Functional Analysis of BcBem1 and Its Interaction Partners in Botrytis cinerea: Impact on Differentiation and Virulence

Abstract

In phytopathogenic fungi the establishment and maintenance of polarity is not only essential for vegetative growth and differentiation, but also for penetration and colonization of host tissues. We investigated orthologs of members of the yeast polarity complex in the grey mould fungus Botrytis cinerea: the scaffold proteins Bem1 and Far1, the GEF (guanine nucleotide exchange factor) Cdc24, and the formin Bni1 (named Sep1 in B. cinerea). BcBem1 does not play an important role in regular hyphal growth, but has significant impact on spore formation and germination, on the establishment of conidial anastomosis tubes (CATs) and on virulence. As in other fungi, BcBem1 interacts with the GEF BcCdc24 and the formin BcSep1, indicating that in B. cinerea the apical complex has a similar structure as in yeast. A functional analysis of BcCdc24 suggests that it is essential for growth, since it was not possible to obtain homokaryotic deletion mutants. Heterokaryons of Δcdc24 (supposed to exhibit reduced bccdc24 transcript levels) already show a strong phenotype: an inability to penetrate the host tissue, a significantly reduced growth rate and malformation of conidia, which tend to burst as observed for Δbcbem1. Also the formin BcSep1 has significant impact on hyphal growth and development, whereas the role of the putative ortholog of the yeast scaffold protein Far1 remains open: Δbcfar1 mutants have no obvious phenotypes.

SymB and SymC, two membrane associated proteins, are required for Epichloë festucae hyphal cell–cell fusion and maintenance of a mutualistic interaction with Lolium perenne

“…P. anserina and B. cinerea NoxD and Nox1 transition from the ER into the vesicle network where they co‐localize with NoxR in older hyphae (Lacaze et al ., ; Siegmund et al ., ). E. festucae NoxR and B. cinerea Bem1 are also associated with septa (Takemoto et al ., ; Giesbert et al ., ), similar to SymB and SymC. Although there is no evidence to date of a direct interaction between SymB and SymC and components of the Nox complex, we have found in a parallel study in P. anserina that the four conserved “extracellular” Cys residues in IDC2 and the two conserved Cys residues in IDC3 are required for functional activity (Lalucque et al ., ).…”

BcNoxD, a putative ER protein, is a new component of the NADPH oxidase complex in Botrytis cinerea

“…Literature suggests a localization of Nox at the plasma membrane, where it can join with the cytosolic regulatory subunits like BcNoxR and possibly Bem1 and Cdc24. A possible connection of the latter proteins with the Nox complexes in B. cinerea was recently discussed (Giesbert et al ., ).…”

Update on Nox function, site of action and regulation in Botrytis cinerea

“…In this situation, the NoxA complex consists of the membrane standing subunits BcNoxA and BcNoxD, which are putatively linked via the scaffold protein BcIqg1 to its cytosolic and regulatory subunits BcRac as well as BcNoxR. Since the deletion mutant of the second scaffold protein BcBem1 displays also defects in the colonization of plant tissue [ 15 ] and in the formation of infection cushions (Additional file 6 : Figure S6B) an association to the NoxA complex is likely. The putative association is supported by Takemoto et al (2011), who linked Bem1 to Nox complexes in Epichloe festucae [ 48 ].…”

“…The putative association is supported by Takemoto et al (2011), who linked Bem1 to Nox complexes in Epichloe festucae [ 48 ]. On the other side, the unaffected stress resistance of Δ bcbem1 , its lacking Nox complex interaction partner in B. cinerea as well as its localization in the cytosol and septa [ 15 ] cast some doubts about its membership in the NoxA complex. Outside the ER, the NoxA complex might be similar composed during the formation of CATs and conidiospores, but slightly different during sclerotia development.…”