Morphological characterization of rho-1, rho-2 and NCU00668 mutants

by the identical phenotype of mutants in the corresponding genes. Conidia (which are multinucleate) of the heterokaryotic deletion strains Δrho-1 (het) or Δ00668 (het) seeded on medium selecting for the presence of the knockout cassette develop in three different ways (Figure 23): Some do not grow at all, indicating complete absence of the hygromycin resistance knockout cassette; some only expand isotropically, suggesting severe defects associated with the mutations that prevent polarized growth altogether; others, which presumably harbor both deletion and sheltering wild type nuclei, form slow-growing hyphae, many of which meander unusually and display swollen tips. In accordance with the defects observed in conidia devoid of sheltering wild type nuclei, ascospores produced in crosses with the wild type that bear the respective gene deletion rarely germinate at all, and if so in an apolar manner, with growth soon ceasing and cell lysis occurring. These observations suggest that, like rho-1 (Vogt and Seiler, 2008), NCU00668 is an essential gene in N. crassa.

76 5. Results

Figure 23: Heterokaryotic deletion mutants of rho-1 and NCU00668 exhibit identical phenotypes, and homokaryotic deletion mutants are not viable. Conidia of the two strains were germinated on VMM supplemented with hygromycin and incubated for 12 hours. Insets show rare apolar ascospore germination.

Hypomorphic conditional mutants of rho-1 generated by RIP mutagenesis (S. Seiler, unpublished) allowed the investigation of fungal growth in the absence of fully functional RHO1 (Figure 24). When grown at room temperature, rho-1(9-1) and rho-1(10-1) exhibit largely normal, albeit slightly slowed, growth (cp. also Figure 27); shifting cultures to 37°C, however, quickly leads to pronounced swelling of apical tips, which resume polarized growth in short time, although extension proceeds at a reduced pace (cp. also Figure 26).

Figure 24: Temperature-sensitive mutant strains of rho-1 exhibit apolar tip growth. When grown under permissive conditions, rho-1(9-1) and rho-1(10-1) display a largely normal morphology; after shift to 37°C (time points indicated), apical tips initially swell in an apolar fashion (compare magnified insets), while later polarized growth is resumed.

Sequencing of the rho-1 coding region amplified from genomic DNA of the two mutant strains revealed several silent mutations and mutations translated to two or three amino acid substitutions in the encoded proteins, respectively (Figure 25). An alignment of N. crassa RHO1 with its homologues in S. pombe and S. cerevisiae shows that the substitutions are all located at conserved positions. Intriguingly, both RHO1(9-1) and RHO1(10-1) have one amino acid substitution each within one of the two highly conserved switch domain regions essential for regulation and function of the GTPase; proline at position 37 replaced by leucine in RHO1(9-1) is even predicted to be a direct interaction site for RhoGAPs by the the NCBI Conserved Domains Database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=cdd). None of the amino acid substitutions coincides with any of those encoded by S. cerevisiae rho1

alleles associated with specific impairment of different Rho1p effector pathways (Saka et al., 2001).

Figure 25: Alignment of N. crassa (N.c.) RHO1 with its homologues from the yeasts S. pombe (S.p.) and S. cerevisiae (S.c.). The alignment was generated by MAFFT (version 6.083b). Conserved features predicted by the NCBI Conserved Domains Database (see text) are indicated with black bars. Amino acid substitutions resulting from the mutations identified in rho-1 mutant alleles (9-1) and (10-1) are given above and their positions are highlighted within the alignment in blue and red, respectively. For comparison, amino acids altered in Rho1p proteins of S. cerevisiae strains forming the two complementation groups rho1A and rho1B, which are characterized by distinct Rho1p effector defects (Saka et al., 2001), are highlighted in varying shades of pink and green, respectively.

N. crassa rho-1 possesses a highly similar putative paralogue, rho-2; identity between the encoded proteins is as high as 64%, as determined by sequence alignment with BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Despite this close relation to RHO1, RHO2 had not been a target of NCU00668 in in vitro GEF activity assays. Nevertheless, I tested the physical interaction of the proteins in copurification experiments (Supplementary Figure 10, p.114). Indeed, just like RHO1, RHO2 could be copurified with all three NCU00668 constructs used; however, it also bound to the GEF-PH domain construct of CDC24, and all NCU00668 constructs were enriched in parallel to the GTPase CDC42, too. Thus, there is obviously a general affinity between RhoGEFs and Rho GTPases, so mere physical interaction cannot serve as evidence for a specific regulatory relationship.

Interestingly, while the rho-2 gene is not essential and the homokaryotic deletion mutant is mostly inconspicuous under standard conditions - although growth speed and directionality are slightly affected -, the double mutant Δrho-2;rho-1(9-1) exhibits strong synthetic growth and polarity defects both at room temperature and at 37°C (Figure 26): From most conidia polarized germ tubes never emerge, and the rare hyphae that are formed resemble pearls on

78 5. Results a string, with several subapical and apical compartments swollen in an apolar manner.

Additionally, growth speed, formation of aerial hyphae and conidiation are highly reduced (data not shown).

Figure 26: Mutations in rho-1 and rho-2 have synthetic effects. While the deletion of rho-2 has little effect on hyphal growth and morphology, a Δrho-2;rho-1(9-1) double mutant exhibits strong synthetic polarity and growth defects. Conidia of the indicated strains were incubated on VMM for 15 hours at 37°C.

The finding that lack of RHO2 markedly aggravates the effects of deficiency in RHO1 hints at overlapping functions of the two Rho GTPases, while RHO1 must possess at least one additional essential function, as indicated by the fact that deletion of rho-1 is lethal, whereas knockout of rho-2 has only mild effects.