A p67Phox-Like Regulator Is Recruited to Control Hyphal Branching in a Fungal–Grass Mutualistic Symbiosis

Abstract

Key requirements for microbes to initiate and establish mutualistic symbiotic interactions with plants are evasion of potential host defense responses and strict control of microbial growth. Reactive oxygen species (ROS) produced by a specific NADPH oxidase isoform, NoxA, regulate hyphal growth in the mutualistic interaction between the fungal endophyte Epichloe¨festucae and its grass host Lolium perenne. Unlike mammalian systems, little is known about the fungal NADPH oxidase complex and its response to differentiation signals. We identify an E. festucae p67 phox -like regulator, NoxR, dispensable in culture but essential in planta for the symbiotic interaction. Plants infected with a noxR deletion mutant show severe stunting and premature senescence, whereas hyphae in the meristematic tissues show increased branching leading to increased fungal colonization of pseudostem and leaf blade tissue. Inhibition of ROS production or overexpression of noxR recapitulates the hyperbranching phenotype in culture. NoxR interacts in vitro with the small GTP binding protein RacA and requires a functional RacA binding site to complement the noxR mutant and restore the wild-type plant interaction phenotype. These results show that NoxR is a key regulator of NoxA in symbiosis, where it acts together with RacA to spatially regulate ROS production and control hyphal branching and patterning.

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

“…In contrast, the C terminus of NoxR lacks the protein-protein interaction domains present in p67 phox , including Src Homology 3 (SH3) and a conventional Phox and Bem1 (PB1), for interaction with p47 phox and p40 phox , respectively (1,14). The absence of these domains in NoxR is consistent with the apparent absence of p47 phox and p40 phox homologs in fungal genome databases (3,13). However, NoxR does possess a nonconventional PB1 domain in the C terminus of the protein, suggesting that fungi have distinct regulatory components that, upon activation, interact with NoxR to translocate this protein from the cytosol to the plasma membrane to assemble and activate the Nox enzyme complex (3,13) (Fig.…”

“…The absence of these domains in NoxR is consistent with the apparent absence of p47 phox and p40 phox homologs in fungal genome databases (3,13). However, NoxR does possess a nonconventional PB1 domain in the C terminus of the protein, suggesting that fungi have distinct regulatory components that, upon activation, interact with NoxR to translocate this protein from the cytosol to the plasma membrane to assemble and activate the Nox enzyme complex (3,13) (Fig. 1A).…”

“…In the symbiotic fungus E. festucae, a noxR mutant has a similar disrupted symbiotic interaction phenotype as ΔnoxA (6,13). In B. cinerea and N. crassa, deletion of noxR (nor-1) resulted in a similar developmental phenotype to the noxA(nox1)/noxB(nox2) double mutant (7,11).…”

“…Although NoxA and NoxB have distinct cellular functions, it is not yet known how NoxR regulates these two different Nox isoforms. The N-terminal domain of NoxR is similar to p67 phox , and includes four tetratricopeptide repeat (TPR) motifs required for Rac binding and a putative NADPH oxidase activation domain (3,13) (Fig. 1A).…”

Coral-associated marine fungi form novel lineages and heterogeneous assemblages

“…For this reason, the fact that many of the fungi detected in our study belong to clades of parasitic fungi, including plant pathogenic fungi such as Pleosporaceae and dermatophytes such as Malassezia, may not necessarily indicate parasitism in this particular environmental context. Lichenic symbioses, for example, show evidence of repeated shifts from symbiosis to pathogen (Arnold et al, 2009), and mutation in a single gene has been shown to convert a plant endosymbiotic fungus from a mutualist to a parasite (Takemoto et al, 2006).…”