Dissecting the function of the different chitin synthases in vegetative growth and sexual development in Neurospora crassa

Abstract

a b s t r a c tChitin, one of the most important carbohydrates of the fungal cell wall, is synthesized by chitin synthases (CHS). Seven sequences encoding CHSs have been identified in the genome of Neurospora crassa. Previously, CHS-1, -3 and -6 were found at the Spitzenkörper (Spk) core and developing septa. We investigated the functional importance of each CHS in growth and development of N. crassa. The cellular distribution of each CHS tagged with fluorescent proteins and the impact of corresponding gene deletions on vegetative growth and sexual development were compared. CHS-2, -4, -5 and -7 were also found at the core of the Spk and in forming septa in vegetative hyphae. As the septum ring developed, CHS-2-GFP remained at the growing edge of the septum until it localized around the septal pore. In addition, all CHSs were located in cross-walls of conidiophores. A partial co-localization of CHS-1-m and CHS-5-GFP or CHS-2-GFP occurred in the Spk and septa. Analyses of deletion mutants suggested that CHS-6 has a role primarily in hyphal extension and ascospore formation, CHS-5 in aerial hyphae, conidia and ascospore formation, CHS-3 in perithecia development and CHS-7 in all of the aforementioned. We show that chs-7/ csmB fulfills a sexual function and chs-6/chsG fulfills a vegetative growth function in N. crassa but not in Aspergillus nidulans, whereas vice versa chs-2/chsA fulfills a sexual function in A. nidulans but not in N. crassa. This suggests that different classes of CHSs can fulfill distinct developmental functions in various fungi. Immunoprecipitation followed by mass spectrometry of CHS-1-GFP, CHS-4-GFP and CHS-5-GFP identified distinct putative interacting proteins for each CHS. Collectively, our results suggest that there are distinct populations of chitosomes, each carrying specific CHSs, with particular roles during different developmental stages.

Regulation of Neurospora crassa cell wall remodeling via the cot-1 pathway is mediated by gul-1

“…In contrast, Δchs-1, Δchs-3 and Δchs-7 exhibited slower growth (80 % of wild type), while Δchs-5 and Δchs-6 grew at only 35 % of the rate measured in the wild type. Excluding the case of the slow growing Δchs-5, results obtained were in line with those recently described (Fajardo-Somera et al 2015).…”

“…The fact that deletion/inactivation of some (but not all) single chs-encoding genes in N. crassa results in various changes in hyphal growth rates has been previously described (Beth Din and Yarden, 1994;Beth Din et al 1996;Sanchez-Leon et al 2011;Fajardo-Somera et al 2015). To verify the differences in growth rates of the different mutants under the conditions performed in this study, we first carried out comparative growth tests in which all chs deletion strains were examined in parallel.…”

“…All of the seven chs genes in N. crassa (Table 1) were found to be nonessential, suggesting redundancy in chs gene function (Beth Din and Yarden 1994;Beth Din et al 1996;Dunlap et al 2007;Riquelme and Bartnicki-Garcia 2008;SanchezLeon et al 2011;Fajardo-Somera et al 2015). Chitinases are also involved in a variety of functions associated with fungal growth (Kuranda and Robbins 1991;Dunkler et al 2005;Baker et al 2009).…”

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

“…, 2015; Sanchez‐Leon et al . , 2011; Takeshita, Yamashita, Ohta, & Horiuchi, 2006; Weber, Assmann, Thines, & Steinberg, 2006).…”

Hyphal ontogeny in Neurospora crassa: a model organism for all seasons

“…This hypothesis has been proven correct in the past few years. In N. crassa , all CHSs (CHS-1 to CHS-7) accumulate at the core of the Spk 58– 60 , whereas FKS-1, GS-1, and RGF-1 (RHO-1 GEF) accumulate at the periphery of the Spk 61– 63 . The localization pattern of CHS and the GSC in the inner and outer regions of the Spk corresponds to the microvesicular (chitosomes) and macrovesicular regions of the Spk ( Figure 1B and Figure 3), revealed by transmission electron microscopy in N. crassa and by electron tomography in A. nidulans 64, 65 .…”