The Glyoxysomal Protease LON2 Is Involved in Fruiting-Body Development, Ascosporogenesis and Stress Resistance in Sordaria macrospora

Abstract

Microbodies, including peroxisomes, glyoxysomes and Woronin bodies, are ubiquitous dynamic organelles that play important roles in fungal development. The ATP-dependent chaperone and protease family Lon that maintain protein quality control within the organelle significantly regulate the functionality of microbodies. The filamentous ascomycete Sordaria macrospora is a model organism for studying fruiting-body development. The genome of S. macrospora encodes one Lon protease with the C-terminal peroxisomal targeting signal (PTS1) serine-arginine-leucine (SRL) for import into microbodies. Here, we investigated the function of the protease SmLON2 in sexual development and during growth under stress conditions. Localization studies revealed a predominant localization of SmLON2 in glyoxysomes. This localization depends on PTS1, since a variant without the C-terminal SRL motif was localized in the cytoplasm. A ΔSmlon2 mutant displayed a massive production of aerial hyphae, and produced a reduced number of fruiting bodies and ascospores. In addition, the growth of the ΔSmlon2 mutant was completely blocked under mild oxidative stress conditions. Most of the defects could be complemented with both variants of SmLON2, with and without PTS1, suggesting a dual function of SmLON2, not only in microbody, but also in cytosolic protein quality control.

Analysis of the Putative Nucleoporin POM33 in the Filamentous Fungus Sordaria macrospora

“…Both fusion constructs (POM33-EGFP and POM33-TagRFP-T) displayed a donut-like localization presumably around the nucleus that differs from the localization of free EGFP or TagRFP-T ( Figure 2 and Figure S2 in Supplementary Materials ). For localization of free EGFP or TagRFP-T, S. macrospora wt was transformed with plasmid p1783-1 [ 62 ] or pTagRFP-T_nat [ 44 ], respectively. To verify the localization of POM33 around the nucleus, we crossed the wildtype (wt) strain expressing POM33-EGFP with a fus1-1 (fus) strain expressing the histone 2B (H2B) fused to tdTomato ( Figure 2 A).…”

“…The resulting fragment was integrated into Bgl II-digested p5′sci1gfp_nat via HR in S. cerevisiae . For plasmids p5′pom33-TagRFP-T_hyg/_nat, a fragment comprised of the pom33 promoter and pom33 ORF was amplified with primer pair Smpom33-f/Smpom33-RFP-r from wt gDNA and a fragment comprised of TagRFP-T and the TtrpC terminator of A. nidulans was amplified with primer pair RFP-f/pRS426GFPrev from pTagRFP-T_nat [ 44 ]. Both fragments were integrated into Xho I-linearized pRS-nat [ 46 ] or pRS-hyg [ 6 ] via HR in S. cerevisiae .…”

Tracking Fungal Growth: Establishment of Arp1 as a Marker for Polarity Establishment and Active Hyphal Growth in Filamentous Ascomycetes

“…To construct the pSmarp1-TagRFP-T_nat/_hyg plasmids, the Smarp1 promoter and coding sequence was amplified with the Arp1_5′f/Arp1_RFP_r primer pair from S. macrospora wt gDNA. The TagRFP-T sequence and the A. nidulans TtrpC were amplified using the primer pairs of RFP-f/RFP-r-trpC from pTagRFP-T_nat [ 61 ] and TrpC_F/pRS426GFPrev from p1783-1 [ 60 ], respectively. The S. cerevisiae strain PJ69-4A was transformed with the three amplicons and Xho I-linearized pRS-hyg [ 62 ] or pRS-nat [ 58 ] to generate recombinant plasmids via homologous recombination [ 45 , 46 ].…”

“…Crosses were performed as described previously [51]. wt::TagRFP-T ectopic integration of ptRFP_nat into DSM997; nat R , ssi, Pccg1::TagRFP-T::TtrpC [61] wt::GG-C-F-mNG ectopic integration of pGG-C-F-mNG in DSM997; nat R , ssi, Pgpd::mNG::3xFLAG::TtrpC…”

Spatiotemporal Dynamic Regulation of Organelles During Meiotic Development, Insights From Fungi

“…Consistently, elimination of Atg24 –a sorting nexin involved in pexophagy, mitophagy and non-selective autophagy–reduces the lifespan of this fungus ( Henkel et al, 2020 ). Furthermore, in the mycelial ascomycete Sordaria macrospora , loss of the pexophagy receptor NBR1 ( Werner et al, 2019 ), or of the peroxisomal protease LON2 ( Werner et al, 2021 ), decreases ascospore formation, suggesting parallel systems for peroxisome quality control during ascospore formation. Still, in Saccharomyces cerevisiae , peroxisomes are segregated meiotically, but changes in their intracellular dynamics, at least concerning their abundance, are less apparent ( Gurvitz et al, 1998 ), suggesting different requirements for peroxisome dynamics in distinct meiotic developmental processes.…”