Purification of Fungal Haustoria from Infected Plant Tissue by Flow Cytometry

Abstract

A hallmark of biotrophy in many fungal plant pathogens is the formation of a specialized pathogenic structure called the haustorium from infectious hyphae. This is the major parasitic structure, where nutrients are taken up from the host and pathogenicity factors are exported to the host tissue. Obligate biotrophic fungi can typically be cultured in vivo only to a limited extent and do not produce haustoria under these conditions. This has hampered the application of classic molecular biology techniques to haustoria-forming pathogens. The lectin Concanavalin A (Con A), which binds specifically to sugars present on the exterior of rust haustoria, was first used in a column-based affinity purification procedure in 1992 (Hahn and Mendgen, Protoplasma 170:95–103, 1992). Here we describe a new technique where we combine initial gradient purification of haustoria with flow-sorting based on labeling of haustoria with fluorescent Con A. Our method allows haustorial isolation with purity above 98 % and yields ten times more isolated haustoria in a single experiment than the previous procedure.