Generation of thermo-sensitive allele of the TPR like protein Nup211by PCR mutagenesis

Motivation: TPR proteins are conserved large coiled-coil proteins that localize at the nucleoplasmic side of the nuclear pore complex and participate in multiple aspects of DNA metabolism. The protein Nup211, fission yeast homolog of Mlp1/Mlp2/Tpr, participate in the mRNA export and is essential for vegetative growth. The aim of this work is to create a collection of thermo-sensitive alleles of nup211.Methods: To create the collection, we have generated a new strain with the nup211 gen tagged with GFP at the amino terminal extreme and confirmed by fluorescent microscopy that the protein Nup211 localized in the nuclear envelop. Then, we have carried out a Taq PCR-based Random Mutagenesis with reduced concentration of dATP. The PCR products were transformed into a wild type strain to generate conditional mutants. The transformants obtained whose growth was impaired at 36ºC were preselected as thermo-sensitive mutants. To confirm the growth deficiency of these clones, a drop assay was performed and the best candidates were selected. These thermo-sensitive mutants were cultivated at 25ºC as well as 36ºC and both cultures were subjected to various experiments in order to study any changes in the localization of Nup211.Results: Up to now, we have demonstrated by fluorescent microscopy that the thermo-sensitive mutants show a modified nuclear distribution of Nup211 and different cellular phenotype, suggesting that the differents clones might represent differents nup211 thermo-sensitive alleles. These alleles are going to be subjected to various experiments to clarify the role of the protein in the mRNA export

MoSfl1 Is Important for Virulence and Heat Tolerance in Magnaporthe oryzae

The formation of appressoria, specialized plant penetration structures of Magnaporthe oryzae, is regulated by the MST11-MST7-PMK1 MAP kinase cascade. One of its downstream transcription factor, MST12, is important for penetration and invasive growth but dispensable for appressorium formation. To identify additional downstream targets that are regulated by Pmk1, in this study we performed phosphorylation assays with a protein microarray composed of 573 M. oryzae transcription factor (TF) genes. Three of the TF genes phosphorylated by Pmk1 in vitro were further analyzed by coimmunoprecipitation assays. One of them, MoSFL1, was found to interact with Pmk1 in vivo. Like other Sfl1 orthologs, the MoSfl1 protein has the HSF-like domain. When expressed in yeast, MoSFL1 functionally complemented the flocculation defects of the sfl1 mutant. In M. oryzae, deletion of MoSFl1 resulted in a significant reduction in virulence on rice and barley seedlings. Consistent with this observation, the Mosfl1 mutant was defective in invasive growth in penetration assays with rice leaf sheaths. In comparison with that of vegetative hyphae, the expression level of MoSFL1 was increased in appressoria and infected rice leaves. The Mosfl1 mutant also had increased sensitivity to elevated temperatures. In CM cultures of the Mosfl1 and pmk1 mutants grown at 30°C, the production of aerial hyphae and melanization were reduced but their growth rate was not altered. When assayed by qRT-PCR, the transcription levels of the MoHSP30 and MoHSP98 genes were reduced 10- and 3-fold, respectively, in the Mosfl1 mutant. SFL1 orthologs are conserved in filamentous ascomycetes but none of them have been functionally characterized in non-Saccharomycetales fungi. MoSfl1 has one putative MAPK docking site and three putative MAPK phosphorylation sites. Therefore, it may be functionally related to Pmk1 in the regulation of invasive growth and stress responses in M. oryzae

RNA metabolism is the primary target of formamide in vivo

The synthesis, processing and function of coding and non-coding RNA molecules and their interacting proteins has been the focus of a great deal of research that has boosted our understanding of key molecular pathways that underlie higher order events such as cell cycle control, development, innate immune response and the occurrence of genetic diseases. In this study, we have found that formamide preferentially weakens RNA related processes in vivo. Using a non-essential Schizosaccharomyces pombe gene deletion collection, we identify deleted loci that make cells sensitive to formamide. Sensitive deletions are significantly enriched in genes involved in RNA metabolism. Accordingly, we find that previously known temperature-sensitive splicing mutants become lethal in the presence of the drug under permissive temperature. Furthermore, in a wild type background, splicing efficiency is decreased and R-loop formation is increased in the presence of formamide. In addition, we have also isolated 35 formamide-sensitive mutants, many of which display remarkable morphology and cell cycle defects potentially unveiling new players in the regulation of these processes. We conclude that formamide preferentially targets RNA related processes in vivo, probably by relaxing RNA secondary structures and/or RNA-protein interactions, and can be used as an effective tool to characterize these processes