Mir1 is Highly Upregulated and Localized to Nuclei During Infectious Hyphal Growth in the Rice Blast Fungus

Abstract

Rice blast, caused by Magnaporthe grisea, is a devastating disease of rice throughout the world. Many recent molecular studies have focused on the early infection stages, but our knowledge about molecular events at the infectious hyphae stage is limited. In this study, 750 hygromycin-resistant transformants were isolated by transforming M. grisea Guyll with a promoterless enhanced green fluorescent protein (EGFP) construct. In one of the transformants, L1320, EGFP signals were observed in the nuclei of infectious hyphae. The transforming vector was inserted in a predicted gene named MIR1 and resulted in a Mir1 1-107-EGFP fusion. Mir1 is a low-complexity protein with no known protein domain and has no homolog in GenBank or other sequenced fungal genomes. Quantitative real-time reverse-transcriptase polymerase chain reaction analysis and expression assays of MIR1-EGFP fusion constructs indicated that the expression of MIR1 was highly induced during plant infection. Deletion analyses identified a 458-bp region that was sufficient for the MIR1 promoter activity. Further characterization revealed that a 96-bp sequence was essential for the enhanced in planta expression. MIR1 is an M. grisea-specific gene that is highly conserved among the field isolates belonging to the M. grisea species complex. The mir1 mutants had no obvious defects in appressorial penetration and rice infection. When overexpressed with the RP27 promoter, nuclear localization of the Mir1-EGFP fusion was observed in conidia and vegetative hyphae. These data suggest that the expression but not the nuclear localization of MIR1 is specific to infectious hyphae and that reporter genes based on MIR1 may be suitable for monitoring infectious growth in M. grisea.