Fajardo-Somera Rosa A, Jöhnk Bastian, Bayram Özgür, Valerius Oliver, Braus Gerhard H, Riquelme Meritxell
Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada, CICESE, Ctra. Ensenada-Tijuana No. 3918, Ensenada, Baja California C.P. 22860, Mexico.
Department of Molecular Microbiology and Genetics, Georg-August Universität Göttingen, Grisebachstrasse 8, 37077 Göttingen, Germany.
Fungal Genet Biol. 2015 Feb;75:30-45. doi: 10.1016/j.fgb.2015.01.002. Epub 2015 Jan 13.
Chitin, 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.
几丁质是真菌细胞壁中最重要的碳水化合物之一，由几丁质合酶（CHS）合成。在粗糙脉孢菌的基因组中已鉴定出七个编码CHS的序列。此前，在顶体（Spk）核心和正在形成的隔膜中发现了CHS-1、-3和-6。我们研究了每种CHS在粗糙脉孢菌生长和发育中的功能重要性。比较了用荧光蛋白标记的每种CHS的细胞分布以及相应基因缺失对营养生长和有性发育的影响。在营养菌丝的Spk核心和正在形成的隔膜中也发现了CHS-2、-4、-5和-7。随着隔膜环的发育，CHS-2-GFP一直保留在隔膜的生长边缘，直到它定位在隔膜孔周围。此外，所有CHS都位于分生孢子梗的横壁中。CHS-1-m与CHS-5-GFP或CHS-2-GFP在Spk和隔膜中部分共定位。缺失突变体分析表明，CHS-6主要在菌丝延伸和子囊孢子形成中起作用，CHS-5在气生菌丝、分生孢子和子囊孢子形成中起作用，CHS-3在子囊壳发育中起作用，CHS-7在上述所有过程中起作用。我们表明，chs-7/csmB在粗糙脉孢菌中发挥有性功能，chs-6/chsG在粗糙脉孢菌中发挥营养生长功能，但在构巢曲霉中并非如此，反之，chs-2/chsA在构巢曲霉中发挥有性功能，但在粗糙脉孢菌中并非如此。这表明不同类别的CHS可以在各种真菌中发挥不同的发育功能。对CHS-1-GFP、CHS-4-GFP和CHS-5-GFP进行免疫沉淀后再进行质谱分析，确定了每种CHS不同的假定相互作用蛋白。总的来说，我们的结果表明存在不同的几丁质体群体，每个群体携带特定的CHS，在不同发育阶段发挥特定作用。
