Source: AGRICULTURAL RESEARCH SERVICE submitted to
MOLECULAR ANALYSIS OF GENES CONTROLLING PATHOGENESIS OF USTILAGO MAYDIS AND M. GRISEA
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0402945
Grant No.
(N/A)
Project No.
3655-22000-013-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Aug 1, 1999
Project End Date
Jan 27, 2003
Grant Year
(N/A)
Project Director
LEONG S A
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
501 WALNUT STREET
MADISON,WI 53726
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121510104056%
2121510110224%
2121530104020%
Goals / Objectives
Determine the role of iron homeostasis in fungal spore germination and survival in the corn smut pathogen Ustilago maydis, and the molecular bases of fungal pathogenicity, race-plant cultivar specificity and fungal genome evolution in the rice blast pathogen Magnaporthe grisea.
Project Methods
We will isolate and characterize the structural and regulatory genes and gene products involved in ferrichrome siderophore biosynthesis in U. maydis and determine the role of siderophores in the long term survival of U. maydis teliospores and sporidia in the environment. We will clone and characterize the M. grisea cultivar-specificity gene avrCO39 and determine the genetic basis of resistance of rice to M. grisea strains carrying avrCO 39. We will continue to characterize the transposable elements MAGGY, Pot2 and MG-SINE like elements in M. grisea and determine their relationship to pathogenic variation in the fungus. Finally, we will map, clone and characterize the M. grisea gene(s) controlling the fungal adherence/penetration of rice tissue.

Progress 08/01/99 to 01/27/03

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Fungal pathogens of plants are the most devastating plant disease agents. We are studying the molecular biology of host recognition of the model plant pathogenic fungus, Magnaporthe grisea, by rice. This fungus is considered a premier example of an ascomycete fungal plant pathogen, which can be manipulated both genetically and with the tools of molecular biology. Likewise, rice is considered to be the model monocot plant with a small, diploid genome that can be readily modified by transformation. The genomes of both organisms have been sequenced. 2. How serious is the problem? Why does it matter? M. grisea causes rice blast disease, one of the most devastating diseases of rice worldwide. In addition, different subspecific groups of M. grisea are known to attack other important cereals and grasses such as wheat causing wheat blast and turf causing grey leaf spot. Recent studies have shown that similar genes are needed for infection of different host plants by Magnaporthe. Thus we can anticipate that information that is broadly applicable to fungal pathogenesis of grasses will be obtained through studies of this model fungus. The evolutionary relationship of fungal host and cultivar specificity genes and their corresponding disease resistance loci is currently unknown and may be exploited to develop new resistance specificities using transgenic and wide hybridization technologies. Usilago causes corn smut disease, a disease which is no longer a major threat to agricultural production in the U.S. as a result of an active breeding effort to maintain disease resistance in commercial varieties. While U. maydis itself is not an economic threat, other related smut and bunt fungi of cereals are serious problems. Basic studies of plant pathogenesis have already revealed similarities in the mating type control system of U. maydis and Ustilago hordei which controls the abilility of these fungi to cause plant disease. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 303, Plant Diseases (100%) This research allows scientists and other customers to better understand mechanisms of plant and field survival and develop new methods for control of these fungi. Approaches for durable resistance to rice blast disease may be gained. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2003: Transcription of the AVR1-CO39 locus of M. grisea was examined; the predicted protein product of the transcript that was discovered was expressed in blast resistant (varieties Drew and CO39) and susceptible (varieties Nipponbare and M201) cells of rice leaves in our laboratory. The resistant plant cells appeared to die when this fungal gene was expressed in them while the susceptible cells were unaffected. This result is consistent with the activation of plant defense response by the fungal protein after its recognition by the resistant plant cell. The availability of this fungal gene will enable us to explore methods to engineer rice plants to resist fungal infection. B. Other significant accomplishment(s), if any: Approximately 80 kb of additional DNA was sequenced and annotated from the Pi-CO39 (t) locus of rice resistant variety CO39 by our laboratory. This completes the DNA sequence analysis of the locus within flanking genetic markers of 0.2 cM. Additional NBS-LRR, protein kinase and serpin genes were found within the sequenced DNA. One or more of these genes may be responsible for conferring blast disease resistance associated with the Pi-CO30 (t) locus. Rice callus culture of susceptible California variety M201 which is suitable for transformation has been established by our laboratory and collaborator Chakradhar Akula, University of Wisconsin. This will allow assessment of which genomic clone from resistant variety CO39 contains the resistance gene. Perennial rye callus culture suitable for transformation has been established by collaborator Mark Farman at the University of Kentucky and a material transfer agreement has been signed for transfer of the cloned genomic DNA containing the rice resistance locus Pi-CO39 (t). The clones have been transmitted to him for transformation. This will allow assessment of which genomic clone from resistant variety CO39 contains the resistance gene and will lead to the development of transgenic perennial rye with major gene resistance to grey leaf spot. Analysis of the Nipponbare genome sequence surrounding a marker tightly linked to blast resistance gene Pi-Kh identified a cluster of NBS-LRR genes by ARS scientist, Robert Fjellstrom, USDA-ARS, Rice Research Unit, Beaumont, Texas. The region has been scanned for additional markers for use in the identification of cosegregating markers. C. Significant activities that support special target populations: None. D. Progress report: Using GFP (green fluorescent protein), rice phytoene desaturase and candidate rice resistance gene COR8 of variety CO39 as test genes, conditions have been standardized for bombardment of rice leaves with gene constructs to express and silence genes in our laboratory. GFP was successfully silenced when coexpressed with the GFP gene silencing construct; however, it was not possible to effectively silence the endogenous phytoene desaturase and COR8 genes. This result underscores the potential problems that may be encountered with transient silencing of endogenous, constitutively active genes and points to the need to provide adequate time for silencing and turnover of the endogenous target protein before phenotypic assay. This approach will be used to establish which candidate resistance gene is functionally important in the recognition of AVR1-CO39 and will provide information needed to develop novel kinds of blast resistant plants. This is research conducted under a specific cooperative agreement between ARS and the University of Wisconsin, Agreement Number 58-3655-2-0103. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The most effective method for control of rice blast is to grow disease resistant plants. Unfortunately, M. grisea is able to overcome this resistance within 1-3 years after resistant plants are cultivated widely. We are trying to understand the molecular details of how the rice blast fungus is recognized by rice plants that are resistant to blast and how the fungus changes in order to overcome this recognition. In order to improve our understanding of this host-parasite interaction, we have cloned a pathogen gene AVR1-C039 that is involved in recognition of the pathogen by the resistant rice plant. This is the second AVR (AviRulence) gene to be cloned from the rice blast fungus. We employed a combination of physical, genetic and molecular genetic methods to obtain the cloned gene. These methods included our laboratory's high density genetic map and molecular karyotype, targeted genome cleavage methods, and a transformation system we developed for M. grisea that is based on drug resistance. Considerable insight was gained about the map-based cloning approaches through this effort. These approaches and insights will likely have broad application to other organisms such as the cereal rusts in which map-based cloning approaches are being used to clone similar genes. In parallel work, we have mapped and cloned the corresponding disease resistance locus Pi-CO39 (t), which allows the host to recognize strains of the fungus that carry AVR1-CO39. This work has shown that resistance is controlled by a dominant locus mapping to chromosome 11 of rice. The complete DNA sequence of the locus from two rice genotypes has been completed, annotated, and analyzed for transcriptional activity. Annotation of 416 kb of DNA sequence from rice variety Nipponbare and 144 kb from variety CO39 linked to Pi-CO39(t) locus, that contains the putative receptor for the M. grisea avirulence gene AVR1-CO39, revealed the presence of several NBS-LRR and Serpin genes in both haplotypes. Our long term goal is to study the molecular biology of the interaction of Avr1-CO39p and the host resistance gene product and/or other host products in order to understand when, where and how the fungus communicates its presence to the host. Based on this information, we hope to develop new strategies for engineering novel kinds of broad- spectrum resistance to M. grisea in rice and other cereals and grasses of economic and ornamental value. Studies on the mating type control system and high affinity iron transport system of U. maydis were conducted to understand how these systems contribute to pathogenesis of this smut fungus. Smut fungi must mate in order to cause plant infection. The mating type loci a and b were cloned and partially characterized. Both loci were shown to be important to pathogenicity through analysis of knock-out mutations. The b locus was further shown to encode a putative transcription factor belonging to the homeodomain family. Different alleles of b showed predicted variable amino acid sequences in their N-terminal domains with a conserved C- terminal domain. Homologs of the a and b loci were identified in other smut fungi through hybridization analysis. This work has been etended by a former postdoctoral researcher of the lab and was discontinued in my laboratory. Studies of high affinity iron transport in U. maydis have resulted in defining through gene cloning for the first time an understanding of the molecular biology of biosynthesis and regulation of biosynthesis of siderophores (microbial iron transport agents) by fungi. In response to iron starvation, we discovered that the U. maydis produces two cyclic hydroxamate siderophores ferrichrome and ferrichrome A. Three genes required for siderophore biosynthesis and regulation have been cloned and characterized: sid1 encodes ornithine-N5-oxygenase, the first enzyme in the ferrichrome biosynthetic pathway; sid2 encodes a putative nonribomal peptide synthetase required for ferrichrome biosynthesis; and urbs1 encodes a GATA family transcription repressor of sid1/sid2. Current efforts are focused on developing an understanding of how iron homeostasis is maintained by Urbs1. urbs1 mutants secrete siderophores constitutively and hyperaccumulate iron and siderophores intracellularly. urbs1 mutants grow poorly when compared to wild type. The process of iron regulation of iron uptake is still poorly understood and the full extent of the function of urbs1 in cells in unknown. Fur, the analog of urbs1 in many bacteria also controls adaptation to low pH and ability to grow on alternate carbon sources such as succinate. We have initiated a new mutant hunt to look for mutants degregulated in iron uptake in order to better understand how iron uptake is regulated and if other genes in addition to urbs1 are required. We are also studying the molecular details of the interaction of urbs1 with the promoter (regulatory) region of sid1/sid2. We have demonstrated that urbs1 binds specifically to GATA sequences in these promoters and that the DNA undergoes dramatic changes in chromatin organization as a function of iron availability to the cell consistent with derepression of these genes. In other microbes as well as plants and man, iron overload is toxic. Furthermore, mammals restrict infection by bacteria through a system referred to as nutritional immunity whereby iron is mobilized to tissue stores thus making it less available to invading microbes. The comparative analysis of the genome sequences of a laboratory strain of Eshcerichia coli K-12 with that of other highly pathogenic strains of E. coli such as 0157 have revealed a propensity of iron acquisition systems and virulence genes known to be regulated by the iron deficiency that is encountered in the mammalian host. In at least one plant pathogen Erwinia chrysanthemi, siderophore systems have been shown to be required for systemic infection of a plant host. However, our limited studies to date in an in vitro system using corn seedlings grown in a growth chamber did not reveal a significant role for sid1 or urbs1 in pathogenicity. Based on the confounding results that Drs. C. Upper and K. Willis have obtained for pathogencity phenotypes of mutants of Pseudomonas inoculated on plants in the field and growth chamber, it would not be prudent to firmly conclude that these two siderophore genes are not essential for pathogencity. The ability of fungal pathogens to acquire iron from the host or its environment may influence spore viability, spore germination, colonization of a habitat as well as virulence. Siderophores are germination factors in many fungi including Ustilago spp. Candida albicans was recently shown to require high affinity iron transport systems to be pathogenic. The field survival and field infectivity of these mutants needs to be studied once an appropriate method is developed for "natural" inoculation of corn with Ustilago. Ongoing work at the University of Minnesota should provide this needed background. The role of siderophore genes in the infection of mammalian hosts has been spurred by our pioneering work in Ustilago. Analogs of urbs1 have been isolated from several other fungi based on the conservation of the DNA binding domain. It is very likely that fungal siderophore genes will be important in an animal model such as mouse and we are anxiously awaiting the results of ongoing work in other labs in Aspergillus fumigatus and Histoplasma capsulata. This opens up the possibility of exploiting siderophore/iron uptake genes as a means of controlling infection in farm animals and man. Three approaches are immediately evident. One is based on an ongoing collaboration with Drs. M. Miller at Notre Dame and H. Von Dohren at the Technical University of Berlin. The approach is to develop novel drug congugates based on siderophores which can be illicitly transported into target cells via the siderophore uptake system. The concept of illicit transport may extend the life of a drug by delivering it through an uptake system for an essential nutrient iron. Dr. Miller's synthetic chemistry work has already demonstrated the potential of several model compounds which are based on the U. maydis ferrichrome siderophore structure for control of Cryptococcus neoformans. Our work with Dr. Von Dohren was aimed at developing a system of overexpression of the enzymes encoded by the sid genes in order to facilitate the development of a biotransformation system to produce precursors needed for large scale production of these drug conjugates. The second approach makes use of the observation that all fungi with the exception of the Zygomycetes, produce delta-N hydroxyornithine-containing. Thus the sid1 gene should be rather ubiquitous among fungi and may be a useful target for drug development if siderophores are found to be virulence factors of fungi and yeasts. The third approach for control of fungi could take advantage of knowledge of the 3-D structure and metal requirement of the DNA binding domain of urbs1 which, based on structural information from other GATA family members, will have a finger structure comprised of zinc or iron tetrahedrally coordinated via four cysteines. For example, anti-HIV agents have been developed that covalently modify the metal-coordinating cysteine thiolates of the retrovial nucleocapsid protein fingers causing metal (zinc) ejection thus disrupting virus replication. These agents did not affect function of other metal-dependent transcription factors including Sp1 or Gata-1, a GATA family transcription factor. By analogy, it may be possible to design chemical agents that can selectively remove metals from the fingers of fungal GATA factors such as urbs1 but not those of the plant or animal host. As a prelude to the work described above, we developed the first transformation system available for U. maydis and pioneered other methods such as electrophoretic karyotyping, gene replacement, gene disruption, and gap repair which are widely used in yeast. This work has been discontinued in order to focus efforts on both host and pathogen in the rice blast system. 6. What do you expect to accomplish, year by year, over the next 3 years? This is the final Report of Progress for this project. Expected accomplishments for the next three FYs are shown in the replacement project, 3655-22000-015-00D. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Cloned genomic DNA of rice variety CO39 that contains the Pi-CO39 (t) locus was transferred for development of perennial rye with major gene resistance to grey leaf spot, a serious problem on turf in the U.S. and Japan. No major gene resistance to this disease is present in perennial rye. M. grisea isolates that infect perennial rye contain the corresponding AVR1-CO39 gene.

Impacts
(N/A)

Publications

  • Leong, S.A., Chauhan, R.S, Lazaro, D. Nipponbare BAC OSJNBa0044D15 DNA sequence, 144544 bp. Genbank. 2002. AC119072. Available from: http://www. ncbi.nlm.nih.gov/Genbank/
  • Leong, S.A., Chauhan, R.S, Lazaro, D. Nipponbare BAC OSJNBa0073N20 DNA sequence, 147289 bp. Genbank. 2002. AC119072. Available from: http://www. ncbi.nlm.nih.gov/Genbank/
  • Leong, S.A., Chauhan, R.S, Lazaro, D. Nipponbare BAC OSJNBa0082N20 DNA sequence, 138826 bp. Genbank. 2002. AC119073. Available from: http://www. ncbi.nlm.nih.gov/Genbank/
  • Leong, S.A., Chauhan, R.S., Farman, M.L., Eto, Y., Durfee, T., Punekar, N. S., Lazaro, D., Nogawa, M., Tosa, Y., Blattner, F., Ronald, P., Zhang, H.- B., Mayama, S., Nakayashi, H. Comparative Genomics of the Magnaporthe grisea AVR1-CO39 Locus and Its Corresponding Resistance Locus Pi-CO39 (t). Rice Technical Working Group. 2002. Abstract p. 11.
  • Farman, M. L., Eto, Y., Nakao, Y., Tosa, Y., Nakayashiki, H., Mayama, S., Leong, S.A,. Analysis of the structure of the AVR1 CO39 avirulence locus in virulent rice-infecting isolates of Magnaporthe grisea. Molecular Plant- Microbe Interactions. 2002. v. 15. p. 6-16.
  • Chauhan, R.S., Leong, S.A. Genomics of Disease Resistance Loci in Cereals. Recent Research Developments in Genetics. 2002. v. 2. p. 1-29.


Progress 10/01/01 to 09/30/02

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Fungal pathogens of plants are the most devastating plant disease agents. We are studying the molecular biology of plant pathogenesis of two model plant pathogenic fungi, Ustilago maydis and Magnaporthe grisea. These fungi represent two of the major classes of phytopathogenic fungi, basidiomycetes and ascomycetes, and are considered the premier examples of fungal plant pathogens which can be manipulated both genetically and with the tools of molecular biology. 2. How serious is the problem? Why does it matter? Usilago causes corn smut disease, a disease which is no longer a major threat to agricultural production in the United States as a result of an active breeding effort to maintain disease resistance in commercial varieties. While U. maydis itself is not an economic threat, other related smut and bunt fungi of cereals are serious problems. Basic studies of plant pathogenesis have already revealed similarities in the mating type control system of U. maydis and Ustilago hordei which controls the abilility of these fungi to cause plant disease. M. grisea causes rice blast disease, one of the most devastating diseases of rice worldwide. In addition, different subspecific groups of M. grisea are known to attack other important cereals and grasses such as wheat causing wheat blast and turf causing grey spot. Recent studies have shown that similar genes are needed for infection of different host plants by Magnaporthe. Thus we can anticipate that information that is broadly applicable to fungal pathogenesis will be obtained through studies of these two model fungi. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program 303, Plant Diseases - This research allows scientists and other customers to better understand mechanisms of plant pathogenicity and field survival and develop new methods for control of these fungi. Approaches for durable resistance to rice blast disease may be gained. 4. What was your most significant accomplishment this past year? A. Single most significant accomplishment during FY 2002: Annotation of 416 kb of DNA sequence from rice variety Nipponbare and 144 kb from variety CO39 linked to Pi-CO39(t) locus, that contains the putative receptor for the M. grisea avirulence gene AVR1-CO39, revealed the presence of several NBS-LRR and Serpin genes in both haplotypes. Seventeen NBS-LRR genes (Nipponbare NBR enes) and four NBS-LRR genes (CO39 CODR genes) were identified in the contiguous equenced regions, respectively. Two orthologous NBS-LRR gene pairs, CODR1 (CO39) and NBR11 (Nipponbare) and CODR2 (CO39) and NBR6 (Nipponbare) are highly identical (>93%), except for deletions of two LRR units in NBR6 and NBR11 compared to CODR2 and CODR1, respectively. Whether these LRR units are determinants of recognition specificity remains to be seen. Comparative analysis of LRR units from selected NBS-LRR genes from both haplotypes revealed that the consensus motif XXLXLXX is conserved in all the LRRs compared to the flanking residues, which is in contrast to analysis done on other NBS-LRR genes, wherein the XXLXLXX motif is highly diverged and predicted to interact with the pathogen ligands. Total RNA was isolated from Nipponbare and CO39 rice leaves inoculated with Guy11 and Guy11 AVR1-CO39 transformant and harvested at different time points (0, 3, 6, 12, 24, 36, 48, 96, 7d). RT-PCR was performed on these RNA templates using gene specific primers designed from NBS and LRR domains of NBS-LRR genes predicted in Nipponbare and CO39 haplotypes at Pi-CO39(t) locus. All the predicted genes are constitutively expressed in both genotypes, except RPR1 gene in Nipponbare, which showed induced expression in response to M. grisea infection. Two serpin genes in CO39 and one in Nipponbare showed induced expression in response to M. grisea infection. Genomic DNA of two resistant rice varieties, CO39 and Drew, and two susceptible lines, Nipponbare and 51583, was digested with HpaII and Msp1 and Southern blots were probed with NBS-LRR gene-specific probes from Nipponbare and CO39 haplotypes to examine the methylation state of the DNA. NBR7 or its ortholog is methylated only in resistant rice lines, CO39 and Drew, both at seedling and adult plant stages, whereas unmethylated in the susceptible genomes. However, Nipponbare and Drew homologues of NBR7 are 99.4% identical in nucleotide sequence. Both homologues differ only for four amino acid residues out of which three are in the LRR domain. NBR16 is methylated in Nipponbare, 51583 and Drew. NBR16 seems to be either deleted or highly rearranged in CO39 genome. Conditions have been standardized for initiating callus cultures from mature seeds of japonica rice, M202, for using in particle bombardment as well as Agrobacterium-mediated transformation for complementation analysis of genes present in CO39 BAC clones. Sufficient numbers of calli are being established to pursue this work. B. Other significant accomplishment(s), if any: In collaboration with David Schwartz, a genome-wide optical map of rice variety Nipponbare having 13.5X coverage has been prepared and alignment of publically available DNA sequence of Nipponbare is being done. Excellent alignment of the 412 Kb of Nipponbare sequence generated at the Pi-CO39 (t) locus was observed. As part of the International Rice Genome Sequencing Project, the DNA sequence of 3 BAC clones of Nipponbare DNA from chromosome 11 at the Pi- CO39 (t) locus have been determined to an error rate of less than 1 per 10,000 bp and deposited in Genbank. A method for overexpression of open reading frame 3 of AVR1-CO39 using the pET system in Escherichia coli was established. C. Significant accomplishments/activities that support special target populations: none. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? The most effective method for control of rice blast is to grow disease resistant plants. Unfortunately, M. grisea is able to overcome this resistance within 1-3 years after resistant plants are cultivated widely. We are trying to understand the molecular details of how the rice blast fungus is recognized by rice plants that are resistant to blast and how the fungus changes in order to overcome this recognition. In order to improve our understanding of this host-parasite interaction, we have cloned a pathogen gene AVR1-C039 that is involved in recognition of the pathogen by the resistant rice plant. This is the second AVR (AviRulence) gene to be cloned from the rice blast fungus. We employed a combination of physical, genetic and molecular genetic methods to obtain the cloned gene. These methods included our laboratory's high density genetic map and molecular karyotype, targeted genome cleavage methods, and a transformation system we developed for M. grisea that is based on drug resistance. Considerable insight was gained about the map- based cloning approaches through this effort. These approaches and insights will likely have broad application to other organisms such as the cereal rusts in which map-based cloning approaches are being used to clone similar genes. In parallel work, we have mapped and cloned the the corresponding disease resistance locus Pi-CO39 (t) which allows the host to recognize strains of the fungus that carry AVR1-CO39. This work has shown that resistance is conferred by a dominant gene mapping to chromosome 11 of rice. Our long term goal is to study the molecular biology of the interaction of Avr1-CO39p and the host resistance gene product and/or other host products in order to understand when, where and how the fungus communicates its presence to the host. Based on this information, we hope to develop new strategies for engineering novel kinds of broad-spectrum resistance to M. grisea in rice and other cereals and grasses of economic and ornamental value. Studies on the mating type control system and high affinity iron transport system of U. maydis were conducted to understand how these systems contribute to pathogenesis of this smut fungus. Smut fungi must mate in order to cause plant infection. The mating type loci a and b were cloned and partially characterized. Both loci were shown to be important to pathogenicity through analysis of knock-out mutations. The b locus was further shown to encode a putative transcription factor belonging to the homeodomain family. Different alleles of b showed predicted variable amino acid sequences in their N- terminal domains with a conserved C-terminal domain. Homologs of the a and b loci were identified in other smut fungi through hybridization analysis. This work has been extended by a former postdoctoral researcher of the lab and was discontinued in my laboratory. Studies of high affinity iron transport in U. maydis have resulted in defining through gene cloning for the first time an understanding of the molecular biology of biosynthesis and regulation of biosynthesis of siderophores (microbial iron transport agents) by fungi. In response to iron starvation, we discovered that the U. maydis produces two cyclic hydroxamate siderophores ferrichrome and ferrichrome A. Three genes required for siderophore biosynthesis and regulation have been cloned and characterized: sid1 encodes ornithine-N5-oxygenase, the first enzyme in the ferrichrome biosynthetic pathway; sid2 encodes a putative nonribomal peptide synthetase required for ferrichrome biosynthesis; and urbs1 encodes a GATA family transcription repressor of sid1/sid2. Current efforts are focused on developing an understanding of how iron homeostasis is maintained by Urbs1. urbs1 mutants secrete siderophores constitutively and hyperaccumulate iron and siderophores intracellularly. urbs1 mutants grow poorly when compared to wild type. The process of iron regulation of iron uptake is still poorly understood and the full extent of the function of urbs1 in cells in unknown. Fur, the analog of urbs1 in many bacteria also controls adaptation to low pH and ability to grow on alternate carbon sources such as succinate. We have initiated a new mutant hunt to look for mutants degregulated in iron uptake in order to better understand how iron uptake is regulated and if other genes in addition to urbs1 are required. We are also studying the molecular details of the interaction of urbs1 with the promoter (regulatory) region of sid1/sid2. We have demonstrated that urbs1 binds specifically to GATA sequences in these promoters and that the DNA undergoes dramatic changes in chromatin organization as a function of iron availability to the cell consistent with derepression of these genes. In other microbes as well as plants and man, iron overload is toxic. Furthermore, mammals restrict infection by bacteria through a system referred to as nutritional immunity whereby iron is mobilized to tissue stores thus making it less available to invading microbes. The comparative analysis of the genome sequences of a laboratory strain of Eshcerichia coli K-12 with that of other highly pathogenic strains of E. coli such as 0157 have revealed a propensity of iron acquisition systems and virulence genes known to be regulated by the iron deficiency that is encountered in the mammalian host. In at least one plant pathogen Erwinia chrysanthemi, siderophore systems have been shown to be required for systemic infection of a plant host. However, our limited studies to date in an in vitro system using corn seedlings grown in a growth chamber did not reveal a significant role for sid1 or urbs1 in pathogenicity. Based on the confounding results that Drs. C. Upper and K. Willis have obtained for pathogencity phenotypes of mutants of Pseudomonas inoculated on plants in the field and growth chamber, it would not be prudent to firmly conclude that these two siderophore genes are not essential for pathogencity. The ability of fungal pathogens to acquire iron from the host or its environment may influence spore viability, spore germination, colonization of a habitat as well as virulence. Siderophores are germination factors in many fungi including Ustilago spp. Candida albicans was recently shown to require high affinity iron transport systems to be pathogenic. We plan to assess the field survival and field infectivity of these mutants once an appropriate method is developed for "natural" inoculation of corn with Ustilago. Ongoing work at the University of Minnesota should provide this needed background. The role of siderophore genes in the infection of mammalian hosts has been spurred by our pioneering work in Ustilago. Analogs of urbs1 have been isolated from several other fungi based on the conservation of the DNA binding domain. It is very likely that fungal siderophore genes will be important in an animal model such as mouse and we are anxiously awaiting the results of ongoing work in other labs in Aspergillus fumigatus and Histoplasma capsulata. This opens up the possibility of exploiting siderophore/iron uptake genes as a means of controlling infection in farm animals and man. Three approaches are immediately evident. One is based on an ongoing collaboration with Drs. M. Miller at Notre Dame and H. Von Dohren at the Technical University of Berlin. The approach is to develop novel drug congugates based on siderophores which can be illicitly transported into target cells via the siderophore uptake system. The concept of illicit transport may extend the life of a drug by delivering it through an uptake system for an essential nutrient iron. Dr. Miller's synthetic chemistry work has already demonstrated the potential of several model compounds which are based on the U. maydis ferrichrome siderophore structure for control of Cryptococcus neoformans. Our work with Dr. Von Dohren aims at developing a system of overexpression of the enzymes encoded by the sid genes in order to facilitate the development of a biotransformation system to produce precursors needed for large scale production of these drug conjugates. Currently we are studying the sid2-encoded peptide synthetase. The second approach makes use of the observation that all fungi with the exception of the Zygomycetes, produce delta-N-hydroxyornithine-containing. Thus the sid1 gene should be rather ubiquitous among fungi and may be a useful target for drug development if siderophores are found to be virulence factors of fungi and yeasts. The third approach for control of fungi could take advantage of knowledge of the 3-D structure and metal requirement of the DNA binding domain of urbs1 which, based on structural information from other GATA family members, will have a finger structure comprised of zinc or iron tetrahedrally coordinated via four cysteines. For example, anti-HIV agents have been developed that covalently modify the metal-coordinating cysteine thiolates of the retrovial nucleocapsid protein fingers causing metal (zinc) ejection thus disrupting virus replication. These agents did not affect function of other metal-dependent transcription factors including Sp1 or Gata-1, a GATA family transcription factor. By analogy, it may be possible to design chemical agents that can selectively remove metals from the fingers of fungal GATA factors such as urbs1 but not those of the plant or animal host. Toward this end we have established working collaborations with several structural biologists on the University of Wisconsin campus in order to study the structure and metal coordination of urbs1 and its DNA binding domain by EXAFS, NMR, and EPR. Our current efforts are focused on producing sufficient quantities of biologically active protein for this work. As a prelude to the work described above, we developed the first transformation system available for U. maydis and pioneered other methods such as electrophoretic karyotyping, gene replacement, gene disruption, and gap repair which are widely used in yeast. 6. What do you expect to accomplish, year by year, over the next 3 years? Our goals for the next year include the completion of the analysis of the AVR1-CO39 to define its transcript and the regulation of this transcript and to demonstrate through specific disruption of this transcript that a virulent strain can be generated when inoculated on rice cultivar CO39. Restoration of gene function in these null mutants will be investigated through transformation with the wild type gene and the cDNA expressed with heterologous promoters. Methods for heterologous overexpression of this transcript/gene product will be used in infection studies of cultivar CO39 with a virulent isolate to see if the peptide can protect plants. The presence of a calcium-binding site in the AVR1- CO39 peptide will be investigated. The function of candidate disease resistance genes found at the Pi-CO39 locus will be assessed through transformation of susceptible rice to resistance. In collaboration with Japanese colleagues at the University of Kyoto, homologs of AVR1-CO39 from rice and Setaria-infecting isolates of M. grisea will be transformed into rice-infecting isolates that are virulent on CO39. The genes will be disrupted in selected Setaria isolates and their infection on Setaria spp. and rice with or without the corresponding known rice resistance will be done. In collaboration with Drs. S. Hittalmani and K. Devos, we will investigate the genetic basis of blast resistance and drought tolerance in Eleucine coracana. Characterization of of AVR1-CO39 and Pi-CO39 homologs in fingermillet-infecting isolates of M. grisea, respectively, and their host will be made. Isolation of SNPs for grass genome mapping using oligonucletide microarray studies will be initiated. A method for isolating iron regulatory mutants based on iron-dependent auxotrophy will be implemented and mutants will be classified phenotypically and genetically. In collaboration with Dr. H. Von Dohren, the enzymatic activity of sid2 will be defined and discussions with industry will begin in order to obtain funding for our proposed biotransformation studies to generate siderophore-drug conjugates. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? A provisional international patent application was submitted (10/01): Leong, S.A., Chauhan,R.S., Farman, M.F., Durfee, T. "Plant Genes That ConferResistance to Strains of Magnaporthe Grisea Having Avr1 C039 Cultivar Specificity Gene."

Impacts
(N/A)

Publications

  • Chauhan, R., Durfee, T., Holt, J., Blattner, F., Leong, S.A. 2002. Sequence for BAC OSJNBa0073N20 from Rice Variety Nipponbare to GenBank. Genbank. Available at http://www.ncbi.nlm.nih.gov/nuccore/20279524.
  • Chauhan, R., Durfee, T., Holt, J., Blattner, F., Leong, S.A. 2002. Sequence for BAC OSJNBA0082N40 from Rice Variety Nipponbare to Genbank. Genbank. Available at http://www.ncbi.nlm.nih.gov/nuccore/20279525.
  • Chaunhan, R., Durfee, T., Holt, J., Blattner, F., Leong, S.A. 2002. Sequence for BAC OSJNBa0044D15 from Rice Variety Nipponbare GenBank. Genbank. Available at: http://www.ncbi.nlm.nih.gov/nuccore/20279523.
  • Chauhan, R.S., Leong, S.A. 2002. Genomics of Disease Resistance Loci in Cereals. In: Recent Research Developments in Genetics 2:1-29.
  • Leong, S.A., Allen, C.A., Triplett, E.W. (Editors). 2002. Biology of Plant-Microbe Interactions. St. Paul, MN: APS Press. 360 p.
  • Farman, M.L., Eto, Y., Tosa, Y., Nakayashiki, H., Mayama, S., Leong, S.A. 2002. Analysis of the Structure of the AVR1-CO39 Avirulence Locus in Virulent Rice-Infecting Isolates of Magnaporthe grisea. Molecular Plant-Microbe Interactions. 15:6-16.
  • Chauhan, R., Farman, M., Zhang, H.B., Leong, S.A. 2002. Genetic and Physical Mapping of a Rice Blast Resistance Locus, PiCO39(t), that Corresponds to the Avirulence Gene AVR1-CO39 of Magnaporthe grisea. Molecular Genetics and Genomics. 267:603-612.
  • Leong, S.A., Chauhan, R.S, Lazaro, D. Nipponbare BAC OSJNBa0044D15 DNA sequence, 144544 bp. Genbank. 2002. AC119072. Available from: http://www.ncbi.nlm.nih.gov/Genbank/
  • Leong, S.A., Chauhan, R.S, Lazaro, D. Nipponbare BAC OSJNBa0073N20 DNA sequence, 147289 bp. Genbank. 2002. AC119072. Available from: http://www.ncbi.nlm.nih.gov/Genbank/
  • Leong, S.A., Chauhan, R.S, Lazaro, D. Nipponbare BAC OSJNBa0082N20 DNA sequence, 138826 bp. Genbank. 2002. AC119073. Available from: http://www.ncbi.nlm.nih.gov/Genbank/
  • Leong, S.A., Chauhan, R.S., Farman, M.L., Eto, Y., Durfee, T., Punekar, N.S., Lazaro, D., Nogawa, M., Tosa, Y., Blattner, F., Ronald, P., Zhang, H.-B., Mayama, S., Nakayashi, H. Comparative Genomics of the Magnaporthe grisea AVR1-CO39 Locus and Its Corresponding Resistance Locus Pi-CO39 (t). Rice Technical Working Group. 2002. Abstract p. 11.
  • Farman, M. L., Eto, Y., Nakao, Y., Tosa, Y., Nakayashiki, H., Mayama, S., Leong, S.A,. Analysis of the structure of the AVR1 CO39 avirulence locus in virulent rice-infecting isolates of Magnaporthe grisea. Molecular Plant-Microbe Interactions. 2002. v. 15. p. 6-16.
  • Chauhan, R.S., Leong, S.A. Genomics of Disease Resistance Loci in Cereals. Recent Research Developments in Genetics. 2002. v. 2. p. 1-29.


Progress 10/01/00 to 09/30/01

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Fungal pathogens of plants are the most devasting plant disease agents. We are studying the molecular biology of plant pathogenesis of two model plant pathogenic fungi, Ustilago maydis and Magnaporthe grisea. These fungi represent two of the major classes of phytopathogenic fungi, basidiomycetes and ascomycetes, and are considered the premier examples of fungal plant pathogens which can be manipulated both genetically and with the tools of molecular biology. 2. How serious is the problem? Why does it matter? Ustilago causes corn smut disease, a disease which is no longer a major threat to agricultural production in the United States as a result of an active breeding effort to maintain disease resistance in commercial varieties. While U. maydis itself is not an economic threat, other related smut and bunt fungi of cereals are serious problems. Basic studies of plant pathogenesis have already revealed similarities in the mating type control system of U. maydis and Ustilago hordei which controls the ability of these fungi to cause plant disease. M. grisea causes rice blast disease, one of the most devastating diseases of rice worldwide. In addition, different subspecific groups of M. grisea are known to attack other important cereals and grasses such as wheat causing wheat blast and turf causing grey spot. Recent studies have shown that similar genes are needed for infection of different host plants by Magnaporthe. Thus we can anticipate that information that is broadly applicable to fungal pathogenesis will be obtained through studies of these two model fungi. 3. How does it relate to the National Program(s) and National Component(s)? This research allows scientists and other customers to better understand mechanisms of plant pathogenicity and field survival and develop new methods for control of these fungi. Approaches for durable resistance to rice blast disease may be gained. All of these are key components of National Program 303, Plant Diseases. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2001: In collaboration with Fred Blattner at the University of Wisconsin, the blast disease resistance gene Pi-CO39 (t) locus in rice was further characterized by determination of the DNA sequence of a 170 kb region at the locus in resistant variety CO39 and 430 kb in the susceptible variety Nipponbare. The two haplotypes are substantially diverged with respect to relative number, size, orientation and location of resistance gene homologs within each cluster as well as insertion of retroelements in the Nipponbare genopme and deletion of resistance gene RPR1 and its flanking genes from the CO39 genome. The Pi-CO39 gene is the putative receptor for the M. grisea avirulence gene AVR1-CO39 that we have cloned. The cloning of both genes will allow the analysis of the molecular basis of pathogen recognition and the eventual engineering of rice and possibly other grass plants with broad spectrum resistance to M. grisea. B. Other Significant Accomplishment(s), if any: In collaboration with Yukio Tosa and colleagues at the Kobe University, the AVR1-CO39 locus was studied by PCR and Southern hybridization analysis in 85 well-characterized isolates of M. grisea having different host specificities. This analysis confirmed the absence of the gene in isolates infecting rice but its presence in other cereal-infecting strains that attack Setaria italica, Setaria viridis, Panicum spp., Eleucine spp., Triticum aestivum, and Avena sativa. These data provide the foundation for studies on the evolution of host and cultivar specificity conferred by the AVR1-CO39 gene in M. grisea and the corresponding Pi-CO39 gene in the grasses. C. Significant Accomplishments/Activities that Support Special Target Populations: Nothing to report. D. Progress Report: In collaboration with David Schwartz, a preliminary optical map of rice variety Nipponbare having 5X coverage has been prepared and alignment of publically available DNA sequence of Nipponbare from chromosomes 1 and 10 has been done. As part of the International Rice Genome Sequencing Project, the DNA sequence of 9 BAC clones of Nipponbare DNA from chromosome 11 have been determined to phase II level of completion and posted at http://www.gcow.wisc.edu/Rice/BAC_sequences.htm. In collaboration with Hans von Dohren at the Technical University of Berlin, the sid2 gene product has been detected as a high molecular weight species in ammonium sulfate-precipitated cell extracts of Ustilago maydis. The eventual purification of the sid2 non ribosomal peptide synthetase will led to its enzymological characterization and provide the biological materials to develop a biotransformation system to create novel siderophore drug-conjugates for control of microbial pathogens. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. The most effective method for control of rice blast is to grow disease resistant plants. Unfortunately, M. grisea is able to overcome this resistance within 1-3 years after resistant plants are cultivated widely. We are trying to understand the molecular details of how the rice blast fungus is recognized by rice plants that are resistant to blast and how the fungus changes in order to overcome this recognition. In order to improve our understanding of this host-parasite interaction, we have cloned a pathogen gene AVR1-C039 that is involved in recognition of the pathogen by the resistant rice plant. These approaches and insights will likely have broad application to other organisms such as the cereal rusts in which map-based cloning approaches are being used to clone similar genes. In parallel work, we are mapping the corresponding disease resistance gene which allows the host to recognize strains of the fungus that carry AVR1-CO39. This work has shown that resistance is conferred by a single dominant gene mapping to chromosome 11 of rice. We have also constructed a genomic DNA library from this resistant rice host as the first step toward cloning and characterization of this gene. Our long term goal is to study the molecular biology of the interaction of Avr1-CO39p and the host resistance gene product in order to understand when, where and how the fungus communicates its presence to the host. Based on this information, we hope to develop new strategies for engineering novel kinds of broad-spectrum resistance to M. grisea in rice and other cereals and grasses of economic and ornamental value. 6. What do you expect to accomplish, year by year, over the next 3 years? Our goals for the next year include the completion of the analysis of the AVR1-CO39 to define its transcript and the regulation of this transcript and to demonstrate through specific disruption of this transcript that a virulent strain can be generated when inoculated on rice cultivar CO39. Restoration of gene function in these null mutants will be investigated through transformation with the wild type gene and the cDNA expressed with heterologous promoters. Methods for heterologous overexpression of this transcript/gene product will be used in infection studies of cultivar CO39 with a virulent isolate to see if the peptide can protect plants. The presence of a calcium-binding site in the AVR1-CO39 peptide will be investigated. The function of candidate disease resistance genes found at the Pi-CO39 locus will be assessed through transcription analysis of the genes and transformation of susceptible rice to resistance. In collaboration with Japanese colleagues at the University of Kyoto, homologs of AVR1-CO39 from rice and Setaria-infecting isolates of M. grisea will be transformed into rice-infecting isolates that are virulent on CO39. The genes will be disrupted in selected Setaria isolates and their infection on Setaria spp. and rice with or without the corresponding known rice resistance will be done. In collaboration with Shailaja Hittalmani and Katrien Devos, we will investigate the genetic basis of blast resistance and drought tolerance in Eleucine coracana. Characterization of of AVR1-CO39 and Pi-CO39 homologs in fingermillet-infecting isolates of M. grisea, respectively, and their host will be made. Fingerprint analysis of 500 fungal isolates will begin. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product? A provisional patent application was submitted: Leong, S. A., Chauhan,R. S., Farman, M. F. "Chromosomal Region Conferring Resistance to Rice Blast." A provisional patent application was submitted: Leong, S. A., Chauhan, R. S., Durfee, T. J. "Complete Sequence of BACs E2P5 and K6P36 isolated from a Genomic Library of Oryza Sativa Variety CO39." The availability of the AVR1-CO39 gene as a protected technology was listed on the web site of the Wisconsin Alumni Research Foundation, http://www.wisc.edu/warf.boi/p98067us.html, Cultivar Specificity Gene from the Rice Pathogen Magnaporthe grisea, Methods and Use. Grower/scientist requests: I received a request for transformation vectors for fungi. I received a request for the AVR1-CO39 gene. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)

Impacts
(N/A)

Publications

  • Chauhan, R., Farman, M., Hirt, J., Durfee, T., Ronald, P. Zhang, H.B., Blattener, F., Leong, S.A. Genomics of blast resistance in rice line CO39 corresponding to avirulence locus AVR1-CO39 of Magnaporthe grisea. Plant and Animal Genome IX, San Diego, CA. 2001. Abstract p. 157.
  • Chauhan, R.S., Durfee, T., Farman, M., Ronald, P., Zhang, H.B., Blattner, F., Leong, S.A. Comparative genome analysis of the Pi-CO39(T) locus in haplotypes of indica and japonica. Rice. 10th International Congress on Molecular Plant-Microbe Interactions, Madison, WI. 2001. Abstract p. 367.
  • Yuan, W.M., Gentil, G., Budde, A.D., Leong, S.A. Characterization of the Ustilago maydis sid2 gene encoding a multidomain nonribosomal peptide synthetase in the ferrichrome biosynthetic gene cluster. Journal of Bacteriology. 2001. v. 183. p. 4040-4051.


Progress 10/01/99 to 09/30/00

Outputs
1. What major problem or issue is being resolved and how are you resolving it? Fungal pathogens of plants are the most devastating plant disease agents. We are studying the molecular biology of plant pathogenesis of two model plant pathogenic fungi, Ustilago maydis and Magnaporthe grisea. These fungi represent two of the major classes of phytopathogenic fungi, basidiomycetes and ascomycetes, and are considered the premier examples of fungal plant pathogens which can be manipulated both genetically and with the tools of molecular biology. 2. How serious is the problem? Why does it matter? Ustilago causes corn smut disease, a disease which is no longer a major threat to agricultural production in the United States as a result of an active breeding effort to maintain disease resistance in commercial varieties. While U. maydis itself is not an economic threat, other related smut and bunt fungi of cereals are serious problems. Basic studies of plant pathogenesis have already revealed similarities in the mating type control system of U. maydis and Ustilago hordei which controls the ability of these fungi to cause plant disease. M. grisea causes rice blast disease, one of the most devastating diseases of rice worldwide. In addition, different subspecific groups of M. grisea are known to attack other important cereals and grasses such as wheat causing wheat blast and turf causing grey spot. Recent studies have shown that similar genes are needed for infection of different host plants by Magnaporthe. Thus we can anticipate that information that is broadly applicable to fungal pathogenesis will be obtained through studies of these two model fungi. 3. How does it relate to the National Program(s) and National Component(s)? This research allows scientists and other customers to better understand mechanisms of plant pathogenicity and field survival and develop new methods for control of these fungi. Approaches for durable resistance to rice blast disease may be gained. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY2000 year: The blast disease resistance gene Pi-CO39 (t) in rice was further mapped to 5.9 cM from marker RG1094 and 2.7 cM from marker R2316 on chromosome 11 in an F2 susceptible population. Three additional markers were found to cosegregate with the Pi-CO39 gene in 400 homozygous susceptible F2 progenies and 200 resistant F2 progenies among 1,100 F2 tested for resistance. These markers were used to isolate BAC clones from the resistant variety CO39 and the susceptible variety Nipponbare. Single BAC clones hybridizing to all cosegregating markers were obtained in the Nipponbare library while clones hybridizing to only a single probe were found in the CO39 library. A contig of 0.5 mb was constructed in this region of the Nipponbare genome. DNA sequencing of these clones is underway. A GC-MS high resolution method for analysis of siderophore constituents in Ustilago cells was successfully implemented. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. The most effective method for control of rice blast is to grow disease resistant plants. Unfortunately, M. grisea is able to overcome this resistance within 1-3 years after resistant plants are cultivated widely. We are trying to understand the molecular details of how the rice blast fungus is recognized by rice plants that are resistant to blast and how the fungus changes in order to overcome this recognition. In order to improve our understanding of this host-parasite interaction, we have cloned a pathogen gene AVR1-C039 that is involved in recognition of the pathogen by the resistant rice plant. This is the second AVR (AviRulence) gene to be cloned from the rice blast fungus. We employed a combination of physical, genetic and molecular genetic methods to obtain the cloned gene. These methods included our laboratory's high density genetic map and molecular karyotype, targeted genome cleavage methods, and a transformation system we developed for M. grisea that is based on drug resistance. Considerable insight was gained about the map- based cloning approaches through this effort. These approaches and insights will likely have broad application to other organisms such as the cereal rusts in which map-based cloning approaches are being used to clone similar genes. In parallel work, we are mapping the corresponding disease resistance gene which allows the host to recognize strains of the fungus that carry AVR1-CO39. This work has shown that resistance is conferred by a single dominant gene mapping to chromosome 11 of rice. We have also constructed a genomic DNA library from this resistant rice host as the first step toward cloning and characterization of this gene. Our long term goal is to study the molecular biology of the interaction of Avr1-CO39p and the host resistance gene product in order to understand when, where and how the fungus communicates its presence to the host. Based on this information, we hope to develop new strategies for engineering novel kinds of broad-spectrum resistance to M. grisea in rice and other cereals and grasses of economic and ornamental value. Studies on the mating type control system and high affinity iron transport system of U. maydis were conducted to understand how these systems contribute to pathogenesis of this smut fungus. Smut fungi must mate in order to cause plant infection. The mating type loci a and b were cloned and partially characterized. Both loci were shown to be important to pathogenicity through analysis of knock-out mutations. The b locus was further shown to encode a putative transcription factor belonging to the homeodomain family. Different alleles of b showed predicted variable amino acid sequences in their N-terminal domains with a conserved C-terminal domain. Homologs of the a and b loci were identified in other smut fungi through hybridization analysis. This work has been extended by a former postdoctoral researcher of the lab and was discontinued in my laboratory. Studies of high affinity iron transport in U. maydis have resulted in defining through gene cloning for the first time an understanding of the molecular biology of biosynthesis and regulation of biosynthesis of siderophores (microbial iron transport agents) by fungi. In response to iron starvation, we discovered that the U. maydis produces two cyclic hydroxamate siderophores ferrichrome and ferrichrome A. Three genes required for siderophore biosynthesis and regulation have been cloned and characterized: sid1 encodes ornithine-N5-oxygenase, the first enzyme in the ferrichrome biosynthetic pathway; sid2 encodes a putative nonribomal peptide synthetase required for ferrichrome biosynthesis; and urbs1 encodes a GATA family transcription repressor of sid1/sid2. Current efforts are focused on developing an understanding of how iron homeostasis is maintained by Urbs1. urbs1 mutants secrete siderophores constitutively and hyperaccumulate iron and siderophores intracellularly. urbs1 mutants grow poorly when compared to wild type. The process of iron regulation of iron uptake is still poorly understood and the full extent of the function of urbs1 in cells in unknown. Fur, the analog of Urbs1 in many bacteria also controls adaptation to low pH and ability to grow on alternate carbon sources such as succinate. We have initiated a new mutant hunt to look for mutants degregulated in iron uptake in order to better understand how iron uptake is regulated and if other genes in addition to Urbs1 are required. We are also studying the molecular details of the interaction of Urbs1 with the promoter (regulatory) region of sid1/sid2. We have demonstrated that Urbs1 binds specifically to GATA sequences in these promoters and that the DNA undergoes dramatic changes in chromatin organization as a function of iron availability to the cell consistent with derepression of these genes. In other microbes as well as plants and man, iron overload is toxic. Furthermore, mammals restrict infection by bacteria through a system referred to as nutritional immunity whereby iron is mobilized to tissue stores thus making it less available to invading microbes. The comparative analysis of the genome sequences of a laboratory strain of Eshcerichia coli K-12 with that of other highly pathogenic strains of E. coli such as 0157 have revealed a propensity of iron acquisition systems and virulence genes known to be regulated by the iron deficiency that is encountered in the mammalian host. In at least one plant pathogen Erwinia chrysanthemi, siderophore systems have been shown to be required for systemic infection of a plant host. However, our limited studies to date in an in vitro system using corn seedlings grown in a growth chamber did not reveal a significant role for sid1 or urbs1 in pathogenicity. Based on the confounding results that Chris Upper and Kyle Willis have obtained for pathogenicity phenotypes of mutants of Pseudomonas inoculated on plants in the field and growth chamber, it would not be prudent to firmly conclude that these two siderophore genes are not essential for pathogenicity. The ability of fungal pathogens to acquire iron from the host or its environment may influence spore viability,spore germination,colonization of a habitat as well as virulence. Siderophores are germination factors in many fungi including Ustilago spp. Candida albicans was recently shown to require high affinity iron transport systems to be pathogenic. We plan to assess the field survival and field infectivity of these mutants once an appropriate method is developed for "natural" inoculation of corn with Ustilago. Ongoing work at the University of Minnesota should provide this needed background. The role of siderophore genes in the infection of mammalian hosts has been spurred by our pioneering work in Ustilago. Analogs of urbs1 have been isolated from several other fungi based on the conservation of the DNA binding domain. It is very likely that fungal siderophore genes will be important in an animal model such as mouse and we are anxiously awaiting the results of ongoing work in other labs in Aspergillus fumigatus and Histoplasma capsulata. This opens up the possibility of exploiting siderophore/iron uptake genes as a means of controlling infection in farm animals and man. Three approaches are immediately evident. One is based on an ongoing collaboration with Drs. Marvin Miller at Notre Dame and Hans Von Dohren at the Technical University of Berlin. The approach is to develop novel drug congugates based on siderophores which can be illicitly transported into target cells via the siderophore uptake system. The concept of illicit transport may extend the life of a drug by delivering it through an uptake system for an essential nutrient iron. Dr. Miller's synthetic chemistry work has already demonstrated the potential of several model compounds which are based on the U. maydis ferrichrome siderophore structure for control of Cryptococcus neoformans. Our work with Dr. Von Dohren aims at developing a system of overexpression of the enzymes encoded by the sid genes in order to facilitate the development of a biotransformation system to produce precursors needed for large scale production of these drug conjugates. Currently we are studying the sid2-encoded peptide synthetase. The second approach makes use of the observation that all fungi with the exception of the Zygomycetes, produce delta-N- hydroxyornithine-containing. Thus the sid1 gene should be rather ubiquitous among fungi and may be a useful target for drug development if siderophores are found to be virulence factors of fungi and yeasts. The third approach for control of fungi could take advantage of knowledge of the 3-D structure and metal requirement of the DNA binding domain of Urbs1 which, based on structural information from other GATA family members, will have a finger structure comprised of zinc or iron tetrahedrally coordinated via four cysteines. For example, anti-HIV agents have been developed that covalently modify the metal-coordinating cysteine thiolates of the retrovial nucleocapsid protein fingers causing metal (zinc) ejection thus disrupting virus replication. These agents did not affect function of other metal-dependent transcription factors including Sp1 or Gata-1, a GATA family transcription factor. By analogy, it may be possible to design chemical agents that can selectively remove metals from the fingers of fungal GATA factors such as Urbs1 but not those of the plant or animal host. Toward this end we have established working collaborations with several structural biologists on the University of Wisconsin campus in order to study the structure and metal coordination of Urbs1 and its DNA binding domain by EXAFS, NMR, and EPR. Our current efforts are focused on producing sufficient quantities of biologically active protein for this work. As a prelude to the work described above, we developed the first transformation system available for U. maydis and pioneered other methods such as electrophoretic karyotyping, gene replacement, gene disruption, and gap repair which are widely used in yeast. 6. What do you expect to accomplish, year by year, over the next 3 years? Our goals for the next year include the completion of the analysis of the AVR1-CO39 to define its transcript and the regulation of this transcript and to demonstrate through specific disruption of this transcript that a virulent strain can be generated when inoculated on rice cultivar CO39. Restoration of gene function in these null mutants will be investigated through transformation with the wild type gene and the cDNA expressed with heterologous promoters. Methods for heterologous overexpression of this transcript/gene product and purchased synthetic peptide will be used in infection studies of cultivar CO39 with a virulent isolate to see if the peptide can protect plants. Once this system is better defined, we will begin to examine the timing of perception of the AVR peptide through the use of mutants of M. grisea which are defective in appressorium development (infection structure needed for pathogen ingress into host). Infections will be monitored cytologically and through the use of rice cDNA clones encoding genes known to be expressed at different times during infection of rice with M. grisea. A rice expression microarray will be constructed for this purpose. The BAC clone(s) mapping to the Pi-CO39 (t) locus will be sequenced and directed subcloning will be done to identify candidate genes and assess their function through transformation of susceptible rice to resistance. In collaboration with Japanese colleagues at the University of Kyoto, homologs of AVR1-CO39 from rice and Setaria-infecting isolates of M. grisea will be transformed into rice-infecting isolates that are virulent on CO39. The genes will be disrupted in selected Setaria isolates and their infection on Setaria spp. and rice with or without the corresponding known rice resistance will be done. A method for isolating iron regulatory mutants based on iron- dependent auxotrophy will be implemented and mutants will be classified phenotypically and genetically. In collaboration with Dr. Von Dohren, the enzymatic activity of sid2 will be defined and discussions with industry will begin in order to obtain funding for our proposed biotransformation studies to generate siderophore-drug conjugates. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product? Invention disclosure filed and development of a patent application is underway : Chromosomal Region Conferring Resistance to Rice Blast. Grower/scientist requests: I received a request for transformation vectors for fungi. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)

Impacts
(N/A)

Publications

  • LEONG, S. A.,...KEARNEY, L.. Iron...Remodeling. In: Iron Chelators: New Development Strategies (D.G. Badman, R. J. Bergeron, G. M. Brittingham, eds.), Saratoga Press, Saratoga. 2000. p.43-65.
  • Chauhan, R. S., Farman, M. L., Wirt, J. E., Ronald, P. C., Zhang, H. B., and Leong, S. A. Genetic analysis and fine mapping of a locus for blast resistance in rice line CO39 and construction of its BAC library. Abstract Book. Plant and Animal Genome IV. 2000. p. 96.