Deletion of scp2 causes an appressorium defect

U. maydis forms non-melanized appressoria on the leaf surface that mediate the entry into the plant tissue. In contrast to Magnaporthe oryzae and Colletotrichum spp. that invade the host plant through melanized appressoria and mechanical force, appressoria of U. maydis seem to penetrate the plant cuticle by locally secreting plant cell wall degrading enzymes (Tucker and Talbot, 2001; Schirawski et al., 2005). Several steps are important for successful appressoria formation and penetration. First, the pathogen needs to locate and perceive the host surface by detecting extracellular signals. These signals are transmitted to intracellular signaling cascades which initialize the reorganization of the fungal cytoskeleton thus allowing the formation of appressoria and the redirection of growth towards the plant surface prior to penetration (Tucker and Talbot, 2001; Patkar et al., 2010). During this process the fungal appressorium has to

adhere tightly to the host surface to allow appressorium mediated entry into the host plant tissue (Tucker and Talbot, 2001). In U. maydis, appressorium formation can be induced in vitro by providing hydroxy-fatty acids and a hydrophobic surface as stimulus (Mendoza-Mendoza et al., 2009).

A qPCR approach revealed an upregulation of scp2 during appressorium formation and penetration at around 20 hours after maize seedling infection. Microscopic analysis showed that filaments of scp2 deletion strains express the appressorial marker am1 on parafilm and on the leaf surface suggesting that the genetic program for appressorium formation has been initiated.

However, scp2 deletion strains showed a significantly lower amount of appressoria on parafilm when compared to the respective progenitor strain sixteen hours after appressorium induction.

This defect could not be attributed to a delayed appressorium initiation since 24 hours after induction no enhanced appressorium formation frequency was observed in the mutant. An impaired sensing of external fatty acid stimuli was excluded by demonstrating the induction of filament formation in liquid culture using HFA. Interestingly, the appressorial defect observed on parafilm was not apparent on the plant surface where appressoria could be frequently detected for scp2 deletion strains.

The appressorium formation defect of scp2 mutants on parafilm coincided with an extension of the cytoplasm-filled part of the filaments. Compared to wild type hyphae in which the cytoplasm filled tip compartment had an average length of 54.0 µm (± 9.6) the tip compartments of scp2 mutants had an average length of 89.6 µm (± 8.0). This extension occurred without the additional integration of septa causing the formation of elongated hyphae and coincided with a higher mortality rate of filaments on parafilm when compared to the progenitor strain.

During the elongation process several filaments appeared to attempt the formation of appressoria by forming a crook-like structure but failed to form an appressorium and continued hyphal growth. In M. oryzae, the loss of the polarized growth regulator Tea4 has been shown to cause a zigzag morphology in aerial hyphae and defects in appressoria formation on inductive surfaces while vegetative growth was mostly unaffected (Patkar et al., 2010). In contrast to the phenotype observed for the polarized growth mutants in M. oryzae, however, U. maydis scp2 deletion strains are able to form appressoria on the maize leaf surface and to penetrate the host plant cuticle to a certain extent. Considering this, it seems unlikely that the observed scp2 appressorium formation phenotype is caused by a defect in polarized growth of forming filaments.

This might indicate that scp2 deletion strains are not impaired in polarized growth of filaments and are capable to initiate appressoria but that these appressoria are not firmly attached to the

hydrophobic parafilm. Such an effect could be caused by a reduction of filament hydrophobicity. A general reduction of hydrophobicity in scp2 deletion mutant filaments, however, seems unlikely. First, the parafilm slides used for the analysis of appressoria were washed in water prior to microscopic examination. In case of a less effective overall attachment washing would have caused a significant loss of filaments. Further, the hydrophobicity of scp2 deletion strain filaments was not significantly reduced. This was demonstrated by a water soaking assay (Müller et al., 2008). For this, water drops were spotted on filamentous growing SG200 and scp2 deletion strains on PD-charcoal solid medium and the time until the water drop was fully absorbed by the colony was measured as an indication for the surface hydrophobicity of the respective strains. No difference of hydrophobicity was observed for SG200 and SG200Δscp2 (not shown).

Furthermore, the secretion of glycolipids such as mannosylerythritol lipids (MELs) leads to a reduced surface tension of U. maydis culture medium (Hewald, 2005). The biosynthesis of MELs takes partially place in peroxisomes and is coupled to peroxisomal fatty acid degradation (Freitag et al., 2014). The surface tension of liquid medium incubated with the U. maydis strains MB215 and MB215Δscp2, however, showed that both strains are able to reduce the surface tension of the medium to a comparable extent (not shown) indicating that Scp2 is not involved in MEL production.

Adhesion of lily pollen tubes has been shown to depend on a lipid transfer-like protein (nsLTP), a class of extracellular cystein-rich proteins with a broad affinity for various lipids (Park et al., 2000). In this system the secreted nsLTP either mediate adhesion of the pollen tube directly or act indirectly as a transporter of lipophilic compounds involved in signaling (Park et al., 2000).

Even though sterol carrier proteins like Scp2 do not share specific motifs of lipid transfer proteins both protein classes contain a hydrophobic cavity that allows the unspecific transport of different lipid species (De Berti et al., 2013; Liu et al., 2015). Interestingly, several fungal species produce an extracellular matrix (ECM) presumably consisting of glycoproteins, polysaccharides and lipids which promote appressorial adhesion (Tucker and Talbot, 2001;

Ahn et al., 2004). This might indicate that components of the fungal ECM depend on nsLTP or sterol carrier protein mediated transport. With respect to this, the U. maydis Scp2 protein could be involved in the generation, transport or secretion of compounds that help the forming appressorium to effectively attach to the parafilm surface. On the maize leaf surface, however, additional factors like topography and structure or the presence of plant derived cuticle waxes and fatty acids could help to overcome the appressorium formation defect.

3.3.1 Scp2 is required for efficient penetration of the host plant surface

A question arising from the observed appressorium formation defect on parafilm was whether the presence of appressoria on planta necessarily correlates with appressorium functionality.

This question was addressed by the quantification of penetration efficiency which showed that appressoria of scp2 deletion strains are less efficient in successful invasion of the epidermal layer of the leaf than wild type appressoria. The penetration defect indicates that although appressoria formation is restored on the plant surface scp2 deletion strain appressoria are less efficient in breaching the plant cuticle. This data was supported by the quantification of fungal biomass which revealed that scp2 deletion strains accumulate significantly less biomass within the maize plant when compared to the SG200 progenitor strain.

A phenotype similar to that of scp2 deletion strains was observed for mutants of the plasma membrane spanning protein Sho1 and the transmembrane mucin Msb2. Both proteins are involved in the perception of surface signals and the induction of the MAP-kinase cascade that induces pathogenic development in U. maydis (Lanver et al., 2010). Single gene deletions of sho1 and msb2 caused a strong reduction of appressorium formation on parafilm. Interestingly, this defect was partially compensated on the plant surface. In contrast to scp2 deletion strains, however, sho1 and msb2 did not express the appressorial marker on parafilm indicating that Scp2 either functions downstream of the signaling cascade which triggers appressorium induction or is involved in a separate pathway.

3.4 Scp2 deletion strains show a misdistribution of peroxisomes and lipid droplets