. A second MFS transporter gene (CFP) provided moderately enhanced cercosporin
resistance when constitutively expressed in the crg1 mutant, however it was unable to restore cercosporin biosynthesis, a result that contrasts to previous reports that CFP is the main cercosporin transporter. In the past year we have cloned and sequenced two ABC transporter genes (ATR1, ATR2) that are down-regulated in the crg1 mutant. ATR1 (ABC Transporter 1) has an ORF of 4,368 bp and one intron of 72 bp and encodes a putative protein of 1,431 amino acids. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis, suggesting that this transporter, and not CFP, is the major transporter responsible for cercosporin transport out of the cell. This conclusion was supported by disruption of ATR1 in the wild type strain where atr1 disruption mutants were significantly reduced in cercosporin biosynthesis. Disruption of ATR1 in the wild type strain significantly reduced growth when disruption mutants were grown on cercosporin-containing medium, and constitutive
expression of ATR1 in the crg1 mutant enhanced resistance to the toxin, suggesting a role for ATR1 also in cercosporin resistance. Cloning and sequencing of ATR2 identified a 3,925 bp ORF with one intron of 58 bp, encoding a putative protein of 1,288 amino acids. Constitutive expression of ATR2 in the crg1 mutant resulted in significantly enhanced resistance to cercosporin , but only slight increases in cercosporin biosynthesis. Efforts to silence ATR2 in the wild type to confirm the overexpression studies are currently in progress. CRG1 shows homology to WAR1, a transcription factor from yeast that affects cellular pH homeostasis. We found that the crg1 mutant shows pH-dependent sensitivity to cercosporin, with highest sensitivity at pH 4-5; wild type was unaffected by pH. Efforts to use fluorescent microscopy and pH-sensitive dyes to assay cytoplasmic pH changes in response to acid were unsuccessful due to dye sequestration and toxicity. Efforts are in progress to use pH-responsive
GFP to monitor the response of the crg1 mutant to acid. These experiments will allow us to study if CRG1 regulates cellular pH homeostasis and in turn, if pH is important for cercosporin resistance.
PARTICIPANTS: The principle investigators for this grant are Dr. Margaret Daub and Dr. Sonia Herrero. Dr. Daub is responsible for oversight of the project, planning, and analysis of the work. Dr. Herrero conducted the subtractive hybridization studies and is taking the lead to characterize the role of pH in cercosporin resistance. Dr.Alongkorn Amnuaykanjanasin is a postdoctoral associate supported from this grant who is characterizing the transporter genes and their role in cercosporin biosynthesis and resistance. Through this grant, Drs. Herrero and Amnuaykanjanasin were both able to present their work at a Fungal Genetics conference in spring 2007.
TARGET AUDIENCES: Dr. Daub co-teaches an advanced course in Fungal Genetics and Physiology for graduate students at NC State University, which is enhanced by the work supported by this grant. She also teaches a professionalism course for graduate students from diverse curricula that conduct research in biotechnology. She participates in training and outreach activities supported by the Center for Integrated Fungal Research at NC State University.
Impacts
The research conducted through this proposal will advance our understanding of the mechanisms used by plant pathogenic fungi to parasitize their hosts, and has the potential to lead to the development of novel methods of disease control. Fungi that produce toxins that damage the cells of their hosts must have biochemical mechanisms to protect themselves against the toxin's damaging activity. This proposal will lead to the identification of genes that are necessary for fungal autoresistance to a toxin essential for plant pathogenesis. If successful, these genes may have utility in engineering crop plants for resistance to damaging fungal pathogens.
Publications
- Herrero, S., Amnuakanjanasin, A., and Daub, M. E. 2007. Identification of genes differentially expressed in the phytopathogenic fungus Cercospora nicotianae between cercosporin toxin-resistant and -susceptible strains. FEMS Microbiol. Lett. 275-236-337.
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Progress 09/01/05 to 09/01/06
Outputs
The goal of this proposal is to identify and characterize genes involved in resistance of Cercospora species to the photoactivated, active-oxygen generating toxin cercosporin which they produce. In previous work, we identified a zinc cluster transcription factor (CRG1) required for normal expression of resistance of Cercospora fungi to cercosporin as well as for normal biosynthesis of cercosporin. By using a subtractive hybridization approach, we recovered transcripts differentially regulated in the wild type and crg1 mutant. To date we have sequenced all of the recovered genes. When comparing the libraries using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database, we determined that the sequences upregulated in the wild type strain were specifically enriched for genes involved in energy metabolism, cofactor and vitamin metabolism, and polyketide metabolism as compared to the library of sequences upregulated in the mutant. We are using quantitative RT-PCR to
confirm differential regulation of transcripts of interest in the wild type and mutant strain. These studies to date indicate that approximately half of the sequences are differentially expressed. The phenotype of the crg1 mutant suggests that it is deficient in transport of cercosporin out of the cell, thus we have focused our initial characterization on two genes that encode major facilitator superfamily (MFS) transporters and four genes that encode ATP-binding cassette (ABC) transporters. All of these genes are significantly down-regulated in the crg1 mutant. By using gene disruption and overexpression experiments, we have eliminated one of the MFS transporter genes (MFS2) as having a defined role in cercosporin resistance or biosynthesis. Complementation of the crg1 mutant with the second MSF transporter gene (CFP) showed moderately enhanced resistance to cercosporin. CFP had previously been described as the transporter responsible for exporting cercosporin out of the mycelium.
Surprisingly, the CFP-complemented crg1 mutants remained unable to synthesize cercosporin. These results suggest either that CFP is not the major transporter or that the crg1 mutant is deficient in other essential biosynthetic genes. We are currently investigating the four ABC transporter genes by gene disruption experiments in the wild type and complementation experiments in the crg1 mutant to define their role in resistance and biosynthesis. In addition, we have discovered that the sensitivity of the crg1 mutant to cercosporin is influenced by pH. We hypothesize that the crg1 mutant may be deficient in its ability to regulate mycelial pH, and that pH regulation is essential for the expression of resistance. We are currently conducting experiments to investigate this hypothesis.
Impacts
The research conducted through this proposal will advance our understanding of the mechanisms used by plant pathogenic fungi to parasitize their hosts, and has the potential to lead to the development of novel methods of disease control. Fungi that produce toxins that damage the cells of their hosts must have biochemical mechanisms to protect themselves against the toxin's damaging activity. This proposal will lead to the identification of genes that are necessary for fungal autoresistance to a toxin essential for plant pathogenesis. If successful, these genes may have utility in engineering crop plants for resistance to damaging fungal pathogens.
Publications
- No publications reported this period
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