In-response to a phytohormone, gibberellic acid, the aleurone layers of barley seeds synthesize and secrete a-amylages, which are coded by a set of stable mRNAs. When aleurone layers are subjected to heat shock treatment, the synthesis of a-imylase is suppressed while heat shock proteins are induced. The suppression of a-amylase synthesis is not the result of translational control as reported in several other systems. Rather, the sequences of a-amylase mRNA are rapidly degraded during heat shock as shown by in vitro translation and dot blot hybridization with a cDNA probe. Upon recovery from heat shock, the tissue resumes the synthesis of a-amylase in 24 hr. However, in the presence of a transcription inhibitor, cordycepin, the resumption of synthesis of ao-amylase does not take place, indicating that new transcription of a-amylase genes is necessary for this recovery process. The degradation of a-amylase mRNAs correlates with the rapid destruction of endoplasmic reticulum as observed by electron microscopy, a phenomenon that has not been reported previously as a heat shock response. Since a-amylase mRNA is associated with the endoplasmic reticulum via membranebound polyribosomes, we suggest that the destruction of the endoplasmic reticulum during heat shock causes the destabilization and the eventual degradation of a-amylase mRNA.

One of the most important plant-fungal symbiotic relationships is that of cool season grasses with endophytic fungi of the genera Epichloë and Neotyphodium. These associations often confer benefits, such as resistance to herbivores and improved drought tolerance, to the hosts. One benefit that appears to be unique to fine fescue grasses is disease resistance. As a first step towards understanding the basis of the endophyte-mediated disease resistance in Festuca rubra we carried out a SOLiD-SAGE quantitative transcriptome comparison of endophyte-free and Epichloë festucae-infected F. rubra. Over 200 plant genes involved in a wide variety of physiological processes were statistically significantly differentially expressed between the two samples. Many of the endophyte expressed genes were surprisingly abundant, with the most abundant fungal tag representing over 10% of the fungal mapped tags. Many of the abundant fungal tags were for secreted proteins. The second most abundantly expressed fungal gene was for a secreted antifungal protein and is of particular interest regarding the endophyte-mediated disease resistance. Similar genes in Penicillium and Aspergillus spp. have been demonstrated to have antifungal activity. Of the 10 epichloae whole genome sequences available, only one isolate of E. festucae and Neotyphodium gansuense var inebrians have an antifungal protein gene. The uniqueness of this gene in E. festucae from F. rubra, its transcript abundance, and the secreted nature of the protein, all suggest it may be involved in the disease resistance conferred to the host, which is a unique feature of the fine fescue–endophyte symbiosis.

Thiamine or vitamin B-1, is an essential constituent of all cells since it is a cofactor for two enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. Thiamine is synthesized by plants, but it is a dietary requirement for humans and other animals. The biosynthetic pathway for thiamine in plants has not been well characterized and none of the enzymes involved have been isolated. Here we report the cloning and characterization of two cDNAs representing members of the maize thi1 gene family encoding an enzyme of the thiamine biosynthetic pathway. This assignment was made based on sequence homology to a yeast thiamine biosynthetic gene and by functional complementation of a yeast strain in which the endogenous gene was inactivated. Using immunoblot analysis, the thi1 gene product was found to be located in a plastid membrane fraction. RNA gel blot analysis of various tissues and developmental stages indicated thi1 expression was differentially regulated in a manner consistent with what is known about thiamine synthesis in plants. This is the first report of cDNAs encoding proteins involved in thiamine biosynthesis for any plant species.

Mutualistic fungal endophytes infect many grass species and often confer benefits to the hosts such as reduced herbivory by insects and animals. The physiological interactions between the endophytes and their hosts have not been well characterized. Fungal-secreted proteins are likely to be important components of the interaction. In the interaction between Poa ampla and the endophyte Neotyphodium sp., a fungal ␤-1,6-glucanase is secreted into the apoplast, and activity of the enzyme is detectable in endophyte-infected plants. Sequence analysis indicates the ␤-1,6-glucanase is homologous to enzymes secreted by the mycoparasitic fungi Trichoderma harzianum and Trichoderma virens. DNA gel-blot analysis indicated the ␤-1,6-glucanase was encoded by a single gene. As a secreted protein, the ␤-1,6-glucanase may have a nutritional role for the fungus. In culture, ␤-1,6-glucanase activity was induced in the presence of ␤-1,6-glucans. From RNA gel blots, similar ␤-1,6-glucanases were expressed in tall fescue (Festuca arundinacea Schreb.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman) infected with the endophyte species Neotyphodium coenophialum and Epichloë festucae, respectively.Fungal endophytes of the genus Neotyphodium (formerly Acremonium; Glenn et al., 1996) infect many grass species, some of which are important turf and forage grasses. The fungi colonize the intercellular spaces of the aerial plant parts but do not invade the plant cells. The endophyte-grass associations are generally considered to be mutualistic symbioses (Clay, 1988). In many associations, the production of alkaloids by the fungus results in reduced herbivory by insects and animals, thus benefiting the host (Breen, 1994;Bush et al., 1997). The fungi benefit from the access to nutrients provided by the plants.Within the past 20 years, considerable knowledge has been gained on the synthesis and effects of alkaloids, the genetics and taxonomic relationships of endophytes, and the ecological effects of endophyte infection (Clay, 1990;Siegel and Schardl, 1991;Schardl, 1996;Bush et al., 1997). The physiological aspects of the endophyte-grass interactions have not, however, been well characterized in any system. We are investigating the physiology of the fungus-grass interaction with the long-range objective of eventually being able to manipulate agriculturally important interactions. We are using the Poa ampla cv Service (big bluegrass)/Neotyphodium sp. interaction as a model system for the grass/fungus interaction (Lindstrom et al., 1993). P. ampla is apomictic, so we have a ready supply of plants of identical genotype. We also have uninfected plants of the identical genotype, which were identified in older seed lots in which the endophyte had lost viability.Almost nothing is known of the proteins relevant to the interaction between the plant hosts and the fungal endophytes. We are interested in fungalsecreted proteins because they are likely to be important components of the mutualistic interaction because they are located at the...

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