Multiple genes evolved for fungal septal pore plugging identified via large-scale localization and functional screenings

Abstract

Multicellular organisms exhibit cytoplasmic exchange using porous structures for cooperation among cells. Fungal multicellular lineages have evolved septal pores for this function. Interconnected hyphal cells possess the risk of wound-related cytoplasmic loss unless the septal pores are plugged. However, the gene evolution of regulatory mechanisms underlying fungal septal pore plugging remains poorly understood. To identify novel septal components, 776 uncharacterized proteins were identified using genomic comparisons between septal pore-bearing and -lacking ascomycete species. We then determined their subcellular localizations, and in total 62 proteins localized to the septum or septal pore. We analyzed the effects of deleting the encoding genes on septal pore plugging upon hyphal wounding. Of the 62 proteins, 23 were involved in regulating septal pore plugging. Here, using orthologous group and phylogenetic analyses, this study suggests that septal pore regulation has evolved either by co-option of preexisting genes or by Pezizomycotina-specific gene acquisition.

Identification of novel proteins regulating lipid droplet biogenesis by reverse genetics

“…Twelve of the 19 proteins were functionally important for LD biogenesis and, uncharacterized of the ten were termed LDR proteins (Fig 2A–C). In addition, SppE and SppO, which participate in septal pore regulation (Mamun et al, 2022 preprint ), were identified here to be associated with LD (Fig 1B).…”

“…To identify novel LD-associated proteins, we employed an EGFP-fused protein library developed by our group with a large set of uncharacterized proteins conserved in Pezizomycotina but absent or diverged in the representatives of Saccharomycotina and Taphrinomycotina (Fig 1A and Mamun et al, 2022 preprint ). A total of 314 strains (Table S2) corresponding to the organelle-like pattern of subcellular localization were analyzed here after culturing them on the minimal medium (Fig 1A).…”

“…However, forward genetics screening is often laborious due to genome-wide manipulation (Guo et al, 2008; Szymanski et al, 2007). Here, we applied a reverse genetics strategy for the first time by utilizing the previously established EGFP-fused library (Mamun et al, 2022 preprint ). Colocalization analysis with LD staining dyes Nile red and BODIPY, and subsequent cross-checking with LD maker proteins Erg6 and Dga1, provided high confidence in our primary dataset consisting of 19 candidate proteins (Figs.…”

“…Recombinant plasmids expressing mCherry-fused proteins were digested with Not I and ectopically inserted into the sC locus of the NSlD1. The resultant strains expressing AoDga1-mCherry and AoErg6-mCherry were further transformed (ectopically) with Not I digested EGFP-fused plasmids (Mamun et al, 2022 preprint ) into the niaD locus by homologous recombination. Strain expressing AoSec63-mCherry was transformed with 12 EGFP-fused LD-associated proteins (Fig S2).…”