Molecular Characterization of the NADPH Oxidase Complex in the Ergot Fungus Claviceps purpurea: CpNox2 and CpPls1 Are Important for a Balanced Host-Pathogen Interaction

Abstract

Reactive oxygen species producing NADPH oxidase (Nox) complexes are involved in defense reactions in animals and plants while they trigger infection-related processes in pathogenic fungi. Knowledge about the composition and localization of these complexes in fungi is limited; potential components identified thus far include two to three catalytical subunits, a regulatory subunit (NoxR), the GTPase Rac, the scaffold protein Bem1, and a tetraspanin-like membrane protein (Pls1). We showed that, in the biotrophic grass-pathogen Claviceps purpurea, the catalytical subunit CpNox1 is important for infection. Here, we present identification of major Nox complex partners and a functional analysis of CpNox2 and the tetraspanin CpPls1. We show that, as in other fungi, Nox complexes are important for formation of sclerotia; CpRac is, indeed, a complex partner because it interacts with CpNoxR, and CpNox1/2 and CpPls1 are associated with the endoplasmatic reticulum. However, unlike in all other fungi, Δcppls1 is more similar to Δcpnox1 than to Δcpnox2, and CpNox2 is not essential for infection. In contrast, Δcpnox2 shows even more pronounced disease symptoms, indicating that Cpnox2 controls the infection process and moderates damage to the host. These data confirm that fungal Nox complexes have acquired specific functions dependent of the lifestyle of the pathogen.

Small-GTPase-Associated Signaling by the Guanine Nucleotide Exchange Factors CpDock180 and CpCdc24, the GTPase Effector CpSte20, and the Scaffold Protein CpBem1 in Claviceps purpurea

“…In addition, the CpDock180 localization at the plasma membrane corresponds with the predicted association of active GTPases with the plasma membrane and could represent the recruitment and subsequent activation of GTPases at the cell periphery. A similar vesicle localization has been observed for CpNoxR and NoxR of B. cinerea (88; D. Buttermann and P. Tudzynski, unpublished data), further strengthening the close connection between Rac and Nox signaling in C. purpurea (47). Another hint for the cross talk between CpRac and Nox signaling is the interaction of the regulatory Nox protein CpNoxR with CpCdc24 and CpDock180.…”

“…The interaction of NoxR and Cdc24 occurs similarly in E. festucae (32) and is mediated by the type I PB1 domain in CpCdc24 and the type I/II PB1 domain in CpNoxR. We recently showed that CpNoxR is able to interact with Rac, in a loading-status-dependent manner, and binds to CpBem1 (47). All these characteristics indicate a role of CpNoxR as another type of downstream effector of CpRac.…”

“…The protein consists of 563 amino acids and contains two SH3 domains, a PX domain, and a C-terminal PB1 domain. It shows 76% sequence identity with BemA of E. festucae and 58% identity with Fusarium oxysporum Scd2 (47).…”

“…For this purpose, both GEFs were fused to either the DNA-binding domain or the activation domain of the Gal4 transcription factor and tested with fusion proteins of CpRac and CpCdc42. The GTP-locked (G14V) and GDP-locked (T22N) forms of both GTPases were included in the assay, as the loading state has been shown to be important for interaction patterns of GTPases of C. purpurea with the p21-activated kinase CpCla4 and the NADPH oxidase regulator CpNoxR (15,47). All cotransformed yeast strains were able to grow on control SD medium lacking leucine and tryptophan, whereas no growth could be detected on SD medium lacking histidine, selecting for interaction.…”

“…Small GTPase signaling in C. purpurea is strongly connected to NADPH oxidase (Nox) complex dynamics (47). Similarly, as in E. festucae (32), the regulatory protein CpNoxR is able to interact with CpRac in a loading-status-dependent manner, identifying it as a downstream factor of CpRac.…”