Development of a GFP-expressing Ustilaginoidea virens strain to study fungal invasion and colonization in rice spikelets

• Transforming the U. virens strain by A. tumefaciens-mediated transformation
• Observing the expression of eGFP in pathogenic fungus U. virens
• Monitoring the infection processes of U. virens in rice flower organs by observing GFP

The fungal pathogen Ustilaginoidea virens is now widespread in most rice producing areas of the world and attacks the seed spikes of rice, causing sterility, reduced seed weight and accumulation of mycotoxins. The lack of appropriate data relating to the mechanisms of infection leaves a gap within the pathosystem, forcing the search for new methodologies for monitoring the disease. Here, we developed an efficient A. tumefaciens mediated transformation system for rice false smut disease. In addition, infection patterns were explored in rice using the green fluorescent protein gene (gfp) transformed U. virens. ATMT uses both the Hygromycin B resistance (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by PCR. Expression of the gfp gene did not affect the pathogenicity of the U. virens transformants. U. virens pathogenicity was examined on the floral structures using a fluorescence microscope. Fluorescence microscopy allowed in vivo imaging of this pathogen during infection of rice spikelets. These studies will allow us to develop strategies to determine the mechanisms of U. virens–rice interaction in greater detail and to apply functional genomics for the characterization of involved genes at the molecular level either by insertional mutagenesis or gene knock-out.