A tether for Woronin body inheritance is associated with evolutionary variation in organelle positioning

Abstract

Eukaryotic organelles evolve to support the lifestyle of evolutionarily related organisms. In the fungi, filamentous Ascomycetes possess dense-core organelles called Woronin bodies (WBs). These organelles originate from peroxisomes and perform an adaptive function to seal septal pores in response to cellular wounding. Here, we identify Leashin, an organellar tether required for WB inheritance, and associate it with evolutionary variation in the subcellular pattern of WB distribution. In Neurospora, the leashin (lah) locus encodes two related adjacent genes. N-terminal sequences of LAH-1 bind WBs via the WB-specific membrane protein WSC, and C-terminal sequences are required for WB inheritance by cell cortex association. LAH-2 is localized to the hyphal apex and septal pore rim and plays a role in colonial growth. In most species, WBs are tethered directly to the pore rim, however, Neurospora and relatives have evolved a delocalized pattern of cortex association. Using a new method for the construction of chromosomally encoded fusion proteins, marker fusion tagging (MFT), we show that a LAH-1/LAH-2 fusion can reproduce the ancestral pattern in Neurospora. Our results identify the link between the WB and cell cortex and suggest that splitting of leashin played a key role in the adaptive evolution of organelle localization.

Figure 1. The leashin mutant is defective in Woronin body inheritance.

(A) The lah mutant accumulates HEX assemblies in the apical compartment. The distribution of HEX assemblies was quantified in apical (a) and sub-apical (sa) hyphal compartments in the indicated strains. (B) HEX assemblies are enveloped by WSC in the lah mutant background. RFP-PTS1 reveals the peroxisome matrix and WSC-GFP reveals assembly of the sorting complex. HEX assemblies can be seen in the DIC channel. Arrowhead points to a pair of aberrantly associated nascent WBs. Bar = 2 µm. (C) Dot plots reveal repetitive sequences R1, R2 and R3 in the predicted LAH polypeptide. The schematic indicates conserved regions (black bars) and repeat regions (light gray bars). Red bars indicate two predicted coiled-coil domains. (D) Alignment of repetitive sequences using Clustal W . R1 contains 18 repeats centered on a core consensus sequence, LPVDEDLDLLPALPES, and R2 and R3 contain 33 repeats of the consensus sequence, PEEVELPASP. Acidic residues are colored blue, basic residues are red, hydrophobic sequences are green and Proline is indicated in yellow.

Figure 2. 3′-sequences of lah are dispensable for WB–associated functions.

Growth rate and WB–segregation were assessed in the indicated strains. Truncations were produced at the indicated positions by integrating stop codons to the chromosome using the hyg^r marker under control of the trpC promoter. The last two cartoons depicted epitope tagged versions of lah produced by marker fusion tagging. Yellow = hyg^r, green = GFP.

Figure 3. N-terminal sequences of LAH associate with WBs via C-terminal sequences of WSC.

(A) The first 344 amino acids of LAH co-localize with WSC at the WB surface. LAH^1–344-RFP was co-expressed with WSC-GFP and visualized by confocal microscopy. Bar = 2 µm. (B) LAH^1–344 association with a dense organellar fraction depends on the presence of WSC. LAH^1–344-HA was expressed in wild-type and wsc mutant background and a crude organellar fraction (T) was separated into supernatant (S) and pellet (P) fractions and the distribution of LAH^1–344-HA was revealed with anti-HA epitope antibodies. (C) C-terminal tail of WSC is required for WB-segregation but not the production of nascent WBs. HEX assembly distribution was assessed in a strain expressing WSCΔC-GFP. Inset shows the distribution of WSCΔC-GFP and RFP-PTS1, which reveals the peroxisome matrix. (D) LAH^1–344 does not associate with the WB in the WSCΔC-GFP expressing strain. WSCΔC-GFP and LAH^1–344-RFP were co-expressed and visualized as in (A). Bar = 2 µm.

Figure 4. Marker fusion tagging (MFT) reveals distinct localization patterns of tagged versions of LAH; evidence for two transcription units.

(A) Confocal microscopy reveals distinct non-overlapping localization patterns of MFTs at position 1 and 2. 1-GFP is localized exclusively to the WB (left panels) and 2-GFP is localized to the hyphal tip and septal pores (right panels). The inset shows 2-GFP ring structures, which can be seen in large hyphae. Arrow indicates the WB, which is not decorated by the 2-GFP tag. Box indicates the region that is magnified in the right most panels. Left bar = 2 µm, right bar = 5 µm. The lower cartoon summarizes results from MFT tags at positions 1, 2, 3 and 4. Yellow and green bars represent hyg^r and eGFP, respectively. (B) HA epitope tags at positions 1 and 2 migrate as distinct polypeptides. 1-HA is detected in WB enriched fractions and 2-HA can be detected in total cell extracts. Molecular size in kDa is indicated. (C) The localization of 2-GFP is impervious to introduction of a stop codon at position 1, using the panB gene from Aspergillus nidulans as a selective marker (magenta bar). Lower panels show GFP localization in the indicated strains. Bar = 5 µm.

Figure 5. Molecular dissection of the leashin locus; identification of the 3′-end of lah-1 and lah-2 promoter.

(A) The cartoon depicts the structure of the lah locus. Thick solid lines are exons and the position of MFT tags is indicated along with their localization to either the Woronin body (WB) or sepal pore (SP). Scale is indicated in kilobases (Kb). Introns are depicted as gaps and numbered. Introns identified in this study are marked with an asterisk. The alternative exon derived from splicing at intron 10 is indicated as a blue bar. (B) Gray arrows depict DNA fragments that were fused to the hyg^r gene and transformed to assess their ability to promote Hygromycin resistance in a stable transformation assay. The graph indicates the transformation efficiency of these fragments compared with the strong ccg-1 promoter. Inset panels show the levels of protein expressed from three randomly picked colonies expressing an epitope tagged version of hyg^r from plah-2 and pccg-1 promoter sequences. (C) An MFT tag (5-GFP) in the alternatively spliced exon is localized to the WB with enrichment between the WB and cell cortex. Bar = 2 µm.

Figure 6. The ancestral pattern of WB localization can be produced in Neurospora by a LAH-1/LAH-2 fusion protein.

(A) The schematic summarizes the two types of WB localization (I and II) in apical and sub-apical compartments. Woronin bodies are continuously formed de novo in the apical compartment (left panels) and are inherited into sub-apical compartments through different modes of cell cortex association (right panels). The lower panel shows a phylogenetic tree based on 18S r-RNA in species where WB localization has been determined. Arrow indicates the ancestor of the Pezizomycotina where WBs are presumed to have evolved. an, Aspergillus nidulans ; ao, Aspergillus oryzae ; cg, Chaetomium globosum ; mg, Magnaporthe grisea ; nc, Neurospora crassa , fo, Fusarium oxysporum ; sc, Saccharomyces cerevisiae; sp, Schizosaccharomyces pombe; sf, Sordaria fimicola (this study). (B) The cartoon shows the structure of the lah locus and MFT produced lah-1/2 fusion. Panels show the localization of the LAH-1/2 fusion protein (GFP) and HEX assemblies (DIC), in apical and sub-apical compartments. Bars = 5 µm. (C) Woronin body function in the indicated strains was determined by measurement of protoplasmic bleeding.

Figure 7. Model for the evolution of Woronin body tethering in the Pezizomycotina.

(A) Legend indicates symbols used to depict domains of the Leashin tether. (B) The minimal events associated with splitting of the ancestral leashin locus are indicated. (C) Model for septal pore associated WB-tethering in most of the Pezizomycotina. (D) Model of WB tethering in Neurospora and Sordaria. The double-headed arrow indicates extensive protoplasmic streaming that can be observed in Neurospora and Sordaria (See Video S1).