Recombinant expression of the coat protein of Botrytis virus X and development of an immunofluorescence detection method to study its intracellular distribution in Botrytis cinerea

Abstract

Botrytis cinerea is infected by many mycoviruses with varying phenotypical effects on the fungal host, including Botrytis virus X (BVX), a mycovirus that has been found in several B. cinerea isolates worldwide with no obvious effects on growth. Here we present results from serological and immunofluorescence microscopy (IFM) studies using antiserum raised against the coat protein of BVX expressed in Escherichia coli fused to maltose-binding protein. Due to the high yield of recombinant protein it was possible to raise antibodies that recognized BVX particles. An indirect ELISA, using BVX antibodies, detected BVX in partially purified virus preparations from fungal isolates containing BVX alone and in mixed infection with Botrytis virus F. The BVX antiserum also proved suitable for IFM studies. Intensely fluorescing spots (presumed to be virus aggregates) were found to be localized in hyphal cell compartments and spores of natural and experimentally infected B. cinerea isolates using IFM. Immunofluorescently labelled sections through fungal tissue, as well as fixed mycelia grown on glass slides, showed aggregations of virions closely associated with fungal cell membranes and walls, next to septal pores, and in hyphal tips. Also, calcofluor white staining of mature cell walls of virus-transfected Botrytis clones revealed numerous cell wall areas with increased amounts of chitin/glycoproteins. Our results indicate that some BVX aggregates are closely associated with the fungal cell wall and raise the question of whether mycoviruses may be able to move through the wall and therefore not be totally dependent on intracellular routes of transmission.