Quantitative Assessment of in Planta Distribution of Metabolic Activity and Gene Expression of Neotyphodium Endophytes

Abstract

Using perennial ryegrass (Nui) infected with the Neotyphodium transformant (FM 13 (Murray et al., 1992) carrying the’ Escherichia coli B-D-glucuronidase gene (gusA) (GUS system (Jefferson, 1987) under control of a heterologous constitutive promoter, we have developed methods for extracting ≥ 85% of endophyte-associated GUS activity from plant material by grinding of frozen tissue followed by sonication. Fluorometric assays of these extracts using the substrate 4-methyl-umbelliferryl glucuronide allow quantitative assessment of the distribution of endophyte-associated GUS activity within single tillers with high resolution; ≤ 1 mg of dry weight of tissue are required for the assessment of GUS activity. Fluorescence microscopy with the dye Imagene Green can in addition visualize individual GUS-expressing hyphae (Fig. 1).