Mitochondrial and peroxisomal Lon proteases play opposing roles in reproduction and growth but co-function in the normal development, stress resistance and longevity of Thermomyces lanuginosus.

The ATP-dependent Lon enzyme is a highly conserved protease with multiple roles in diverse species such as fungi; however, there are few reports on Lon enzymes in filamentous fungi. Thermomyces lanuginosus, a typical thermophilic fungus, has been widely studied in physiology and cell biology; thus, studies on Thermomyces Lons are important. Two Lons were bioinformatically deduced in T. lanuginosus. Subcellular localization analysis showed that one is present in mitochondria (MLon), while the other is found in peroxisomes (PLon). Although both Lon enzymes were activated by H2O2, they were not induced by heat shock; instead, they were induced by low temperatures. Two single-deletion Lon mutants (ΔMLon and ΔPLon) were generated. Biological analysis demonstrated that ΔMLon decreased the production of conidia but increased the growth of mycelia. By contrast, ΔPLon increased the production of conidia but decreased the growth of mycelia. The lifespan was measured in time and in length of continuous growth. The wild-type strain showed continuous linear growth for 60days, whereas growth was impeded at 30 and 50days for ΔPLon and ΔMLon mutants, respectively, suggesting that PLon is more important for longevity than MLon. Interestingly, ΔPLon, which accumulated larger amount of H2O2 was not only more sensitive to exogenous H2O2 but also much more sensitive to other selected stressors. Taken together, our data indicate that mitochondrial and peroxisomal Lons play opposite roles in controlling growth and development, but exhibit synergistic effects on the normal states of vegetative growth, asexual development, stress resistance and longevity in T. lanuginosus.

Genetic Analysis of Four Sexual Differentiation Process Proteins (isp4/SDPs) in Chaetomium thermophilum and Thermomyces lanuginosus Reveals Their Distinct Roles in Development.

Fungal sexual development requires the involvement of a large number of functional genes. Fungal genes encoding sexual differentiation process proteins (SDPs), isps, have been known for decades. isp4/SDP and its homologs function as oligopeptide transporters (OPTs), yet their roles in reproduction are unknown. Here, we genetically analyzed all four isp4/SDP homologs in the sexual species Chaetomium thermophilum and asexual species Thermomyces lanuginosus. Using single gene deletion mutants, we found that T. lanuginosus SDP (TlSDP) participated in asexual sporulation, whereas the other homologs participated in sexual morphogenesis. In complementary tests, C. thermophilum SDPs (CtSDP1-3) restored sporulation defects in TlSDP deletion strains (ΔTlSDP), and their translated proteins, which were localized onto the cytomembrane, possessed OPT activity. Interestingly, CtSDP2 accumulated at the top of the hyphae played a distinct role in determining the sexual cycle, glutathione transport, and lifespan shortening. A unique 72nt-insertion fragment (72INS) was discovered in CtSDP2. Biological analysis of the 72INS deletion and DsRED-tagged fusion strains implied the involvement of 72INS in fungal growth and development. In contrast to TlSDP, which only contributes to conidial production, the three CtSDPs play important roles in sexual and asexual reproduction, and CtSDP2 harbors a unique functional 72INS that initiates sexual morphogenesis.

ANXC7 Is a Mitochondrion-Localized Annexin Involved in Controlling Conidium Development and Oxidative Resistance in the Thermophilic Fungus Thermomyces lanuginosus.

Annexins (ANXs) are widely expressed and structurally related proteins which play multiple biological roles in animals, plants, and fungi. Although ANXs have been localized to the cytosol and the cell membrane and the molecular basis of the four annexin repeats is well established, the in vivo roles of these proteins are still far from clear, particularly with regard to the filamentous fungi. Thermomyces lanuginosus, a thermophilic fungus, is widely used in the fermentation industry; however, the role of ANX in this organism is unknown. In this study, a single ANX homologue (ANXC7) was identified and characterized in T. lanuginosus. The expression pattern indicated that ANXC7 is closely associated to conidium development, and it accumulated in the mitochondria of the forming conidia. The deletion of ANXC7 (ΔANXC7) resulted in no obvious phenotype related to colony growth on solid CM medium. However, when ΔANXC7 was grown in CM liquid culture, the mycelium masses appeared to be larger and looser compared to the wild-type. Additionally, the dry weight of the mutant mycelia was significantly increased. Under conditions that compromise cell-wall integrity, ΔANXC7 was less vulnerable than the wild-type with regard to such damage. Moreover, based on a surface hydrophobicity test, the ΔANXC7 strain was clearly less hydrophobic. The growth of ΔANXC7 was inhibited when grown under selected stress conditions, particularly with regard to salt stress; however, the oxidative resistance to exogenous H2O2 in ΔANXC7 was increased, and endogenous H2O2 levels within the ΔANXC7 were lower than in the wild-type, thereby suggesting that the ANXC7 specifically controls oxidative resistance. Based on microscopic observation, 4-day-conidia were more prevalent than 5-day conidia on the conidiophore stalk of ΔANXC7, even though the ΔANXC7 demonstrated an increased production of conidia during these days, indicating precocious conidial maturation and shedding from the conidiophore stalk in this strain. Taken together, our data indicate that ANXC7 localizes to the mitochondria and is involved in controlling conidium development and oxidative resistance in T. lanuginosus.