Source: UNIV OF WISCONSIN submitted to
MOLECULAR ANALYSIS OF GENES CONTROLLING PATHOGENESIS OF USTILAGO MAYDIS AND MAGNAPORTHE GRISEA
Sponsoring Institution
State Agricultural Experiment Station
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0150774
Grant No.
(N/A)
Project No.
WIS03355
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jan 9, 1989
Project End Date
Sep 30, 2010
Grant Year
(N/A)
Project Director
Leong, S. A.
Recipient Organization
UNIV OF WISCONSIN
(N/A)
MADISON,WI 53706
Performing Department
PLANT PATHOLOGY
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
2124020116040%
2121510116030%
2121530116030%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4020 - Fungi; 1510 - Corn; 1530 - Rice;

Field Of Science
1160 - Pathology;
Goals / Objectives
The tools of recombinant DNA technology will be employed to begin to clone and analyze genes required for pathogenic growth of Ustilago maydis and Magnaporthe grisea.
Project Methods
We will identify mutants of U. maydis which are defective in iron transport and mating. The respective genes will be cloned and their structure, regulation and function studied. A genetic map will be constructed for M. grisea using molecular markers and genes controlling race-cultivar specificity and pathogenic variability will be cloned by chromosome walking from linked molecular markers. The structure, regulation and function of the genes will be analyzed.

Progress 01/09/89 to 09/30/10

Outputs
OUTPUTS: Collaborative work with Mark Farman at the University of Kentucky on transformation of Pi-CO39 candidate genes was abandoned due to a lack of funding and confounding results obtained in the prior year. In collaboration with Mark Farman, a new focus on localization of AVR1-CO39 gene product in rice plants is underway with the fusion of a fluorescent probe and a nucleus localization signal to the gene. PARTICIPANTS: Professor Mark Farman, University of Kentucky TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The cloned region containing the rice blast resistance locus Pi-CO39 (t) is a complex region extending over nearly 200 kb and contains many candidate genes, which may function singly or as a complex to confer disease resistance to Magnaporthe oryzae carrying the corresponding avirulence gene AVR1-CO39. Efforts to determine which gene(s) contribute to disease resistance have been hampered by technical problems leading to misinterpretation of disease resistance in untransformed lines earlier. Further apparently transformed lines of susceptible variety Nipponbare did not yield resistant plants although confirmation of the functional state of the transforming DNA was not fully assessed. Lack of funding has not permitted further studies of the function of this region. Studies on the mechanism of action of AVR1-CO39 are important to understand its utility in management of grey leaf spot and rice blast disease in grasses. An understanding of the site of function of this gene product will provide needed information for deployment of the corresponding resistance gene(s) in a durable fashion.

Publications

  • No publications reported this period


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Through collaboration with Mark Farman at the University of Kentucky, continuing efforts were advanced to obtain transgenic plants containing candidate genes for the Pi-CO39 (t) resistance locus in rice variety CO39. A BAC clone carrying the Cor8 candidate gene was transformed into two rice varieties Kitake and Nipponbare. Kitake is already resistant to strains of Magnaporthe grisea carrying AVR1-CO39 while Nipponbare is not. Pathogenicity tests have not confirmed that the BAC clone tested confers resistance in Nipponbare to M. grisea carrying AVR1-CO39. However, the integrity of the BAC clone remains to be confirmed in transgenic plants. PARTICIPANTS: Mark Farman, University of Kentucky TARGET AUDIENCES: Rice growers, Rice industry including millers, food processing, beer; Rice extension specialists; Cereal scientists; Rice consumers worldwide PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
AVR1-CO39 has been patented and has been shown to induce a resistance response when transiently expressed in rice lines carrying the corresponding resistance gene locus. Identification of the corresponding cloned resistance gene (s) remains illusive, as transformation tests have not conclusively identified a specific rice gene to be responsible for resistance. The DNA region being tested is complex and contains three families of candidate resistance genes as well as potentially important accessory genes such as serpin genes. More than one rice gene at the Pi-CO30 locus may be required to confer resistance to M. grisea carrying AVR1-CO39.

Publications

  • Leong, S. A. 2008. The ins and outs of host recognition of Magnaporthe oryzae, In: The Genomics of Disease (P. Gufstason, Taylor, J., and Stacey, G., eds.) Springer Press, New York. pp. 119-216.
  • Heidenreich, M. L., Budde, A. D., An, Z., and Leong, S. A. 2008. Disruption of an ADE6 homolog in Ustilgo maydis. Fungal Genetics Reports 55:40-43.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: AVR1-CO39 of Magnaporthe oryzae elicits a PiCO39 (t)-dependent defense response in rice. Two genes have been tentatively identified at the PiCO39 (t) locus of rice that function in the disease resistance response in rice. Transformants containing both candidate genes are now available in two cultivars of rice and pathogenicity tests are underway with M. oryzae strains transformed with the AVR1-CO39. Efforts to overexpress the AVR1-CO39 in Escherichia coli for structural determination were not successful. An alternate strategy to express the mature form of the protein is under investigation. We are exploring the use of oligonucleotide microarray technology as a means of rapidly discovering Single Feature Polymorphisms (SFPs) in rice and finger millet that can be used as DNA-based markers for genetic mapping. Genome alignment between the two sequenced rice lines previously identified identity blocks and the coordinates of orthologous SwaI restriction sites and 5,490 SwaI site polymorphisms. Three of these polymorphic regions were amplified from the two rice genomes using the polymerase chain reaction and the polymorphisms were confirmed by SwaI digestion. SwaI was used to generate an optical map of the rice genome. The use of SwaI site polymorphisms allows cross reference to this physical map, which remains the most comprehensive genome map of rice available to date. PARTICIPANTS: Collaborators: David Schwartz, UW Madison; Jacob Kitzman,bioinformatics contractor; Robert Fjellstrom, USDA, ARS, Dale Bumpers Rice Research Center; Yulin Jia, USDA, ARS, Dale Bumpers, Rice Research Center; John Markley, UW Madison; Mark Farman, University of Kentucky TARGET AUDIENCES: Rice growers, Rice industry including millers, food processing, beer; Rice extension specialists; Cereal scientists; Rice consumers worldwide PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Identification of the functional resistance gene will allow transfer of this gene to other cereals and grasses of economic importance where blast disease is important. Grey leaf spot caused by M. oryzae is an important problem in the U. S. and Japan and strains of M. oryzae causing this disease have functional copies of AVR1-CO39. Therefore, the corresponding disease resistance locus of rice may be useful in perennial rye which has no resistance to this disease. The rice genome alignment based on highly similar blocks of ordered rice DNA sequence is valuable for the identification DNA polymorphisms in rice. These polymorphisms are useful in breeding and genetic studies of plants.

Publications

  • No publications reported this period


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: AVR1-CO39 of Magnaporthe oryzae elicits a PiCO39 (t)-dependent defense response in rice. Two genes have been tentatively identified at the PiCO39 (t) locus of rice that function in the disease resistance response in rice. Transformants containing both candidate genes are now available in two cultivars of rice and pathogenicity tests are underway with M. oryzae strains transformed with the AVR1-CO39. Efforts to overexpress the AVR1-CO39 in Escherichia coli for structural determination were not successful. An alternate strategy to express the mature form of the protein is under investigation. We are exploring the use oligonucleotide microarray technology as a means of rapidly discovering Single Feature Polymorphisms (SFPs) in rice and finger millet that can be used as DNA-based markers for genetic mapping. Genome alignment between the two sequenced rice lines previously identified identity blocks and the coordinates of orthologous SwaI restriction sites and 5,490 SwaI site polymorphisms. Three of these polymorphic regions were amplified from the two rice genomes using the polymerase chain reaction and the polymorphisms were confirmed by SwaI digestion. SwaI was used to generate an optical map of the rice genome. The use of SwaI site polymorphisms allows cross reference to this physical map, which remains the most comprehensive genome map of rice available to date. PARTICIPANTS: Collaborators: David Schwartz, UW Madison; Jacob Kitzman,bioinformatics contractor; Robert Fjellstrom, USDA, ARS, Dale Bumpers Rice Research Center; Yulin Jia, USDA, ARS, Dale Bumpers Rice Research Center; John Markley, UW Madison; Mark Farman, University of Kentucky; TARGET AUDIENCES: Rice growers, Rice industry including millers, food processing, beer; Rice extension specialists; Cereal scientists; Rice consumers worldwide

Impacts
Identification of the functional resistance gene will allow transfer of this gene to other cereals and grasses of economic importance where blast disease is important. Grey leaf spot caused by M. oryzae is an important problem in the U. S. and Japan and strains of M. oryzae causing this disease have functional copies of AVR1-CO39. Therefore, the corresponding disease resistance locus of rice may be useful in perennial rye which has no resistance to this disease. The rice genome alignment based on highly similar blocks of ordered rice DNA sequence is valuable for the identification DNA polymorphisms in rice. These polymorphisms are useful in breeding and genetic studies of plants.

Publications

  • Zhou, S., Bechner, M. C., Place, M., Churas, C.P., Pape, L., Leong, S. A., Runnheim, R., Forrest, D. K., Goldstein, S., Livny, M., and Schwartz, D. C. 2007. Validation of rice genome sequence by optical mapping. Biomed Central Genomics, http://www.biomedcentral.com/1471-2164/8/278


Progress 01/01/06 to 12/31/06

Outputs
AVR1-CO39 of Magnaporthe grisea elicits a PiCO39 (t)-dependent defense response in rice. Two genes have been tentatively identified at the PiCO39 (t) locus that function in the disease resistance response in rice. Transformants containing both candidate genes are now available in rice and seed production is underway. Informatic studies have revealed candidate orthologues with >80 % identity in barley. We are exploring the use oligonucleotide microarray technology as a means of rapidly discovering Single Feature Polymorphisms (SFPs) in rice and finger millet that can be used as DNA-based markers for genetic mapping. Methods were established to increase signal to noise and insure probe saturation by optimizing the labeling reaction conditions, removing small products less than 30 bp, increasing the amount of probe in the hybridization and assessing the temperature of hybridization. Genome alignment between the two sequenced rice lines was used to generate a series of identity blocks separated by non identical DNA. A total of 4,413,096 polymorphisms including 16,834 indels >= 25 bp and 5,490 SwaI site polymorphisms were located within identity blocks.

Impacts
Identification of the functional resistance gene will allow transfer of this gene to other cereals and grasses of economic importance where blast disease is important. Optimized methods for hybridization of genomic DNA targets to oligonucleotide arrays will reduce the number of false positives identified and allow better detection of polymorphisms in higher plants. The genome template based on highly similar blocks of ordered rice DNA sequence is valuable for the identification DNA polymorphisms in rice. These polymorphisms are useful in breeding and genetic studies of plants.

Publications

  • No publications reported this period


Progress 01/01/05 to 12/31/05

Outputs
Optimization and characterization of a novel and efficient tissue culture method for the generation of callus directly from rice leaves for rice plastid transformation was made. Drug sensitivity was evaluated and two drugs were found to be of possible use. Leaf callus was obtained from additional rice cultivars showing that the method is of broad utility. This technique will allow the development of novel blast resistant lines of rice, which have lower risk for transgene flow into the environment. One candidate blast resistance gene at the PiCO39 (t) resistance locus was successfully introduced into susceptible US rice variety M202. Transformation of the other was not as successful; however, plantlets have now been generated from transgenic callus. Recent progress suggests a medium modification will overcome past difficulties. The DNA sequence of the candidate resistance gene shows a second potential promoter that could be contributing to this growth inhibition and will be removed in future transformation experiments. Gene silencing studies of the one candidate gene were initiated using a rice-infecting strain of Brome Mosaic Virus. A lesion mimic phenotype was observed. Quantative resistance to grey leaf spot disease was identified on chromosomes 2, 3, 4 and 6 in a cross involving perennial rye and annual rye grasses. These regions are not syntenic to the rice resistance Pi-CO39 (t). We have shown that strains of the M. grisea that cause grey leaf spot have functional homologs of AVR1-CO39 indicating that the Pi-CO39 (t) may be effective for grey leaf spot management if introduced into rye. A genome scan of Nipponbare and 93-11 against the rice probe set of the Affymetrix rice gene expression array was conducted and revealed that about 170,000 single feature polymorphisms in the 93-11 genome might be detected with this array. Pilot studies using total genomic DNA as a probe labeled using random priming with biotin dCTP revealed the the protocol used for a similar application with Arabidopsis was not optimized for rice.

Impacts
New technology for the culture of rice to give rice callus at high levels was developed further to a high efficiency. This method will be useful for maintenance of plant tissue without seed and for exploration of methods for transformation of nuclear and plastid genomes. The functional analysis of candidate resistance genes at the Pi-CO39 (t) locus has progressed. One gene adversely affects plant growth and when introduced into BMV, causes a lesion mimic phenotype consistent with a role in both disease resistance and host growth and development. Methods to improve the detection of single feature polymorphisms on oligonucleotide arrays were developed which will allow better detection of polymorphisms in higher plants.

Publications

  • Curley, J., Sim, S. C., Warnke, S., Leong, S., Barker, R., and Jung, G. 2005. Genetic variability and quantitative trait loci mapping of resistance to gray leaf spot in ryegrass. Theor Appl Genet. 111:1107-1117.
  • The International Rice Genome Sequencing Project. 2005. The map-based sequence of the rice genome. Nature 436:793-800.
  • Tosa, Y. Osue, J., Eto, Y., Nakayashiki, H., Mayama, S., and Leong, S. A. 2005. Evolution of an avirulence gene AVR1-CO39 in relation to the evolution and differentiation of Magnaporthe oryzae. Mol. Plant-Microbe Interactions 18:1148-1160.


Progress 01/01/04 to 12/31/04

Outputs
Transient expression studies of BAC and subcloned genes thereof from the PiCO39 (t) rice blast locus have identified two candidate genes on one BAC clone as the possible resistance determinant(s). This result sets the stage for the development of durable blast resistance US germplasm using the Pi and Avr genes that have been identified and cloned through support of this CRIS project, the University of Wisconsin, and the Rockefeller Foundation. Alternatively, expression of the Avr gene can be explored using epiphytic and endophytic microorganisms of rice lines carrying the Pi-CO39 (t) resistance locus.

Impacts
These results will allow the development of a new approach to protect rice plants from blast disease based on expression of orf3 of AVR1-CO39 directly in resistant rice or in epiphytic or endophytic microbes of rice.

Publications

  • Peabody, P. Bolstering rice against Blast. Agricultural Research, August 2004. pp18-19
  • Leong, S. A. 2004. Biotechnological approaches in plant protection: achievements, new initiatives and prospects. In: Fungal Biotechnology in Agricultural, Food, and Environmental Applications, Volume 21, (D. Aurora, ed.) Marcel Dekker, New York. pp.1-17
  • Leong, S. A. and Farman, M. L. Cultivar specificity gene from the rice pathogen Magnaporthe grisea, and methods of use, Patent No. 6,794,176, September 21, 2004.


Progress 01/01/03 to 12/31/03

Outputs
A particle bombardment system was used to introduce constructs for the production of orf3 of AVR1-CO39 ::GFP with and without its signal sequence under control of the 35S promoter into resistant and susceptible germplasm. A difference in particle number/leaf as measured by a GFP reporter was detected in resistant plants relative to the independently expressed control reporter GUS. No GFP expressing cells were detected using full length constructs of Orf3. This suggests that: 1)the mature form of orf3 is sufficient for signaling inside plant cells and 2)the unprocessed form of orf3 is not expressed sufficiently into protein or is targeted for the cell vacuole and destroyed (PSORT prediction). Expression of the full length construct into mRNA was confirmed by RTPCR. Analysis of the transcription profile of AVR1-CO39 was done by RACE. The data obtained indicate that a transcript includes orfs 3 and 5 is made in infected susceptible rice plants. The full length transcript was confirmed by RTPCR.

Impacts
These results will allow the development of a new approach to protect rice plants from blast disease based on expression of orf3 of AVR1-CO39 directly in resistant rice.

Publications

  • No publications reported this period


Progress 01/01/02 to 12/31/02

Outputs
We tested the pathogenicity of a Brome Mosaic Virus strain from Dr. Paul Ahlquist, on fingermillet genotypes from Dr. Devos by sap inoculation. The idea was to use BMV-based vectors for gene silencing analogous to PVX in tobacco. Viral infection was analyzed both by observing the presence of visual mosaic symptoms as well as determining the presence of viral RNA by RT-PCR. No visual symptoms were observed. For RT-PCR, total RNA was isolated using an Ambion miniprep kit and 1st strand cDNA synthesized using olig(dT)18 primer. These cDNAs were used as templates for PCR using primers specific to BMV sub-genomes 1, 2, and 3. None of the genomes amplified on cDNA from BMV-inoculated finger millet plants, whereas all amplified on cDNA templates obtained from BMV-inoculated barley. We are also pursuing transient gene silencing on leaves using biolistic gene delivery. To explore this avenue we are trying to silence the phytoene desaturase gene (PDS), which protects plants from photo-bleaching. The gene has been silenced successfully in potato and tobacco using PVX and TRV vectors. We amplified a gene fragment (300 bp) from genomic DNA of fingermillet genotype IE1012 using rice, gene-specific primers. Sequencing of the fingermillet and rice gene fragments indicated 100% sequence identity at nucleotide level. The finger millet gene fragment has been cloned in inverted orientation in a RNAi vector, pCA157- received from Paul Schulze-Lefert (JIC). We shot etiolated seedlings of fingermillet and rice with PDS-RNAi construct along with a GFP (green fluorescent protein) reporter as an independent marker to check silencing of the PDS gene. The seedlings were then transferred to light. Specific inhibition of leaf greening was not observed in bombarded zones. The PDS gene may be expressed in the dark and not translated until plants are exposed to light. An RNAi construct of GFP was also tested and shown to silence GFP indicating that the approach can be used to silence genes in both rice and finger millet.

Impacts
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 AVR1-CO39 homologs in fingermillet-infecting isolates of Magnaporthe grisea and their host resistance genes will be made. Isolation of SNPs for grass genome mapping using oligonucletide microarray studies will be initiated. Identification of cDNA AFLPs segregating with these traits will be identified. Gene silencing will be used to test the role of candidate cDNAs in blast resistance as leaves or the necks of panicles can be inoculated with the fungus. These studies will provide new information for marker-assisted breeding of blast resistance, and drought tolerance into popular germplasm for resource-poor farmers in India.

Publications

  • No publications reported this period


Progress 01/01/01 to 12/31/01

Outputs
The map position of the Pi-CO39 (t) disease resistance gene of rice was further defined with flanking markers mapping at 1.0 and 0.2 cM on either side of the gene. The DNA sequence of the region in a susceptible genome Nipponbare (420 kb) and the resistant genome CO39 (160 kb) was determined to a phrap score of greater than 30. Gene annotation lead to the discovery of many candiate NBS-LRR-type genes in both genomes, however, more genes were found in the Nipponbare genome. Clearly the CO39 genome contains deletions and transversions of the corresponding DNA in Nipponbare. Lack of a high degree of synteny at this locus has apparently suppressed recombination among these candidate genes. A particle bombardment system for introduction of transgenes into rice leaves was optimized using a GUS reporter under 35S promoter control. Constructs for the production of the Magnaporthe grisea AVR1-CO39 with and without its signal sequence under control of 35S promoter were constructed.A production strategy for isolation of AVR1-CO39 orf3 with an HA tag using a pET expression system in Escherichia coli was developed. Progress on the purification of sid2 of Ustilago has been made.

Impacts
The isolation of the disease resistance gene locus will soon provide the cloned gene for introduction into susceptible rice varieties and provide opportunities for engineering rice and other grasses with novel forms of resistance involving both the AVR and R gene. Isolation of the sid2 gene product will allow its biochemical characterization and determine what peptide product it can produce. This work is essential to the ultimate goal of engineering novel siderophore drug conjugates using a biotransformation approach.

Publications

  • Yuan, M. W., Gentil, G. D., Budde, A. D. and S. A. Leong. 2001. Characterization of the Ustilago maydis sid2 gene encoding a multidomain peptide synthetase in the ferrichrome biosynthetic gene cluster. J. Bacteriol. 183:4040-4051
  • Farman, M. L., Eto, Y., Nakao, Y., Tosa, Y., Nakayashiki, H., Mayama, S., and S. A. Leong. 2002. Analysis of the structure of the Avr1-CO39 avirulence locus in virulent rice-infecting isolates of Magnaporthe grisea. Mol. Plant-Microbe Interactions 15(1):6-16.


Progress 01/01/00 to 12/31/00

Outputs
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 and has revealed the presence of a number of disease resistance gene homologs. Interestingly, the number, class and distribution of these genes in both genomes is very different.

Impacts
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.

Publications

  • Leong, S.. A., Farman, M. L., Chauhan, R. S., Punekar, N., Mayama, H., Nakayashi, H., Eto, Y., Tosa, Y., and Ronald, P. 2000. Progress on understanding the molecular basis of cultivar specificity in the interaction of the Magnaporthe grisea AVR1-CO39 with rice variety CO39. In: Proceedings of the 2nd International Conference on Rice Blast Disease. Kluwer, Dordrecht. pp 308-315
  • Leong, S. A., Yuan, W. M., and Kearney, L. 2000. Iron, DNA and Chromatin Remodeling. In: Iron Chelators: New Development Strategies (D.G. Badman, R. J. Bergeron, G. M. Brittingham, eds.), Saratoga Press, Saratoga. pp. 43-65


Progress 01/01/99 to 12/31/99

Outputs
Homologs of AVR1-CO39 have been cloned from rice and Setaria-infecting isolates of M. grisea and their DNA sequence determined. The DNA sequences of these amplicons revealed considerable conservation (>99%) of the DNA sequences across the 1 kb region containing the AVR1-CO39 gene. Based on mutagenesis studies and gene structure criteria, orf3 is the most probable open reading frame with function for avirulence. Comparison of the orf3 amino acid sequences using the Clustal program of Megalign ( DNASTAR, Madison, WI) showed between 94.2 and 97.8% sequence conservation when compared with that of laboratory strain 2539. Interestingly, the data indicate that the allele from the rice-infecting strain 033 was the most distantly related to that of laboratory strain 2539. The AVR1-CO39 allele that was cloned from 2539 is most likely derived from the weeping love grass parent of 2539. The extensive changes in the amino acid sequence of orf3 from strain 003 and the paucity of rice isolates found to contain AVR1-CO39 would support the notion that rice-infecting strains have evolved to loose a functional copy of AVR1-CO39. To test this hypothesis, efforts are underway to assess the biological activity of the alleles of AVR1-CO39 through transformation and gene disruption. The corresponding disease resistance gene Pi-CO39 (t) in rice was mapped 5 cM from marker RM202 and 11 cM from marker RG247 on chromosome 11 in an F2 susceptible population.

Impacts
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. 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.

Publications

  • No publications reported this period


Progress 01/01/98 to 12/31/98

Outputs
Mapping of the disease resistance gene corresponding to avirulence gene AVR1-CO39 was begun. Fifteen mirosat (simple sequence repeats) markers, mapping to four rice chromosomes 1, 6, 11 & 12, known to carry disease resistance genes, were tested for polymorphism between CO39(resistant) and 51583(susceptible). Co-segregation of polymorphic RM markers with resistance/susceptibility was tested using bulked segregant analysis as well as individual F3 families. Microsat marker, RM202, located on chromosome 11, co-segregated with blast resistance in resistant/susceptible F3 families as well as bulks. Linkage relationship between RM202 and resistance gene in CO39 is being established by testing the response of segregating F2 populations. RG247 on chromosome 11 which is tighly linked to RM202 also showed cosegregation with the target gene in the progeny tested. Structural homologies between R (resistance) genes from different plant species have facilitated the cloning of R gene from monocots using degenerate oligos from dicot R genes. This strategy is also being used for cloning the R gene in CO39. Degenerate oligonuceotide primers from the conserved regions of both the leucine-rich repeats (LRR) and serine-threonine kinase domains of Xa21, a bacterial blight resistance gene from rice, have amplified a 850bp fragment in CO39 and 51583. DNA sequence analysis of the cloned amplicon has revealed novel R-gene-like homologs. Our working hypothesis is that iron modulates siderophore gene expression in U. maydis at one or more levels. Iron may act as a corepressor of genes regulated by Urbs1 and as a physiological effector that indirectly affects expression of siderophore genes by altering the structure and/or cellular location of Urbs1. As with Fur, the procaryotic analog of Urbs1, iron may directly activate Urbs1 in DNA binding. Urbs1 may repress expression of sid1sid2 through positioning of nucleosomes and/or by formation of a DNA loop in the sid1 upstream region. We have adapted a protocol developed for Aspergillus to the direct analysis of chromatin in Ustilago sporidia disrupted with sand. We have determined that the nucleosome repeat length is 174 +/- 7 bp. Studies of chromatin from wild type and the urbs1 mutant grown under iron replete and iron-starvation conditions revealed discrete and iron-dependent, nuclease hypersensitivity sites in the sid1/sid2 intervening promoter region. These data tentatively support the presence of an organized nucleosome array in the repressed state originating from the GATA sites currently known to be important for iron-mediated, and Urbs1-dependent regulation of these sid genes. A DNA construct containing the sid1 promoter linked in transcriptional fusion with the pyr6 gene of Ustilago was made for development of an iron-dependent auxotrophy selection scheme to isolate new deregulated mutants. A highly efficient transformation system based on electroporation of intact Ustilago sporidia was established.

Impacts
We can anticipate information that is broadly applicable to fungal pathogenesis will be obtained through studies of these two model fungi.

Publications

  • Chauhan, R.S. Farman, M.L. Ronald, P. and Leong, S.A. 1998. An Effort to Clone a Rice Blast Resistance Gene Corresponding to Avirulence Gene AVR1-CO39 of Magnaporthe grisea. Phytopathol. Farman, M.L. and Leong, S.A. 1998. Chromosome Walking to the AVR1-CO39 Avirulence Gene of Magnaporthe grisea: Discrepancy between the Physical and Genetic Maps. Genetics 150:1049-1058
  • Leong, S. Farman, M.L. Punekar, N. Yuan, W.M. Yukiko, E. Mayama, S. and Tosa, Y. 1998. AVR1-CO39, A Cultivar Specificity Gene from Magnaporthe grisea. 2nd International Rice Blast Conference, Abstr. p. S4 O-15. September 1998.
  • Chauhan, R.S. Farman, M.L. Ronald, P. and Leong, S.A. 1998. Molecular geneic analysis of blast resistance in CO39 Corresponding to Avirulence Gene AVR1-CO39 of Magnaporthe grisea. 2nd International Rice Blast Conference, Abstr. p. S1 O-3. September 1998.s
  • Leong, S.A. and Farman, M.L. 1998. A provisional U.S. patent for a "Cultivar specificity gene from the Rice Pathogen Magnaporthe grisea: Methods and Use." (February 1998)


Progress 01/01/97 to 12/31/97

Outputs
A homolog of yeast SNF2 was discovered immediately downstream of the Ustilago maydis sid2 gene. The sequence comparison of umsnf2 with yeast SNF2 showed 37.2% identity and 49.5% similarity. The gene has been disrupted and preliminary phenotypic analysis has revealed an unusal filamentous phenotype with filaments emanating into the solid culture medium. The proximity of umSnf2 to the sid1/2 genes may indicate that it plays a role in transcription activation of these genes. A protocol for rapid chromatin analysis in Ustilago was adapted from a method recently described from Aspergillus. Nucleosomal DNA was detected after micrococcal nuclease digestion and the unit size was estimated to be about 177 +/- 9 bp. Site-directed mutations were created in the 4 open reading frames found in the Magnaporthe grisea AVR1-CO39 gene in order to assess which one(s) may play a role in function of the gene. Mutations in ORF1 and 3 had the strongest impact on function of the gene with those in ORF3 being more significant. ORF3 was also expressed in a pET vector in Escherichia coli and used to spray plants carrying the corresponding resistance gene. Plants inoculated with a virulent strain Guy11 showed reduced lesion size and number when compared to control inoculations with Guy11 and an E. coli extract in which the AVR1-CO39 insert was absent in the vector. These data support the role of ORF3 in conferring avirulence in M. grisea. A survey of genomic DNA of 250 M. grisea isolates with the AVR1-CO39 gene was done to assess distribution of the gene in various host-specific forms. The gene was largely absent from rice-infecting isolates but was ubiquitous in Setaria-infecting isolates.

Impacts
(N/A)

Publications

  • Li, W., Yourman, L. F., Leong, S. A., Spear, R. N. and J. H. Andrews. 1997. Assay of B-glucuronidase activity in intact transformed fungal cells. Fungal Genetics Newsletter. 44:29-33.
  • An, Z., Zhao, Q., McEvoy, J., Yuan, W., Markley, J. and S. A. Leong. 1997. The C-terminal finger domain of Urbs1 is required for iron-mediated regulation of siderophore biosynthesis in Ustilago maydis. Proc. Natl. Acad. Sci. U.S.A. 94:5882-5887.
  • An, Z., Mei, B., Yuan, W. and S. A. Leong. 1997. The distal GATA sequences of the sid1 promoter mediate repression of siderophore production and interact directly with Urbs1, a GATA family transcription factor. EMBO J. 16:1742 - 1750.
  • Kachroo, P., Ahuja, M., Leong, S. A. and B. B. Chattoo. 1997. Organization and evolution of repeated DNA sequences in the rice blast fungus Magnaporthe grisea. Curr. Genet. 31: 361-336.
  • Nitta, N., Farman, M. and S. A. Leong. 1997. Genome organization of Magnaporthe grisea: Integration of genetic maps, clustering of transposable elements, and identification of genome duplications and rearrangements. Theor. Appl. Genetics 95:20-32.
  • Leong, S. A. and Z. An. 1997. Biosynthetic and molecular aspects of fungal siderophores. In: Iron and Related Transition Metals in Microbial Metabolism (G. Winkelmann and C. Carrano, eds.) Harwood, Chur. pp.51-82.
  • Leong, S. A. and G. Winkelmann. 1997. Molecular biology of iron transport in fungi. In: Metal Ions in Biological Systems, Vol 35, Iron Transport and Storage in Microorganisms, Plants and Animals (A. Sigel and H. Sigel, eds.) Marcel Dekker, New York.
  • Miller, M., Darwish, I, Ghosh, A., Ghosh, M., Hansel, J., Hu, J., Niu, C., Ritter, A., Scheidt, K., Suling, C., Sun, S., Zhang, D., Budde, A., De Clercq, E., Leong, S., Malouin, F. and U. Moellmann. 1997. Design, syntheses and studies of new antibacterial, antifungal and antiviral agents. In: Antiinfectives. Recent Advances in the Chemistry of Antiinfective Agents (Bentley, P. H. and O'Hanlon, P. J, eds), Royal Society of London, UK. pp.116-138
  • Leong, S. A., Gentil, G., Yuan, W. M., Zhao, Q., An, Z., Von Dohren, H. and J. Markley. 1997. Organization and regulation of a siderophore biosynthetic gene cluster in Ustilago maydis. In: Proceedings from the 1966 Genetics and Molecular Biology of Industrial Microorganism (Baltz, R., Hegeman, G. and Skatrud, P., eds.), American Society of Microbiology, Washington D. C. pp.89-91.


Progress 01/01/96 to 12/30/96

Outputs
The DNA sequence of the cyclic peptide synthetase gene (sid2) of Ustilago maydisfor ferrichrome biosynthesis revealed three domains for amino acid activation and peptide synthesis as well as a second putative motif per domain for phosphopantetheine binding. Mutational studies of the U. maydis transcription factor Urbs1 confirmed that the C-terminal finger domain but not the N-terminal domain are required for DNA binding. Mutational studies have revealed that two GATA sequences 1.6 kb upstream of the transcription initiation site of sid1 of U. maydis are required for iron-mediated regulation of sid1 in vivo. The AVR1-C039 cultivar specificity gene of Magnaporthe grisea was cloned by map-based cloning. DNA sequence analysis has revealed three small open reading frames within the 1 kb region containing the gene. A highly variable relationship of physical to genetic distance, ranging from 10 to 150 kb per cM, was observed in the 600 kb region surrounding AVR1-CO39. Genetic analysis of host resistance to AVR1-CO39 suggested a single dominant gene conditions resistance.

Impacts
(N/A)

Publications

  • Farman, M. L. and S. A. Leong. 1996. The AVRCO39 locus of the rice blast fungus.8th International Congress of the Society for Molecular Plant-Microbe Interactions, July 14-19, 1996. p. J-27.
  • An, Z., Farman, M. L., Taura, S. and S. A. Leong. 1996. New cosmid vectors for library construction, systematic chromosome walking and rapid restriction mapping in filamentous fungi. Gene 176:93-96.
  • Leong, S. A. 1996. Molecular genetic approaches to the study of fungal pathogenesis revisited. In: Biology of Plant-Microbe Interactions (G. Stacey, B.Mullin, and P. M. Gresshoff, eds.) ISMPI, St. Paul. pp. 219-222.
  • Leong, S. A., Farman, M. and Nitta, N. 1996. Genetic and molecular analysis of a cultivar specificity locus from the rice blast fungus Magnaporthe grisea. In: Rice Genetics III, Proceedings of the Third International Rice Genetics Symposium Kachroo, P.,
  • Leong, S. A. and B. B. Chattoo. 1996. Molecular analysis of repeated DNAs in the rice blast fungus. In: Rice Genetics III, Proceedings of the Third International Rice Genetics Symposium, Island Harbor Press, Manila. pp.279-291.
  • Leong, S. A., An, Z., Mei, B., McEvoy, J., Zhao, Q. and J. Markley. 1995. Sequences and proteins required for iron-regulated expression of sid1 of Ustilago maydis. Can. J. Bot. 73 (Suppl. 1):S140-S147.


Progress 01/01/93 to 12/30/93

Outputs
Promoter deletion studies of the sid1 (ornithine-N(superscript 5)-oxygenase) gene have identified a region which is required for iron regulated expression of the gene. Two of the urbs1 mutant alleles were sequenced and found to contain single base pair mutations in the second finger of the protein coding region suggesting that this motif is required for function of the protein. Antibiotic conjugates of siderophores, synthesized by Marvin Miller and coworkers at the University of Notre Dame, were evaluated against several plant pathogenic fungi. Norneoenactin and its hydroxamate siderophore conjugate were growth inhibitory to Ophiostoma ulmi. A new transposable element Pot1 which is structurally related to Fot1 of Fusarium oxysporum and Tc1 of Ceanorhabditis elegans was isolated and characterized from Magnaporthe grisea. The element encodes a single open reading frame of 544 amino acids which is bounded by perfect, inverted repeats of 44 bp. The element is present at approximately 100 copies per haploid genome of an Indian rice-infecting isolate and is found at high copy number in both rice and grass-infecting isolates.

Impacts
(N/A)

Publications


    Progress 01/01/92 to 12/30/92

    Outputs
    A genetic map was constructed for a cross between the rice isolate Guyll and thelaboratory strain 2539 of Magnaporthe grisea. Segregation data for RFLP markers, isoenzyme loci and the mating-type locus was analyzed using sixty-one progeny of this cross. Segregation of telomeric RFLPs allowed twelve telomeres to be genetically and/or physically mapped. Linkage groups were assigned to corresponding chromosomes by hybridization of selected markers to Southern blots of electrophoretically-separated chromosomes. A single gene that controls cultivar specificity on rice cultivar CO39 was mapped to one end of chromosome one. The Alu-like repetitive DNA element from M. grisea contains Long Terminal Repeat (LTR) sequences and the internal region shows homology, at the peptide level, to reverse transcriptases of the Gypsy class of retrotransposons. DNA sequence analysis combined with Northern hybridization analysis and primer extension analysis studies has shown that the ornithine-N5-oxygenese gene of Ustilago maydis is complex consisting of one large open reading frame with amino acid sequence similarity to iucD of the Escherichia coli aerobactin operon and three more smaller exonic regions. Two methods for tagging single-copy DNA in nuclei have been successfully applied in sporida of Ustilago maydis. One utilizes a fluorochrome-tagged antibody that recognizes a digoxygenin-labeled DNA probe while the other involves primer-induced sequence-specific labelling with fluorescein-12-dUTP.

    Impacts
    (N/A)

    Publications


      Progress 01/01/91 to 12/30/91

      Outputs
      This project was supported by NIH grant GM and Rockfeller Foundation Grant. Genes of the high affinity iron transport system of Ustilago maydis have been further characterized by DNA sequence analysis, gene distruption, Northern hybirdization analysis and cDNA analysis. The ornithine-N-oxgenase encodes a 67 Kd protein which is translated from an iron-regulated mRNA of 2.3 kb and urbsl (Ustilago regulator or biosynthesis of siderophores) encodes a 950 amino acid protein that contains two putative Zn finger motis similar to those found in other activators of transcription. This latter gene is expressed constitutively. Partial purificaation of the oxygenase has resulted in a 200 fold increase in specific activity of the enzyme. Purification of the enzyme will facilitate the design of inhibitors of enzyme activity. These may be useful in control of fungal diseases. Hybridization of the clone b and a mating-type alleles of U. maydis to genomic DNA from other smut fungi, as well as other basidiomycetes and ascomycetes has demonstrated the presence of homologues of these genes in U. hordei and possible other closely related smut fungi. Cleavage of the E. coli lac operator sequence within the genome of Magnaporthe grisea has been achieved. However, complete cleavage has not been observed at all chromosomal locations. Improved methods for karyotype analysis have been developed; routine separation of chromosomes 2, 3, and 4 has been achieved. These techniques will facilitate studies on genome organiztion.

      Impacts
      (N/A)

      Publications

      • VOISARD, C., WANG, J., XU, P. and LEONG, S.A. 1991. Cloning of a gene involved in the regulation of siderophore biosynthesis in Ustilago maydis. 16th Fungal Genetics Conference, Asilomar, California, April 1991.
      • MEI, B., BUDDE, A.D. and LEONG, S.A. 1991. Characterization of an ornithine-N_5-oxygenase gene from Ustilago maydis. 16th Fungal Genetics Conference, Asilomar, California, April 1991.
      • LEONG, S., FARMAN, M., BUDDE, A., TAYLOR, P., TOOLEY, P., LEUNG, H., SMITH, R. and CANTONE, F. 1991. Molecular analysis of pathogenesis in Magnaporthe grisea. Rockfeller Rice Biotechnology Conference, Tuscon, Arizona, October 1991.
      • FARMAN, M. and LEONG S.A. 1991. Molecular techniques for analysis of pathogensis in Magnaporthe grisea. 16th Fungal Geetics Conference, Asilomar, California, April 1991.
      • FROELIGER, E. and LEONG, S.A. 1991. The a mating-type alleles of Ustilago maydis are idiomorphs. Gene 100:113-122.


      Progress 01/01/90 to 12/30/90

      Outputs
      A cosmid vector useful for chromosome walking experiments and a genomic DNA library having 5500 clones were constructed for M.grisea, the cause of rice blast disease. The Escherichia coli lacO sequence and a genomic DNA fragment useful in detecting RFLPs in M. grisea were incorporated into a transformation vector for M. grisea. The resultant vector was targeted to the RFLP locus in the M. grisea genome. We are currently investigating whether we can cleave the genome using the "Achilles Heel Cleavage" scheme. The two alleles of the (alpha) and six alleles of the (beta) mating type loci of Ustilago maydis, the cause of corn smut, have been cloned and their structure was studied. The six (beta) alleles were each found to encode an open reading frame of 410 amino acids. The different (beta) alleles showed considerable sequence similarity; the N-proximal portion of the amino acid sequence (110 amino acids) was more variable between alleles than the C-terminal portion (300 amino acids). By contrast, restriction mapping studies and DNA hybridization analysis of the two (alpha) alleles indicated that they are dissimilar. Disruption of (beta) alleles in haploid cells gave rise to sterile, nonpathogenic mutants; disruption of a /beta/1 allele in a pathogenic diploid ((beta)1/(beta)2) produced a nonpathogenic mutant. These data indicate that the (beta) locus acts in a positive manner to control sexual development and pathogenicity.

      Impacts
      (N/A)

      Publications