Neurospora crassa Protein Arginine Methyl Transferases Are Involved in Growth and Development and Interact with the NDR Kinase COT1 Daria Feldman, Carmit Ziv, Rena Gorovits, Michal Efrat, Oded Yarden* Department of Plant Pathology and Microbiology, The R.H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel Abstract The protein arginine methyltransferaseas (PRMTs) family is conserved from yeast to human, and regulates stability, localization and activity of proteins. We have characterized deletion strains corresponding to genes encoding for PRMT1/3/5 (designated amt-1, amt-3 and skb-1, respectively) in Neurospora crassa. Deletion of PRMT-encoding genes conferred altered Arg-methylated protein profiles, as determined immunologically. Damt-1 exhibited reduced hyphal elongation rates (70% of wild type) and increased susceptibility to the ergosterol biosynthesis inhibitor voriconazole. In namt-3, distances between branches were significantly longer than the wild type, suggesting this gene is required for proper regulation of hyphal branching. Deletion of skb-1 resulted in hyper conidiation (2-fold of the wild type) and increased tolerance to the chitin synthase inhibitor polyoxin D. Inactivation of two Type I PRMTs (amt-1 and amt-3) conferred changes in both asymmetric as well as symmetric protein methylation profiles, suggesting either common substrates and/or cross-regulation of different PRMTs. The PRMTs in N. crassa apparently share cellular pathways which were previously reported to be regulated by the NDR (Nuclear DBF2-related) kinase COT1. Using co-immunprecipitation experiments (with MYC-tagged proteins), we have shown that SKB1 and COT1 physically interacted and the abundance of the 75 kDa MYC::COT1 isoform was increased in a Dskb-1 background. On the basis of immunological detection, we propose the possible involvement of PRMTs in Arg- methylation of COT1. Citation: Feldman D, Ziv C, Gorovits R, Efrat M, Yarden O (2013) Neurospora crassa Protein Arginine Methyl Transferases Are Involved in Growth and Development and Interact with the NDR Kinase COT1. PLoS ONE 8(11): e80756. doi:10.1371/journal.pone.0080756 Editor: Michael Freitag, Oregon State University, United States of America Received August 6, 2013; Accepted October 11, 2013; Published November 19, 2013 Copyright: ß 2013 Feldman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research project was supported by DFG research grant SE1054/3-2, by The Israel Science Foundation and by the German-Israel Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Oded.Yarden@mail.huji.ac.il Introduction Methylated derivatives of arginine were identified 45 years ago [1]. Since then, protein methylation has been recognized as a post translational modification that plays regulatory roles in signal transduction, nucleic transport, activation and repression of genes and mRNA splicing [2]. Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) that since being identified [3,4] have been found in the genomes of many eukaryotic organisms [5]. The PRMTs are divided into four major types. Type I and II (conserved from yeast to human) are responsible for monomethylation. They catalyze asymmetric (aDMA) and symmetric (sDMA) dimethylarginine, respectively. Type III and IV PRMTs catalyze the monomethylation of arginine residues and the formation of monomethylarginine on guanidinium nitrogen, respectively. So far, Type III and IV PRMTs have only been found in higher eukaryotes [2]. In human cells there are nine genes considered to encode for PRMTs. Among them, only PRMT1 and PRMT3 (Type I), and PRMT5 (Type II) are conserved through eukaryotic evolution, including members of the fungal kingdom [6]. In fungi, PRMTs have been shown to be involved in several cellular functions. For example, PRMT1 has been shown to be important for coping with oxidative stress. In Aspergillus nidulans,a concentration of 3 mM H 2 O 2 resulted in more than 50% growth retardation of DrmtA. At 6 mM H 2 O 2, conidiation was completely abolished [7]. PRMT1 was also shown to be important for hyphal development in Coprinopsis cinerea [8] and for pathogenicity in Fusarium graminearum, where DON mycotoxin production and virulence on flowering wheat heads were reduced, and increased sensitivity to oxidative and membrane stresses was observed [9]. PRMT5 was found to play a role in cell morphology in Schizosaccharomyces pombe [10] and Saccharomyces cerevisiae [11], in the increased sensitivity to oxidative stress and elevated temper- ature in A. nidulans [7] and required for full virulence in F. graminearum [9]. So far, deletion strains of PRMT3 have not been shown to affect morphology in any of the fungi analyzed [7,9]. The NDR (Nuclear Dbf2-related) protein kinases are part of the AGC kinase family and belong to the Ser/Thr kinase subgroup [12]. NDR kinases are essential components of important cellular processes such as morphological switches, cell proliferation, mitotic exit and apoptosis in a variety of eukaryotic organisms, and are regulated by co-activators of the MOB (Mps1-one binder) family [13]. N. crassa COT1 is the founding member of the NDR kinase family, and a single amino acid substation (within the catalytic domain) results in slow hyphal elongation accompanied by hyperbranching at temperatures above 32uC [14–16]. COT1 kinase was shown to be regulated both transcriptionally [17] as well as post transcriptionally, where phosphorylation was shown to PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e80756