Functional characterization of genes encoding putative nuclear movement and positioning - associated proteins in Magnaporthe oryzae

Abstract

Rice blast pathogen, Magnaporthe oryzae, causes serious damage to global rice production and has been emerged as a model organism for the characterization of molecular mechanisms relevant to pathogenic development in host plants. Nuclei migration and distribution are known as important processes in development of infection structures called appressorium in M. oryzae. Two genes homologous to ApsA and ApsB in Aspergillus nidulans were selected to understand nuclear migration in M.oryzae. In A. nidulans, ApsA and ApsB proteins are involved in the regulation of asexual reproduction and apsA and apsB deletion mutants showed defects in nuclear migration and positioning of the fungus.(Veith et al., 2005) However, little is known about the molecular mechanisms involved in nuclear distribution during conidiation in M. oryzae. Two genes were named Abnormal Nuclear Distribution (MoAND1 and MoAND2, respectively) and gene deletion mutant of MoAND1 was obtained by homologous recombination. The ΔMoand1 mutant showed defects in mycelial growth and conidiation. Observation of nuclei and septa after staining with Hoest33342 and Calcofluor White indicated that the ΔMoand1 mutant produced abnormal conidia such as one-, two-, four-, and five-celled conidia compared to three-celled wild-type conidia. To elucidate nuclei movement and positioning, RFP-tagged histones were introduced into both wild-type and ΔMoand1 strains. Microscopic observation of the ΔMoand1 strains with RFP-tagged histones revealed that nuclei distributed unevenly in hyphae and even some cells had no nucleus. Pathogenicity of ΔMoand1 was significantly reduced and this might be due to defects of appressorium formation and invasive growth in host cells. Furthermore, ΔMoand1 and double KO strain of MoAND1 and MoAND2 were more sensitive to microtubule-depolymerizing agent Benomyl, indicating that the mutants are defective in microtubule function. Taken together, these results represent that MoAND1 and MoAND2 are essential for pathogenicity as well as nuclei distribution in M. oryzae.