The Neurospora crassa colonial temperature sensitive 2, 4 and 5 (cot-2, cot-4 and cot-5) genes encode regulatory and structural proteins required for hyphal elongation and branching

The morphology and the genetic defects of theNeurospora crassa colonial temperature-sensitive-2, -4 and -5 mutants were analyzed. cot-2 is allelic to gs-1 and encodes a component of the glucan synthesis process. cot-4 encodes the catalytic subunit of a type 2B phosphatase and is allelic to calcineurin (cna-1). cot-5 encodes a homologue of the S. cerevisiae ALG2 manosyltransferase-encoding gene, a component of the dolichol pathway. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol55/iss1/8 32 Fungal Genetics Reports The Neurospora crassa colonial temperature sensitive 2, 4 and 5 (cot-2, cot-4 and cot-5) genes encode regulatory and structural proteins required for hyphal elongation and branching Zipi Resheat-Eini, Alex Zelter, Rena Gorovits, Nick D. Read and Oded Yarden* Department of Plant Pathology and Microbiology, The Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, 76100, Israel and Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK. *corresponding author, email: Oded.Yarden@huji.ac. il Fungal Genetics Reports 55:32-36 The morphology and the genetic defects of the Neurospora crassa colonial temperature-sensitive-2, -4 and -5 mutants were analyzed. cot-2 is allelic to gs-1 and encodes a component of the glucan synthesis process. cot-4 encodes the catalytic subunit of a type 2B phosphatase and is allelic to calcineurin (cna-1). cot-5 encodes a homologue of the S. cerevisiae ALG2 manosyltransferase-encoding gene, a component of the dolichol pathway. A group of five non-allelic Neurospora crassa colonial temperature sensitive (cot) mutants was described by Garnjobst and Tatum (1967). The cot-1 gene was found to encode a Ser/Thr protein kinase (Yarden et al. 1992) which is the founding member of the NDR kinase family. The nature of the cot-3 defect has also been analyzed and the cot-3 gene was found to encode protein elongation factor 2 (Propheta et al. 2001). In order to expand our understanding of the genetic defects that can confer abnormal hyphal elongation/branching patterns, we have performed morphological and genetic analyses of the three remaining cot mutants isolated by Garnjobst and Tatum. We found that even though they all exhibit compact temperaturesensitive macroscopic colonial features, their microscopic hyphal morphology and branching patterns differ. Furthermore, the genetic defects involved in conferring their phenotypes include both regulatory as well as structural factors, all of which are required for maintaining proper hyphal elongation and branching patterns. Confocal microscopic examination, using the membrane-selective dye FM4-64 (as described by Hickey et al. 2005) of Neurospora crassa wild-type (74-OR23-1A; FGSC987), cot-1 (FGSC 4065), cot-2 (FGSC 1512), cot-3 (FGSC 1517), cot4 (FGSC 3600) and cot-5 (FGSC 1362), revealed significant morphological differences between the different strains (Fig. 1). As the morphological features of cot-1 and cot-3 have been studied in depth (Collinge and Trinci, 1974; Collinge et al. 1978; Propheta et al. 2001), we focused on the quantification of the observed differences on cot-2, cot-4 and cot-5. Hyphal extension rates of cot-2 cot-4 and cot-5 were measured on a standard solid medium at permissive and restrictive conditions. All of the mutants exhibited a significant reduction (75 to 99%) in elongation rates (to 0.15±0.03 mm/h, 0.27±0.05 mm/h and negligible elongation for cot-2, cot-4 and cot-5, respectively) and an increase in branching rates when cultured at the restrictive temperature. Even though the mutant's phenotypes are clearly temperature sensitive, we found that their branching rates were significantly higher (60 to 160%) even at the permissive temperatures (Table 1). For the most part, the hyperbranching patterns observed are lateral, rather than apical (Watters et al. 2000). Nonetheless, some apical/dichotomous branching was evident in the cot-5 strain (regardless of temperature; Fig. 1j-k). Figure 1. Morphology of wild type and colonial temperature sensitive strains of N. crassa at permissive (24 C) and restrictive (37 C) temperatures. Fungi were stained with FM4-64 and imaged using a confocal microscope. (a) cot-1 grown at 24 C; (b) cot-1, 37 C; (c) wild type, 37 C; (d) cot-2, 24 C; (e) cot-2, 37 C; (f) cot-3, 24 C; (g) cot-3, 37 C; (h) cot4, 24 C; (i) cot-4, 37 C; (j) cot-5, 24 C; (k) cot-5, 37 C. Bars are 50 um. Published by New Prairie Press, 2017