Use of Green Fluorescent Protein Expressing Colletotrichum falcatum, the Red Rot Pathogen for Precise Host–Pathogen Interaction Studies in Sugarcane

Abstract

Productivity in sugarcane, a vegetatively propagated crop, is affected by various diseases. Red rot caused by the fungal pathogen Colletotrichum falcatum Went is a devastating disease of sugarcane causing serious yield losses in India and in many other countries. Understanding the pathogenesis of this hemibiotrophic fungal pathogen within host tissue will deepen our knowledge on host–pathogen interaction and helps to develop effective disease management strategies. Green fluorescent protein (GFP) is an effective tool for exploring fungal biology during host–pathogen interactions. In the present study we have conducted detailed investigations using GFP transformed strain of C. falcatum to establish the pathogenesis, colonization and spread of this fungus inside host tissues. We transformed C. falcatum with eGFP using Agrobacterium-mediated transformation using hygromycin B as a selectable marker and the transgene was stably integrated with mitotic stability. The C. falcatum transformants maintained morphological characters and growth parameters as like the wild type and the virulence nature was not altered as compared to the wild-type C. falcatum. GFP-tagged C. falcatum pathotypes clearly demonstrated the variations in C. falcatum colonization during compatible and incompatible interactions with sugarcane. These variations were comparable to in planta studies conducted using non GFP-tagged C. falcatum and using calcofluor staining. This was further validated with histological assays with calcofluor staining. The study is first of its kind in sugarcane and C. falcatum and will give a better understanding of pathogenesis inside the host in a spatio-temporal manner.