9603439 Nicholson The project deals with the biochemical basis of disease resistance in sorghum. The overall goal is to determine the route of synthesis of the sorghum phytoalexins which are 3-anthocyanidins. Phytoalexin synthesis will be studied as it occurs in response to infection by the fungi Colletotrichum sublineolum and Bipolaris maydis. The work will include investigation of the route of biosynthesis of the phytoalexins, analysis of enzymes essential for phytoalexin synthesis, comparison of the fungal infection process in mature leaves with that which occurs in juvenile leaves, and determining if specific members of the phenylalanine ammonia-lyase and chalcone synthase gene families are associated with phytoalexin synthesis. In the first objective, metabolic intermediates in phytoalexin synthesis will be identified. This will be done by trapping intermediates using 14C as a radio label tracer. Compounds will be identified by high performance liquid chromatography in combination with Plasma Desorption Mass Spectrometry and Fast Atom Bombardment mass spectrometry. The second objective will be to determine whether the levels of activity of two enzymes essential for phytoalexin synthesis increase in response to infection. Although the enzymes are necessary for the synthesis of the two most fungitoxic phytoalexins, they have never been assayed in sorghum. The first enzyme is flavonoid-3'-hydroxylase which is required for hydroxylation at the 3' position to form luteolinidin. The second is an O-methyltransferase which is essential for the formation of the most fungitoxic phytoalexin, 5-methoxyluteolinidin. The enzymes will be assayed in time course studies that follow phytoalexin synthesis. In the third objective, elements of the C. sublineolum infection process will be studied to determine if the expression of host resistance is associated with the ability of the fungus to penetrate and colonize leaves of different ages. This is important because juvenile plants are uniformly resistant t o C. subIineolum. However, when plants of a susceptible genotype mature they lose their ability to express resistance and become susceptible to the fungus. Methods to be used will include scanning electron microscopy in combination with micromanipulation and fluorescence microscopy to determine in vivo fungal growth and development and to identify the exact time of penetration. Patterns of phytoalexins synthesized as well as the time of synthesis in resistant and susceptible cultivars will be determined as a function of leaf age, and the information will be correlated with events in the infection process. In the final objective, gene-specific probes for members of the PAL and CHS gene families will be used to study the expression of these genes in response to fungal infection. This work will have a significant impact on understanding how grain crops protect themselves from diseases caused by fungi. Some plants, such as sorghum, produce unique compounds called phytoalexins when they are attacked by fungal pathogens. The phytoalexins are often very toxic to the pathogens and thus help the plant to resist disease. The aim of this project is to discover how the sorghum plant makes these important resistance compounds. The research will contribute substantially to understanding what controls the synthesis of these unique compounds. The work should enable the development of environmentally benign ways to protect important crops such as the grains.
