Effector pathways regulated by RHO1 and RHO2 in N. crassa

To distinguish between these proposed shared and distinct cellular functions of RHO1 and RHO2, mutant strains were treated with inhibitors of putative downstream effectors inferred from yeast models (Figure 27). In comparison to the wild type, the temperature-sensitive rho-1 and the rho-2 deletion mutant strains display no markedly increased sensitivity towards latrunculin A, which disrupts the actin cytoskeleton (Spector et al., 1999). However, they are clearly hypersusceptible towards cell wall damage by lysing enzymes or caspofungin, an irreversible inhibitor of β-1,3-glucan synthase (Hoang, 2001). While cell wall integrity thus seems to be equally compromised as a result of deficiency in RHO1 or RHO2, only rho-1(9-1), but not Δrho-2, shows reduced sensitivity towards cercosporamide, a selective inhibitor of protein kinase C (Sussman et al., 2004). Hyposensitivity pointing towards increased activity of PKC1 is even more pronounced in the double deletion strain, possibly suggesting the existence of an alternative, RHO1/2-independent PKC1 activation pathway when cell wall integrity is compromised.

Figure 27: Cell wall integrity is compromised in mutants of both rho-1 and rho-2, while only mutation of rho-1 or the double mutation effects hyposensitivity towards inhibition of PKC1. Actin organization is not clearly affected in any of the mutants tested. Wild type and mutant strains were incubated for 24 hours at room temperature on VMM supplemented with the actin-depolymerizing drug latrunculin A, cell wall damaging agents lysing enzymes or caspofungin (kindly provided by MSD Sharp & Dohme GmbH, Germany) or the protein kinase C inhibitor cercosporamide.

Next, I sought to directly analyze physical interaction between the two GTPases and their putative effectors and regulators by yeast two-hybrid analyses (Figure 28).

The RHO1 and RHO2 constructs expressed in the two-hybrid tests are presumed to represent dominant active (DA) or negative (DN) forms of the proteins, as homologous proteins of other organisms bearing analogous changes at conserved amino acid positions have been shown to have altered signalling properties: Equivalent mutations have been reported to significantly decrease GTPase activity (Marshall et al., 1991; Longenecker et al., 2003) or are thought to interfere with effector interaction by perturbing the switch I region (D g vá e 1996; T mić e 2007), respectively; moreover, analogous rho1 alleles affect polar growth and cell wall integrity in various fungi (Guest et al., 2004).

80 5. Results Initially, bait and prey proteins were expressed as fusions to the GAL4 DNA-binding (BD) or activation (AD) domain from the vectors of the Matchmaker™ Two-Hybrid System 3 (Clontech, USA). In this way, however, I was not able to detect reproducible interaction between any pair of fusion proteins, presumably because expression of the dominant activated form of RHO1 and some other fusion proteins was harmful to yeasts, as evident in very poor transformation efficiency and growth.

To overcome this problem, I generated vectors in which fusion protein expression was no longer controlled by the full-length and intermediately truncated ADH1 promoters associated with high expression levels but by a shorter promoter fragment which results in low expression (for comparison of promoter versions and expression strength, see (Ammerer, 1983; Ruohonen et al., 1991, 1995) and unpublished data by Clontech, USA, presented at http://www.clontech.com/images/pt/PT3024-1.pdf).

Yeast cells tolerated expression of all fusion proteins from the new vectors. None of the fusion proteins used in the assay exhibited autoactivation, i.e. interaction with the respective other GAL4 domain alone (data not shown). General yeast two-hybrid competency of all HO1 d HO2 f si p ei s s ve ified by hei bi i y i e c i h ΔN-RanBPM (see Supplementary Figure 11, p.115).

Figure 28: Overview of combinations of fusion proteins tested for interaction in yeast two-hybrid assays.

All possible pairwise combinations between GAL4 BD and AD fusion constructs (left and right column, respectively) were coexpressed; only the two pairs connected by lines exhibited weak (fine line) or strong (thick line) interaction. All proteins were expressed at low levels under control of a short truncated version of the ADH1 promoter except those denoted by a plus sign, whose expression was controlled by the full-length promoter associated with high expression. See text for details.

As evident in Figure 29, the dominant active version of RHO1, but not its dominant negative counterpart or any RHO2 construct, interacted strongly with the N-terminus of the formin BNI1, which comprises the predicted GTPase binding and FH3 domains but lacks the actin-binding FH2 and autoregulatory domains. RHO1DA was also the only GTPase construct

engaging in weak interaction with PKC1; interestingly, the protein kinase had to be expressed at high levels for detectable interaction.

No interaction of any of the GTPase constructs with the largest intracellular domain of the glucan synthase NCU06871 (which coincides largely with its predicted catalytic domain) or any of the NCU00668 fragments was observed (data not shown).

Figure 29: An activated form of RHO1, but not RHO2, interacts specifically with PKC1 and the N-terminus of BNI1 in yeast two-hybrid experiments. The Rho GTPase and putative effector constructs indicated where coexpressed as fusions to the GAL4 DNA binding (BD) or activation (AD) domain in the reporter yeast strain AH109. Growth of colonies on medium lacking histidine and adenine and formation of a blue galactosidase reaction product from X-α-Gal reveal interaction between fusion protein pairs. Fusion proteins for positive and negative controls were expressed under control of the same ADH1 promoter versions as the corresponding test pairs. Note that growth on selective medium for the combination RHO1DA-PKC1 was poor and only visible after prolonged incubation at 30°C (eight days instead of three).

Dilution factors of cell suspensions used for inoculation are indicated.

Intending to confirm and expand the findings of the yeast two-hybrid analyses, I attempted to identify interaction partners of RHO1 and RHO2 by coimmunoprecipitation experiments using N. crassa strains which coexpress epitope-tagged versions of the potential binding partners.

For this purpose, I have already created plasmids encoding FLAG-tagged BNI1, PKC1, NCU06871 and NCU00668 and generated auxotrophic N. crassa strains stably expressing these constructs (data not shown; cp. sections 4.4.7 and 4.5). Likewise, I have constructed plasmids for expression of HA- and 3xmyc-tagged RHO1 and RHO2 and used them to generate complementary auxotrophic strains. Unfortunately, expression levels of the GTPase constructs, in particular that of RHO2, have turned out to be very low; indeed, GTPase fusion proteins are almost immunologically undetectable in lysates of the forced heterokaryon strains coexpressing both putative interactor constructs, even after enrichment (data not shown). Therefore, I am currently working on improving the detectability of the epitope-tagged GTPase proteins by testing new vector constructs (section 4.4.7).

Inhibitor tests and yeast two-hybrid studies presented above suggest that RHO1 influences PKC1 signalling in N. crassa, while RHO2 only plays a minor, if any, role. In yeasts, protein

82 5. Results kinase C has been established as a regulator of the cell wall integrity MAPK pathway (reviewed in (Levin, 2005)). A homologous tripartite kinase cascade consisting of MIK1, MEK1 and MAK1 has been described in N. crassa (Park et al., 2008). Activating signals are thought to be passed on along the cascade by sequential phosphorylation of the three component kinases, and phosphorylation of the MAPK MAK1, the last kinase in the row, can be detected using a phospho-specific p42/44 antibody (ibid.).

Figure 30: Mutants of the RHO1/RHO2 GTPase module exhibit reduced basal activity of the MAPK MAK1, while they are still able to respond to cell wall stress by activation of the cell wall integrity pathway.

(A) Summary of mean relative MAK1 activity levels in the different strains. Cell lysates prepared from unstressed or stressed N. crassa cultures of the indicated strains were subjected to SDS PAGE and ensuing Western blotting, and phosphorylated MAK1 was detected using a phospho-specific p42/44 antibody. MAK1 phosphorylation levels were quantified by densitometric analysis of MAK1 band intensity and normalized to the values obtained for the unstressed wild type (“wild type -“). Error bars are confidence intervals (p=68%). See section 4.7.5 for details on data acquisition and evaluation.

(B) Immunoblot illustrating mean MAK1 phosphorylation levels. Lysate samples of the indicated strains corresponding to 150 or 50 µg total protein were subjected to SDS PAGE, and phospho-MAK1 and -MAK2 were immunodetected as described in (A). Bands corresponding to the two MAPK proteins (predicted MW=47/41 kD), as verified by their absence in lysate samples of the respective deletion mutants (data not shown), are indicated. Equal loading is confirmed by Ponceau S staining.

B

A

As seen in Figure 30, basal MAK1 activity of mutants affected in components of the RHO1/RHO2 GTPase module is reduced compared to that of wild type. On average, reduction is most pronounced in the two temperature-sensitive rho-1 mutants and especially the Δrho-2; rho-1(9-1) double mutant, while Δrho-2 and ΔNCU00668 (het) exhibit a MAK1 posphorylation status more similar to that of the wild type.

In wild type, the pathway is reliably induced by addition of lysing enzymes to the culture medium. Such cell wall stress still elicits MAK1 activation in all of the mutants analyzed;

unfortunately, further conclusions about differences regarding the strength of the response are prevented by the high variability of the assay.

5.3.4 Analysis of the subcellular localization of NCU00668, RHO1 and RHO2