Mysterious gene functionally decoded: How a fungal gene influences the formation of a plant hormone

These findings have been published online in the scientific magazine Eukaryotic Cell.

BEM46 proteins are evolutionary conserved, but their functions remain elusive. It was reported previously that the BEM46 protein in the fungus Neurospora crassa is targeted to the ER, and is essential for ascospore germination. "In the present study, we established a bem46 knock-out strain of N. crassa. This bem46 mutant exhibited ascospore germination lower than wild type, but much higher than the previously characterized bem46-overexpressing and RNAi lines," says Professor Kempken, senior scientist of the current study. Reinvestigation of the RNAi transformants revealed two types of alternative spliced bem46 mRNA, with expression of either type leading to loss of ascospore germination. The results indicate that the phenotype was not due to bem46 mRNA downregulation or loss, but caused by the alternative spliced mRNAs and their encoded peptides.

Using the N. crassa ortholog of the eisosomal protein PILA from Aspergillus nidulans, the scientists further demonstrated co-localization of BEM46 with eisosomes. Employing the yeast two-hybrid system, they identified a single interaction partner: the anthranilate synthase component two (trp-1). This interaction was confirmed in vivo by a split-YFP approach. The ∆trp-1 mutant showed reduced ascospore germination and increased indole production; bioinformatic tools were used to identify a putative auxin biosynthetic pathway. The involved genes exhibited varying transcriptional regulation among the different bem46 transformant and mutant strains. The team also investigated the strains' indole production in different developmental stages. "Our findings suggest that indole biosynthesis gene regulation was influenced by bem46 overexpression," says Professor Kempken. "Furthermore, we uncovered evidence of co-localization of BEM46 with the neutral amino acid transporter MTR."