The BEM46 protein, auxin and eisosomes
BEM46 proteins are evolutionarily conserved, but their functions remain elusive. We reported previously that
the BEM46 protein in Neurospora crassa is targeted to the endoplasmic reticulum (ER) and is essential
for ascospore germination. In the present study, we established a bem46 knockout strain of N. crassa.
This delta-bem46 mutant exhibited a level of ascospore germination lower than that of the wild type but much
higher than those of the previously characterized bem46-overexpressing and RNA interference (RNAi) lines.
Reinvestigation of the RNAi transformants revealed two types of alternatively spliced bem46 mRNA; expression
of either type led to a loss of ascospore germination. Our results indicated that the phenotype was not due
to bem46 mRNA downregulation or loss but was caused by the alternatively spliced mRNAs and the peptides they
encoded. Using the N. crassa ortholog of the eisosomal protein PILA from Aspergillus nidulans, we further
demonstrated the colocalization of BEM46 with eisosomes. Eisosomes are fungal-specific structures close to the plasma membrane of unknown function.
Employing the yeast two-hybrid system, we identified
a single interaction partner: anthranilate synthase component II (encoded by trp-1). This interaction was
confirmed in vivo by a split-YFP (yellow fluorescent protein) approach. The delta-trp-1 mutant showed reduced
ascospore germination and increased indole production, and we used bioinformatic tools to identify a
putative auxin biosynthetic pathway. The genes involved exhibited various levels of transcriptional
regulation in the different bem46 transformant and mutant strains. We also investigated the indole
production of the strains in different developmental stages. Our findings suggested that the regulation
of indole biosynthesis genes was influenced by bem46 overexpression. Furthermore, we uncovered evidence
of colocalization of BEM46 with the neutral amino acid transporter MTR.
Recently, we were able to elucidate major parts of the genetic network of auxin production in N. crassa.
SARDAR P, KEMPKEN F (2018) Characterization of indole-3-pyruvic acid pathway mediated biosynthesis of auxin in Neurospora crassa. PLoS ONE 13: e0192293 doi: 10.1371/journal.pone.0192293
KOLLATH-LEISS, KEMPKEN F (2018) The fungal MCC/eisosome complex: An unfolding story. The Mycota XV. Physiology and Genetics: Selected Basic and Applied Aspects. 2nd edition, Springer Verlag, New York, Dordrecht, Heidelberg, London, pp119-130
KOLLATH-LEISS K, BOENNIGER C, SARDAR P, KEMPKEN F (2014) BEM46 shows eisosomal localization and association with tryptophan-derived auxin pathway in Neurospora crassa. Euk Cell 13:1051-1063
KUMAR A, KOLLATH-LEISS K, KEMPKEN F (2013) Characterization of bud emergence 46 (BEM46) protein: sequence, structural, phylogenetic and subcellular localization analyses. Biochem Biophys Res Comm 438:526-532
MERCKER M, KOLLATH-LEISS K, ALLGAIER S, WEILAND N, KEMPKEN F (2009) The BEM46-like protein appears to be essential for hyphal development upon ascospore germination in Neurospora crassa and is targeted to the endoplasmic reticulum. Curr Genet 55:151-161
BEM46:GFP expression in N. crassa