Systematic functional analysis of calcium-signalling proteins in the genome of the rice-blast fungus, Magnaporthe oryzae, using a high-throughput RNA-silencing system.

We developed an RNA-silencing vector, pSilent-Dual1 (pSD1), with a convergent dual promoter system that provides a high-throughput platform for functional genomics research in filamentous fungi. In the pSD1 system, the target gene was designed to be transcribed as a chimeric RNA with enhanced green fluorescent protein (eGFP) RNA. This enabled us to efficiently screen the resulting transformants using GFP fluorescence as an indicator of gene silencing. A model study with the eGFP gene showed that pSD1-based vectors induced gene silencing via the RNAi pathway with slightly lower efficiency than did hairpin eGFP RNA-expressing vectors. To demonstrate the applicability of the pSD1 system for elucidating gene function in the rice-blast fungus Magnaporthe oryzae, 37 calcium signalling-related genes that include almost all known calcium-signalling proteins in the genome were targeted for gene silencing by the vector. Phenotypic analyses of the silenced transformants showed that at least 26, 35 and 15 of the 37 genes examined were involved in hyphal growth, sporulation and pathogenicity, respectively, in M. oryzae. These included several novel findings such as that Pmc1-, Spf1- and Neo1-like Ca(2+) pumps, calreticulin and calpactin heavy chain were essential for fungal pathogenicity.