BEM46 Shows Eisosomal Localization and Association with Tryptophan-Derived Auxin Pathway in Neurospora crassa

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. 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.