Polarity proteins Bem1 and Cdc24 are components of the filamentous fungal NADPH oxidase complex

Abstract

Regulated synthesis of reactive oxygen species (ROS) by membrane-bound fungal NADPH oxidases (Nox) plays a key role in fungal morphogenesis, growth, and development. Generation of reactive oxygen species (ROS) by the plant symbiotic fungus, Epichloë festucae , requires functional assembly of a multisubunit complex composed of NoxA, a regulatory component, NoxR, and the small GTPase RacA. However, the mechanism for assembly and activation of this complex at the plasma membrane is unknown. We found by yeast two-hybrid and coimmunoprecipitation assays that E. festucae NoxR interacts with homologs of the yeast polarity proteins, Bem1 and Cdc24, and that the Phox and Bem1 (PB1) protein domains found in these proteins are essential for these interactions. GFP fusions of BemA, Cdc24, and NoxR preferentially localized to actively growing hyphal tips and to septa. These proteins interact with each other in vivo at these same cellular sites as shown by bimolecular fluorescent complementation assays. The PB1 domain of NoxR is essential for localization to the hyphal tip. An E. festucae Δ bemA mutant was defective in hyphal morphogenesis and growth in culture and in planta . The changes in fungal growth in planta resulted in a defective symbiotic interaction phenotype. Our inability to isolate a Δ cdc24 mutant suggests this gene is essential. These results demonstrate that BemA and Cdc24 play a critical role in localizing NoxR protein to sites of fungal hyphal morphogenesis and growth. Our findings identify a potential shared ancestral link between the protein machinery required for fungal polarity establishment and the Nox complex controlling cellular differentiation.

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“…Filamentous fungi possess up to three Nox enzymes. Nox 1 (or NoxA) and Nox2 (NoxB) are similar to gp91 phox , whereas Nox3 (NoxC) enzymes have EFhand domains that putatively bind Ca 2+ in the same way as plant respiratory burst oxidases (8,9) and human Nox5 (3). Fungal Nox complexes seem to involve interactions with cytosolic proteins, previously implicated in the regulation of polarity (1).…”

“…A transcriptional profiling database (http://cogeme.ex.ac.uk/supersage/) (38) was interrogated to determine the expression of NOX1, NOX2, and NOXR in Guy11 during appressorium development (4,6,8,14, and 16 h after spore germination). Expression was plotted relative to gene expression in M. oryzae mycelium (38).…”

“…In animal cells, Nox enzymes are implicated in cell proliferation, cell signaling, and apoptosis (1-3), whereas in plants Nox are necessary for programmed cell death (4), the response to environmental stresses (4), pathogen infection (5), and polarized growth of root hairs (6). In filamentous fungi, Nox are necessary for cellular differentiation during sexual reproduction and for developmental processes that involve transitions from nonpolarized to polarized cell growth, such as tissue invasion by mutualistic and pathogenic fungi, and fungal virulence (7)(8)(9). The underlying function of Nox enzymes in these diverse developmental processes remains unclear.…”

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“…For example, cdc42, a gene central for the establishment of polarity, is up-regulated in Δnox1 protoperithecia compared to wild-type protoperithecia. The activity of CDC42 is regulated by the guanine nucleotide exchange factor (GEF) CDC24 and the scaffold protein BEM1, both of which are able to interact with the NOR1 homolog of E. festucae in a yeast-two-hybrid assay (Takemoto et al 2011). In N. crassa, Bem1 is actively recruited around the forming fusion pore of germlings, and bem1 deletion leads to a drastic reduction of germling fusion, indicating a cross-talk between polarity establishment and hyphal fusion (Schürg et al 2012).…”

“…RacA) (Kawahara and Lambeth 2007;Tanaka et al 2008). In Epichloë festucae, the putative scaffold protein Bem1 was reported to associate with NOR1 and CDC24 (Takemoto et al 2011). Another candidate NOX regulatory protein is tetraspanin Pls1.…”