2007 Annual Report 1a.Objectives (from AD-416) The goal of this project is to elucidate recognition mechanisms in the early phases of plant/pathogen interactions for the purpose of either revealing new opportunities for disease control or improving current control measures through increased understanding of these mechanisms. This study focuses on the role of oxidative metabolism, i.e. the production and regulation of prooxidants and antioxidants, in the earliest phases of the plant/pathogen interaction. This project specifically addresses the role/effect of extracellular or apoplastic antioxidants on the early events of the host/pathogen interaction. Objective 1: Determine the role/effect of plant apoplastic antioxidants in susceptibility or resistance during the early events in plant/bacterial interactions. Objective 2: Describe the role of prooxidants and antioxidants in the apoplast-infection droplet microenvironment in preventing successful penetration of the host by fungal spores. 1b.Approach (from AD-416) For bacterial pathogens, the first interaction with the plant generally occurs in the apoplast once the inoculum is absorbed into the tissue; for fungal pathogens the first interaction is often on the plant epidermis where, in many interactions, the apoplast interacts with the infection droplet. In this microenvironment a complex series of exchanges occurs between the host and pathogen affecting the oxidative balance - with prooxidants generally favoring the host (often causing localized cell death) and antioxidants favoring the pathogen. First we will use a bacteria/cell suspension model system, which mimics in planta recognition of Pseudomonas syringae by soybean, potato and tobacco, and is highly manipulable. Techniques and information gained from this system will be applied to examine the influence of the apoplast/infection-droplet microenvironment on spore germination of Magnaporthe grisea on rice leaves. A new noninvasive technique will allow quantification of the total oxidative burst as well as extracellular antioxidants. In addition to revealing the kinetics and key constituents of oxidative metabolism in the apoplast, we expect to gain quantitative and determinative information regarding its influence on bacterial and fungal interactions, apoplast redox status and cellular prooxidant-tolerance mechanisms. 3.Progress Report Determined the mechanism by which a widely used biological buffer interfered with and altered biochemical reactions involving peroxidase and phenolic radicals. When the buffer was present in much higher proportions, such as millimolar versus micromolar, in certain peroxidase assays in which phenolics radicals were produced, the pattern of utilization of extracellular phenolics drastically changed. Since these phenolics have bioactive properties this phenomenon could cause biochemical as well as physiological artifacts in model systems. This finding could be used by other scientists to reinterpret previous studies and avoid problems with future studies. 4.Accomplishments In the presence of plant cells, the combination of certain chemicals, which individually act as antioxidants, can create reactive oxygen species (ROS). When two phenolics commonly found in the extracellular environment were added to soybean cell suspensions, a burst of hydrogen peroxide was produced. A possible mechanism was suggested based on the results. This supports the existence of an active ROS generating mechanism other than the traditional membrane bound NADPH oxidase; the energy for the alternate cell wall mechanism would come from cell wall phenolics rather than NADPH. This finding could be used by other scientists to further examine resistance mechanisms in plant-pathogen interactions. NP303, Component 2. Extracellular phenolics have bioactivity. This demonstrates that these phenolics are able to regulate the interaction of the plant and pathogen. The identification of acetosyringone (MPPL, PQSL), a known bioactive phenolic in wounded tissue, suggested these phenolics were bioactive towards the pathogen. Further testing of this compound demonstrated that it was able to facilitate plant recognition of bacterial pathogens. The use of these bioactive phenolics to regulate plant pathogen interactions could lead scientists to new strategies for controlling plant diseases. NP303, Component 2. Extracellular phenolics are redox sensitive and inducible. This demonstrates that these compounds are produced upon contact with the pathogens and their concentration and bioactivity could play important roles in regulating the plant pathogen interaction. Individual compounds were quantified during a time period after contact with pathogens. In resistant interactions the compounds were oxidized and removed negating their bioactivity. This could be a significant mechanism to help the plant resist bacterial pathogens. NP303, Component 2. We had previously found that a pool of phenolic antioxidants buffered the extracellular environment of the plant from oxidative bursts produced during pathogenesis. We have identified, in cooperation with the Produce Quality and Safety Lab and the Environmental Quality Lab, a unique class of antioxidants that are induced in the extracellular space upon contact with pathogens. These compounds have properties that may enhance their ability to provide both chemical and physical protection from oxidants. NP303, Component 2. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Review Publications Costanzo, S., Ospina-Giraldo, M., Deahl, K.L., Baker, C.J., Jones, R.W. 2006. Gene duplication event in family 12 glycosyl hydrolase from phytophthora spp. Fungal Genetics and Biology. 43:707-714. Panina, Y., Fravel, D.R., Shcherbakova, L.A., Baker, C.J. 2007. Biocontrol and plant pathogenic fusarium oxysporum-induced changes in phenolic compounds in tomato leaves and roots. Journal of Phytopathology. 155:475-481.