The Smnbr1 deletion mutant displays developmental defects

To elucidate the function of SmNBR1, a S. macrospora Smnbr1 deletion mutant was generated by homologous recombination with a hph-deletion cassette flanked by upstream and downstream sequences of the Smnbr1 ORF (2.2.6.1). The deletion of Smnbr1 was confirmed by PCR and Southern blot analysis (Figure 19). Macroscopic and microscopic investigations of the ∆Smnbr1 strain revealed several developmental defects in comparison to the wild type and to a complemented strain expressing a Smnbr1-egfp fusion gene ectopically (∆Smnbr1::Smnbr1-egfp^ect) (Figure 20). S. macrospora wild type and the complementation strain completed the life cycle within seven days under laboratory conditions. It starts with a germinating ascospore developing into a haploid vegetative mycelium. After three days, sexual development begins with the formation of female gametangia (ascogonia) which are protected by sterile hyphae that develop into a spherical fruiting-body precursor (protoperithecia). Cell

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differentiation leads to pigmentation after 5 days followed by development of the mature fruiting bodies (perithecium). During perithecia development after karyogamy, meiosis and a postmeiotic mitosis takes place, giving rise to eight linearly-ordered black ascospores per ascus (approximately 200 asci per perithecium). The ascospores are discharged through an apical pore (ostiole) at the neck of the fruiting body (Teichert et al. 2014). Microscopic analysis of the sexual reproductive structures of the ∆Smnbr1 strain showed defects at late stages of fruiting-body development. The mutant formed ascogonia, protoperithecia and perithecia but in a reduced number and after a prolonged time (Figure 20A+B).

Figure 19: Generation and analysis of a ∆Smnbr1 knockout strain.

(A) Schematic representation of the Smnbr1 (SMAC_07844, dark grey arrow) genomic region and its flanking regions including adjacent genes SMAC_07843 and SMAC_07845 (white arrows). The verified intron is indicated with a white bar. (B) Gene locus after homologous recombination of the hph-resistance gene under the control of the trpC promoter of A. nidulans (PtrpC and hph, grey arrow). The positions of primers to construct and verify homologous recombination at the Smnbr1 locus are indicated as small black arrows. PstI restriction sites are labeled. (C) Southern blot analysis of wt and ∆Smnbr1 strain. Genomic DNA was digested with PstI and hybridized with the probe indicated in A and B. Hybridization resulted in a 4828-bp band for the wt and a 3565-bp band for the knockout strain. (D) PCR analysis with primer pairs Smnbr1-v5f/Smnbr1-vORF5-r (1425 3565-bp) and Smnbr1-vORF3-f/Smnbr1-v3r (1133 bp) to verify the absence of the Smnbr1 gene in ∆Smnbr1 and the presence in wt. The presence of the hph-resistance cassette at the desired gene locus was verified with primer pairs Smnbr1-v5f/tC1 (1166 bp) and h3/Smnbr1-v3r (873 bp) (PCR not shown). Additionally, the primer pair Smnbr1-ko-5f/Smnbr1-ko-3r was used to verify the presence of the hph gene (3254 bp) in comparison to wt (4433 bp) (PCR

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not shown). Position of the primers are indicated in (A) and (B). (E) PCR analysis of the cDNA (2592 bp) and gDNA (2684 bp) of Smnbr1 which showed a bandshift induced by the splicing of the 92-bp intron. The primer pair Nbr1ORF-f/GFPNbr1r was used indicated in (A).

The ascus rosettes of the mutant harbored incomplete asci with few mature, melanized ascospores (Figure 20A,C,D). In the complemented strain these defects are partially rescued.

Quantitative analysis of fruiting bodies showed a 97.5 % reduction of mature perithecia per cm² in the knockout strain in comparison to the wild type (Figure 20B). Consequently, the number of discharged mature ascospores, which are essential for reproduction, was also drastically reduced (99 % reduction in comparison to wild type) (Figure 20C).

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Figure 20: Phenotypic analysis of S. macrospora wt, ∆Smnbr1 mutant strain and complementation strains expressing S. macrospora or H. sapiens NBR1 proteins.

(A) Microscopic investigation of sexual development of ΔSmnbr1 compared to wt and the complemented strains

∆Smnbr1::Smnbr1-egfp^ect and ∆Smnbr1::Hsnbr1-Dsred^ect. Strains were grown on slides covered with fructification medium (SWG), and photographs were taken at indicated days. (B) The perithecia were counted per cm² after eight days (wt, ∆Smnbr1::Smnbr1-egfp^ect) or ten days (∆Smnbr1). Number of perithecia shown are averages from nine independent measurements of three independent experiments (n=27). (C) Discharged ascospores, after ten days of growth, from the lid of a petri dish (56.7 cm²) for the different strains. (D) Ascus rosettes were analyzed at the same time point as in (B). Microscopic pictures represent a part of representive ascus rosettes. All strains were grown on fructification medium (SWG). Wt was set to 1. Results of (C) and (D) are averages from 25 independent measurements of four independent experiments (n=100). Standard deviations are indicated by error bars (B) and (C) and scale bars are shown in the images. (*) Asterisks indicate significant differences in the deletion mutant in comparison to the wt according to Student´s t-test (p<0.0000001).

85 Microscopic analysis showed that the mature perithecia of the knockout strain contained a high number of undeveloped asci and unviable ascospores in comparison to the wild type (Figure 20D). Thus, we showed that the deletion of Smnbr1 decreased the production of mature perithecia, with an increased number of defective asci and ascospores.

The SmNBR1 homolog shares only 22 % identity to the NBR1 protein from H. sapiens.

However, many domains of the H. sapiens NBR1 protein (HsNBR1) are conserved in SmNBR1. Therefore, we analyzed whether the HsNBR1 protein can complement the ∆Smnbr1 phenotype by expressing the protein in the ∆Smnbr1 strain. For this, ∆Smnbr1 was transformed with the plasmid pHsnbr1-Dsred. Subsequently, 20 ∆Smnbr1::Hsnbr1-Dsred^ect transformants were phenotypically analyzed. After ten days growth on SWG medium 14 of 20 transformants show complementation by producing mature perithecia and ascospores as the S. macrospora complemenation strain. The six other transformants were not able to rescue the phenotypical defects of ∆Smnbr1 perhaps due to the ectopic integration of the heterologous gene (data not shown). Three of the 14 complementation strains expressing the HsNBR1 protein were tested phenotypically in more detail. Strains were able to form ascogonia, protoperithecia and perithecia. The maturation of the late developmental stages are more delayed in comparison to the wild type because the pigmented protoperithecia and perithecia looked smaller. In contrast to the mutant ∆Smnbr1, the H. sapiens complementation strain shows more mature ascospores in ascus rosettes after ten days of growth. In summary, the developmental defects of the mutant

∆Smnbr1 can be partially rescued by the human NBR1 homolog (Figure 20A). The heterologous production of HsNBR1-DsRED was verified by a Western-blot experiment (Supplement 4).

Due to the fact that SmNBR1 lacks an obvious UBA domain we tested the enrichment of ubiquitinated proteins in total protein extracts of wild type, ∆Smnbr1 (two independent mutants) and a control strain ∆Smubp3 (Herzog pers. comm.). The deubiquitination enzyme SmUBP3 is able to bind ubiquitinated proteins to remove the ubiquitin tag for their autophagosomal degradation (Kraft et al. 2008). The Western-blot analysis showed that the

∆Smnbr1 strains behave like the wild type strain whereas the ∆Smubp3 strain showed a high accumulation of ubiquitinated proteins, however several bands seem to be more intensive in the

∆Smnbr1 strain as in the wild type (Figure 21).

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Figure 21: Western blot using anti-ubiquitin to detect ubiquitinated proteins.

Western blot using equal amounts of protein extracts of wt, ∆Smnbr1 and ∆Smubp3 (control) strain. Ubiquitinated proteins were detected with an ubiquitin antibody. Detection of actin was used as loading control to show the protein levels. One of three Western-blot experiments is represented. Quantification of Western blots was done using the program ImageJ. For this the ubiquitin protein amount was quantified to the actin loading control. Wt was set to 100 %. Data in this diagram are represented for three independent experiments. Error bars are indicated.