Chemotropism and Cell Fusion in Neurospora crassa Relies on the Formation of Distinct Protein Complexes by HAM-5 and a Novel Protein HAM-14

Abstract

In filamentous fungi, communication is essential for the formation of an interconnected, multinucleate, syncytial network, which is constructed via hyphal fusion or fusion of germinated asexual spores (germlings). Anastomosis in filamentous fungi is comparable to other somatic cell fusion events resulting in syncytia, including myoblast fusion during muscle differentiation, macrophage fusion, and fusion of trophoblasts during placental development. In Neurospora crassa, fusion of genetically identical germlings is a highly dynamic and regulated process that requires components of a MAP kinase signal transduction pathway. The kinase pathway components (NRC-1, MEK-2 and MAK-2) and the scaffold protein HAM-5 are recruited to hyphae and germling tips undergoing chemotropic interactions. The MAK-2/HAM-5 protein complex shows dynamic oscillation to hyphae/germling tips during chemotropic interactions, and which is out-of-phase to the dynamic localization of SOFT, which is a scaffold protein for components of the cell wall integrity MAP kinase pathway. In this study, we functionally characterize HAM-5 by generating ham-5 truncation constructs and show that the N-terminal half of HAM-5 was essential for function. This region is required for MAK-2 and MEK-2 interaction and for correct cellular localization of HAM-5 to "fusion puncta." The localization of HAM-5 to puncta was not perturbed in 21 different fusion mutants, nor did these puncta colocalize with components of the secretory pathway. We also identified HAM-14 as a novel member of the HAM-5/MAK-2 pathway by mining MAK-2 phosphoproteomics data. HAM-14 was essential for germling fusion, but not for hyphal fusion. Colocalization and coimmunoprecipitation data indicate that HAM-14 interacts with MAK-2 and MEK-2 and may be involved in recruiting MAK-2 (and MEK-2) to complexes containing HAM-5.KEYWORDS cell fusion; Neurospora crassa; MAP kinase signaling; protein complexes; chemotropism F USION between genetically identical cells exists in many organisms and plays an important role in different developmental processes, for example, myoblast fusion during the formation of muscle tissue, macrophage fusion, or fusion of trophoblasts in placental development (Chen and Olson 2005;Aguilar et al. 2013). In fungi, vegetative fusion enables a fungal colony to share resources and has implications for fitness and virulence (Craven et al. 2008;Fricker et al. 2009;Eaton et al. 2011;Richard et al. 2012;Simonin et al. 2012;Roper et al. 2013;Chagnon 2014). In the filamentous ascomycete fungus, Neurospora crassa, genetically identical germinated asexual spores (germlings) undergo fusion to form a syncytium that is an interconnected network of fused cells Leeder et al. 2013;Herzog et al. 2015). An important pathway required for both germling and hyphal fusion is a conserved mitogen-activated protein kinase (MAPK) cascade that includes the MAPK kinase kinase NRC-1, the MAPK kinase MEK-2, the MAPK MAK-2, and the scaffold protein, HAM-5 (Pandey et al. 2004;Dettmann et al. 2012Dettm...

Transcription factor PRO1 targets genes encoding conserved components of fungal developmental signaling pathways

“…As displayed in Table , NCU00938 , NCU07192 and NCU07238 were required for fruiting body formation in at least one crossing experiment. NCU07238 was recently described as ham (hyphal anastomosis)−14 involved in the fusion of conidial germlings (Jonkers et al ., ) and appeared to be sterile in homozygous crosses only.…”

“…Among the identified PRO1 target genes we noticed an elevated number of genes with homology to N. crassa ham genes ( ham‐5 , ‐6 , ‐7 , ‐8 , ‐9, ‐14 ), which were initially described as being essential for hyphal and/or germling fusion (Glass et al ., ; Fleißner et al ., ; Fu et al ., ; Jonkers et al ., ). As mentioned above, HAM‐5 is a scaffold protein of the kinases of the PS pathway, which are probably recruited to HAM‐5 by HAM‐14, and HAM‐6 is the homolog of PRO41 (Fu et al ., ; Jonkers et al ., ; Dettmann et al ., ; Jonkers et al ., ).…”

“…Among the identified PRO1 target genes we noticed an elevated number of genes with homology to N. crassa ham genes (ham-5, -6, -7, -8, -9, -14), which were initially described as being essential for hyphal and/or germling fusion Fleißner et al, 2008;Fu et al, 2011;Jonkers et al, 2016). As mentioned above, HAM-5 is a scaffold protein of the kinases of the PS pathway, which are probably recruited to HAM-5 by HAM-14, and HAM-6 is the homolog of PRO41 (Fu et al, 2011;Jonkers et al, 2014;Dettmann et al, 2014;Jonkers et al, 2016). HAM-7 was recently shown to work as a membrane receptor for the CWI pathway during cell-to-cell signaling and hyphal fusion in N. crassa (Maddi et al, 2012).…”

“…As displayed in Table 2, NCU00938, NCU07192 and NCU07238 were required for fruiting body formation in at least one crossing experiment. NCU07238 was recently described as ham (hyphal anastomosis)214 involved in the fusion of conidial germlings (Jonkers et al, 2016) and appeared to be sterile in homozygous crosses only. When we analysed the combined data set of all GFP-PRO1_OE ChIP-seq experiments, we found a large number of genes that have been published to be involved in sexual development in S. macrospora and other filamentous ascomycetes (Table 3).…”

TheNeurosporaTranscription Factor ADV-1 Transduces Light Signals and Temporal Information to Control Rhythmic Expression of Genes Involved in Cell Fusion

“…During cell fusion, chemotropic growth is associated with alternating oscillatory recruitment of the MAK-2 MAPK module (including ADV-1 targets MEK-1, MAK-1, and the scaffold protein HAM-5) and SO to opposing tips of the communicating cells prior to fusion (Jonkers et al 2014, 2016; Dettmann et al 2014, 2012; Fleissner et al 2009b; Fleissner and Herzog 2016). This alternating pattern of MAK-2 complexes and SO at opposing tips occurs about every 4–5 min, and is thought to play an important role in signaling and response during fusion (Goryachev et al 2012).…”