2002 Annual Report 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 and host recognition in the model plant pathogenic fungus, Magnaporthe grisea with grass hosts, in particular rice. This fungus is considered a premier example of fungal plant pathogens which can be manipulated both genetically and with the tools of molecular biology. Likewise rice has become the model plant for monocots. 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 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 and host recognition will be obtained through 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: Transient expression of introduced genes in plants is being used to study gene function in many plants. We are exploring both established (particle bombardment) and new (BMV) methods for transient expression of genes in grasses at the Plant Disease Resistance Research Unit. Routine transient transformation of rice leaves using plasmid vectors containing marker genes introduced by particle bombardment has been established. These methods will allow rapid functional analysis of genes in grass plants. B. Other Significant Accomplishment(s), if any: In cooperation with Tom German (University of Wisconsin), the host range of BMV (Brome mosaic virus) was examined by infecting different grasses using a leaf rubbing inoculation protocol at the Plant Disease Resistance Research Unit. Total RNA was isolated from inoculated plants two weeks post infection and the presence of the three BMV genomes was tested by RT-PCR. Infection and symptoms were observed as expected on barley varieties Hazen and Morex No infection or symptoms were observed on rice varieties Nipponbare, CO39, 51583 or fingermillet varieties Okhale-1 (E. coracana), KNE 100007 (coracana) and GBK 030647 (africana). D. Progress Report: This report serves to document research under a specific cooperative agreement between ARS and the University of Wisconsin. Additional details of research can be found in the report for the parent project, 3655-22000-013-00D, Molecular Analysis of Genes Controlling Pathogenesis of Ustilago Maydis and Magnaporthe Grisea. Methods for rapid analysis of genes introduced into plants using plasmid and plant viral vectors are being assessed with cooperator Tom German (University of Wisconsin). Gene complementation and gene silencing effects are under study. Preliminary data on the infection of barley, rice and finger millet with BMV RNAs was obtained. Methods for routine transformation of rice leaves using particle bombardment have been established. 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. To facilitate these studies, we are exploring the use of gene silencing and gene complementation in plants using viral vectors such as Brome Mosaic Virus as well as plasmid-based vectors. 6.What do you expect to accomplish, year by year, over the next 3 years? In collaboration with Tom German, our goals for the next year are to expand the number of rice and finger millet varieties in inoculation studies by BMV. We will examine infection by the traditional leaf rubbing assay and viral replication in protoplasts. In the following and subsequent years will examine the function of different candidate NBS-LRR genes at the Pi-CO39 (t) locus by BMV infection of barley and rice to either silence or complement gene function. Leaves and protoplasts will be examined as suitable host material. In parallel work, we will transform rice and barley leaves with the NBS-LRR gene candidates cloned on plasmids into leaves by particle bombardment with the AVRi-CO39 gene. Longterm stable transformation studies will begin once candidate genes are identified. 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? None. 8.List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) None.