The fungus Neotyphodium lolii is an endophytic symbiont. It grows in the intercellular spaces of the perennial ryegrass Lolium perenne, producing secondary metabolites which enhance the fitness of the association over that of uninfected L. perenne. We report that the average number of hyphal strands in a given section of a leaf remains constant during the life of a leaf, indicating synchrony of leaf and hyphal extension, including cessation of hyphal extension when leaf extension ceases. We used a constitutively expressed reporter gene as an indicator of the mycelium's metabolic activity during and after hyphal extension. Reporter gene activity decreased when the mycelium stopped extending in liquid culture but not in planta. This indicates that in planta endophyte hyphae remain metabolically highly active when extension has ceased and throughout the life of the leaf they are colonizing. The behavior of the fungus in planta indicates the existence of signaling pathways which (i) synchronize the extension of leaf and hypha by regulating hyphal extension, (ii) suppress hyphal branching, and (iii) stop apical extension of fungal hyphae, without reducing the mycelium's metabolic activity. These signals may be crucial for the symbiosis, by allowing the endophyte to switch the focus of its metabolic activity from extension to the production of secondary metabolites.Neotyphodium endophytes are mutualistic symbionts of grasses, living in their intercellular spaces. Their presence confers upon the grass a number of physiological characteristics such as drought resistance, enhanced growth, and protection from herbivores, through the synthesis of feeding deterrents and toxins. In return, the plant provides nutrients and propagates the endophyte via its seeds (17,28,34,35,46,47).We have recently used a Neotyphodium endophyte, transformed with the Escherichia coli ␤-glucuronidase gene (GUS reporter gene system [14]), under the control of a heterologous constitutive promoter, to estimate the in planta distribution of endophyte metabolic activity (as GUS activity) (12). We found that GUS activity followed well-defined patterns, established during the growth of ryegrass leaves (12). The endophyte metabolic activity in plant tissue measured in such experiments depends on (i) the number of endophyte hyphae present, (ii) the rate of gene expression in these hyphae, and (iii) the half-life of the GUS enzyme. Thus, while the existence of these patterns was intriguing, these experiment allowed only limited conclusions as to how they are generated. We have now determined directly the in planta distribution of endophyte hyphal biomass. In parallel, we have determined the endophyte's constitutive GUS expression in the same ryegrass tillers. We have also determined GUS activity in exponentially growing and stationary laboratory cultures of the same endophyte strain. Together, these data allow us to draw conclusions regarding the endophyte's metabolic state in different parts of the ryegrass tiller. MATERIALS AND METHODSStrains and plant ...

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