The V. dahliae oleate ∆12-fatty acid desaturase Ode1 contributes to

Different signaling molecules contribute to the crosstalk between fungi and their host plants and decide about the outcome of the interaction. Besides the secretion of effector proteins, which can be regulated by the UPR (Hampel et al., 2016; Pinter et al., 2019), lipid metabolites can act as signaling molecules (Christensen & Kolomiets, 2011). Since linoleic acid and the derived oxylipins were described to be able to modulate fungal differentiation and the development of disease symptoms (Calvo et al., 2001; Brodhagen et al., 2008; Brodhun et al., 2009; Reverberi et al., 2010; Scala et al., 2014; Fischer &

Keller, 2016; Patkar & Naqvi, 2017), the impact of the oleate ∆12-fatty acid desaturase Ode1 on differentiation and virulence of V. dahliae was analyzed.

Due to the protein domain structure, the similarity to the corresponding desaturase in A. nidulans, and the positive impact of medium supplementation with linoleic acid on growth of the ODE1 deletion strain (Figure 24, 26), V. dahliae Ode1 can be assumed to catalyze the conversion of oleic acid to linoleic acid. In A. nidulans and A. parasiticus, a depletion of linoleic acid and the derived products as well as an increase in oleic acid following disruption of the homologous ODE1 gene were observed (Calvo et al., 2001;

Chang et al., 2004).

The construction of strains with Ode1 either N- or C-terminally fused to GFP has shown, that the N-terminus of the protein is important for its function. C-terminally GFP tagged Ode1 is more stable than N-terminally GFP tagged Ode1 since diffuse GFP signals in vacuoles were observed for GFP-ODE1, which were absent in ODE1-GFP (Figure 25C).

The vacuolar localization of diffuse GFP suggest an accelerated degradation of the membrane protein by autophagosomes in the vacuoles (Liu et al., 2012). The presence of an additional larger protein version of Ode1, specifically present for ODE1-GFP in

immunoblots (Figure 25A), supports possible posttranslational modification of Ode1 as part of the mechanism controlling Ode1 activity.

V. dahliae Ode1 localizes to membranes of cell organelles, presumably ER membranes, and in irregular patterns in the plasma membranes, often close to growing hyphal tips or branch points (Figure 25B, C). The protein sequence predicted that Ode1 is a transmembrane protein, which is a typical feature of desaturases (Uttaro, 2006). A signal peptide sequence predicting ER localization was not identified, however

∆12-desaturases in plants, animals, and lower eukaryotes are generally described to be located either in plastids or in ER membranes (Uttaro, 2006). Even if few fungal

∆12-desaturases have been described, their localization has not yet been shown (Passorn et al., 1999; Sakuradani et al., 1999; Calvo et al., 2001; Watanabe et al., 2004;

Chang et al., 2004; Wilson et al., 2004; Wei et al., 2006). The detection of Ode1 at sites of hyphal growth and branch points suggests a directed vesicle transport. The product of the fatty acid desaturase is linoleic acid, which is an important component of fungal plasma membranes with roles in adjusting membrane rigidity (Gostinčar et al., 2009).

The presence of the desaturase close to hyphal tips could facilitate growth by serving one of the products required for extension of hyphae. During growth, liquid uptake supports the increase of the cell volume and exocytosis expands the plasma membrane and cell wall (Money, 2016). In related fungi such as Aspergilli and N. crassa, growth at hyphal tips or branch points was dependent on a structure close to these locations, named the Spitzenkörper as vesicle supply center, consisting of cytoskeletal elements and vesicles which are distributed to extend the hyphal tips by exocytosis (Harris et al., 2005). In N. crassa, chitin and glucan synthases localize to the Spitzenkörper to build up the cell wall (Su et al., 2012). Besides, linoleic acid might not only provide a building block for the membrane. On the other hand, it could increase the membrane fluidity and facilitate exocytosis. In yeast, it has been suggested that increased ergosterol contents in the membrane promote vesicle fusion (Munn et al., 1999).

Even if V. dahliae contains more than one gene encoding potential oleate ∆12-fatty acid desaturases in its genome, this study could show that Ode1 provides an important contribution to fungal growth and differentiation (Figure 33). This observations suggests that not all encoded ∆12-fatty acid desaturases might have additive or only partially-redundant functions. Reciprocal BLAST search has unraveled the presence of a second potential homolog to A. nidulans odeA with only slightly lower conservation of the deduced amino acid sequence than Ode1, almost the same size (479 aa), and similar domain structure (VDAG_JR2_Chr2g10170a; FAD domains: 75-114 aa IPR021863, 142-427 aa IPR005804; four transmembrane helices, Phobius).

Besides, another gene is predicted to encode an OdeA non-homologous ∆12-fatty acid desaturase with 402 aa in length and different domain structure, which was clearly predicted to be localized to the ER membrane using DeepLoc-1.0 and with its N-terminus directed to the ER lumen (VDAG_JR2_Chr4g12440a; FAD domain: 85-367 aa IPR005804; N-terminus located in ER 1-53 aa, Phobius; four transmembrane helices, Phobius). Deletion of ODE1 as a single of these desaturases resulted in a decrease in the colony diameter about 50% in comparison to wild type. This was qualitatively similar on every tested medium including media supplemented with osmotic or cell wall stressors. The only more favorable condition was caused by substitution of linoleic acid (Figure 26).

Figure 33: V. dahliae Ode1 displays a strong impact on vegetative growth, but is dispensable for induction of disease symptoms. The oleate ∆12-fatty acid desaturase Ode1 catalyzes the conversion of oleic acid into linoleic acid. The ODE1 defective strain displays impaired vegetative growth and microsclerotia formation, which is partially restored on medium supplemented with linoleic acid. This corroborates a positive impact of Ode1 or Ode1-derived products on growth and microsclerotia formation (indicated by arrows with green plus). The ODE1 deficiency displays only a minor impact on fungal virulence and the deletion strain is able to induce severe disease symptoms in planta. The availability of plant-derived linoleic acid might complement the observed growth defect (indicated by arrow with dashed line and green plus) and promote fungal propagation or enable the biosynthesis of linoleic acid-derived products with unknown impact on the plants defense responses.

Additional oleate ∆12-fatty acid desaturases (∆12) in V. dahliae could compensate the defect in ODE1 and allow modulation of plant defense responses (indicated by arrows with dashed line with red minus).

Similarly, a general decrease in growth was observed for A. nidulans and A. parasiticus upon deletion of the homologous oleate ∆12-fatty acid desaturases (Calvo et al., 2001;

Chang et al., 2004; Wilson et al., 2004). BLAST and domain searches of A. nidulans OdeA revealed that the genome of this organism harbors an additional gene encoding a non-homologous protein with predicted ∆12-fatty acid desaturase domain and cytosolic N-terminus (XP_664808.1) similar to the Ode1 non-homologous oleate ∆12-fatty acid desaturase predicted in V. dahliae, suggesting that also here other proteins with potentially overlapping functions exist.

With regard to the unaltered phenotype of the V. dahliae ODE1 deletion strain on stress media, one can conclude that Ode1 and its products are not especially required for the response to stresses even if one could expect that the adaptation of the membrane fluidity might influence stress responses. An increased membrane content of unsaturated fatty acids increased the sensitivity to salt stress in yeast for example (Gostinčar et al., 2009). It is possible that different desaturases are upregulated in response to certain environmental conditions or at certain developmental stages. FAD2, a single of a multitude of desaturases in A. thaliana, is activated upon sensing of ER stress and increases the ER stress tolerance by its positive impact on membrane fluidity (Nguyen et al., 2019).

Additionally to the impact on growth, V. dahliae Ode1 contributes to microsclerotia formation (Figure 27). Whereas here only a reduced resting structure formation was observed, deletion of the ODE1 homolog in A. parasiticus resulted in a complete loss of sclerotia development (Chang et al., 2004; Wilson et al., 2004). Since the ODE1 deletion strain displayed no specific stress-dependent phenotype one can assume that the decrease in microsclerotia formation can be seen as a result from the general growth defect and the altered polyunsaturated fatty acid metabolism.

4.4.2 Plant-derived unsaturated fatty acids might replace products synthesized