Woronin bodies (WBs) are dense-core organelles that are found exclusively in filamentous fungi and that seal the septal pore in response to wounding. These organelles consist of a membrane-bound protein matrix comprised of the HEX protein and, although they form from peroxisomes, their biogenesis is poorly understood. In Neurospora crassa, we identify Woronin sorting complex (WSC), a PMP22/MPV17-related membrane protein with dual functions in WB biogenesis. WSC localizes to large peroxisome membranes where it self-assembles into detergent-resistant oligomers that envelop HEX assemblies, producing asymmetrical nascent WBs. In a reaction requiring WSC, these structures are delivered to the cell cortex, which permits partitioning of the nascent WB and WB inheritance. Our findings suggest that WSC and HEX collaborate and control distinct aspects of WB biogenesis and that cortical association depends on WSC, which in turn depends on HEX. This dependency helps order events across the organellar membrane, permitting the peroxisome to produce a second organelle with a distinct composition and intracellular distribution.

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.

A new gene-tagging method (marker fusion tagging [MFT]) is demonstrated for Neurospora crassa and Magnaporthe oryzae. Translational fusions between the hygromycin B resistance gene and various markers are inserted into genes of interest by homologous recombination to produce chromosomally encoded fusion proteins. This method can produce tags at any position and create deletion alleles that maintain N-and C-terminal sequences. We show the utility of MFT by producing enhanced green fluorescent protein (EGFP) tags in proteins localized to nuclei, spindle pole bodies, septal pore plugs, Woronin bodies, developing septa, and the endoplasmic reticulum.The use of homologous recombination to delete or tag chromosomally encoded genes is a key tool in modern biology. Where homologous recombination has been exploited, integrating DNA fragments are selected using marker genes under the control of their own constitutive promoters. As a result, the production of chromosomally encoded fusion proteins has largely been restricted to tagging genes at their 3Ј ends, where selectable markers are unlikely to have a major impact on gene expression (5, 6, 15).We reasoned that expressed genes might be tagged at any position by integrating a promoterless tag-fused selectable marker directly into the gene of interest to produce a chromosomally encoded fusion protein. We began by creating an inframe fusion of the hygromycin B resistance gene (hyg r ) and the enhanced green fluorescent protein (EGFP) (Fig. 1a). Fusion PCR was then used to generate integration fragments ( Fig. 1b and c) flanked by in-frame sequences from a set of Neurospora target genes encoding proteins selected for their distinctive localization patterns and loss-of-function phenotypes. We tagged the fungal spindle pole body (SPB) protein ApsB (12) at the N terminus [apsB::hyg r -gfp(N)], the soft protein (SO), which localizes to septal plugs (3), between residues 40 and 68 [so::hyg r -gfp(⌬40-68)], and the VIB-1 nuclear protein, which has been associated with programmed cell death (2) at the C terminus [vib-1::hyg r -gfp(C)]. Six primers (P1 to P6) are required for PCR synthesis of fusion cassettes (Fig. 1b and c). Primer P1 is located about 700 bp upstream of the integration site, while P6 is located 700 bp downstream from the integration site. P2 and P5 are located immediately at the integration site, with a 20-bp overlap with the hyg r -gfp fragment. P3 and P4 are complements of P2 and P5, respectively. Primers 2 and 3 and 4 and 5 define the reading frame of the translational fusion. In the first step, 3 fragments are produced using the Expand long template PCR kit (Roche Bioscience) with the following PCR protocol: step 1, initial denaturation (94°C for 2 min); step 2, denaturation (94°C for 30 s); step 3, annealing (52°C for 30 s); step 4, elongation (68°C for 1 min); step 5, repeat from step 2 to step 4 30 times; and step 6, fragment finishing (72°C for 5 min). PCR products were checked and gel purified using the GFX gel purification kit (GE Healthcare). Fusion PCR...