V. dahliae Hac1 is essential for fungal colonization of the host plant

In this study, it could be shown that the V. dahliae UPR regulator HAC1 is involved in colonization of the root surface, but not required for the penetration and initial invasion of the root cortex (Figure 22). Further, HAC1 is involved in successful propagation within the host plant (Figure 23). Less fungal growth on the root surface was observed for the HAC1 deletion strain in comparison to wild type and the VTA2 deletion strain in root colonization experiments. This can be either caused by impaired growth or by an inability to adhere to the surface. Similar to Hac1, the transcription factor Som1 was found to be required for proliferation on the root surface correlated to its impact on adhesin gene expression (Bui et al., 2019). Therefore, not only the similar impact on microsclerotia formation, but also the potential role of HAC1 in adhesion could hint into direction of an interplay of the UPR and Som1. However, the impact of HAC1 on adhesion requires further investigation. Different to HAC1, SOM1 is essential for penetration of the root

surface (Bui et al., 2019). However, target genes of Som1 were found to control different steps of plant root penetration and colonization including the transcription factors Vta2 and Vta3 (Bui et al., 2019).

Similar to V. dahliae, the initial penetration of the plant surface was unaffected in the appressorium-forming fungus A. brassicicola in absence of the UPR regulator AbHacA (Joubert et al., 2011). In the dimorphic fungus U. maydis the Hac1 orthologue is required to induce biotrophic growth within the plant after formation of appressoria and penetration of the plant surface (Heimel et al., 2010; Heimel et al., 2013). In contrast, UPR components are involved in the initial penetration of the plant surface by M. oryzae (Yi et al., 2009; Tang et al., 2015; Jiang et al., 2018b).

Since fungal re-isolation from stems was not possible for the V. dahliae HAC1 deletion strain in this study, one can assume that the fungus is hardly able to propagate in the vascular system and primarily remains in the roots or lower parts of the plant or does not survive after initial colonization of the root cortex. The absence of HAC1 resulted in severely reduced conidiation in simulated xylem medium (Figure 21), which might disable fungal spreading in the vascular system. Besides the decrease in conidiation, increased sensitivity to plant phytoalexins was observed in the A. brassicicola UPR regulator-deficient strain. An altered sensitivity to plant antimicrobial compounds remains to be elucidated in the V. dahliae HAC1 deletion strain, but could comprise an explanation for the observed in planta phenotype. Tomato plants treated with the HAC1 deletion strain displayed severely decreased disease symptoms, however, in ~30% of the plants rather mild symptoms could be observed (Figure 23A). The induction of these symptoms could be a result from damage caused by the fungus or from the energy costs of successful immune defense responses in the plants. The impairment in adapting to increased protein secretion demands upon host colonization might also be an explanation for the less pathogenic outcome of the interaction of the HAC1 deletion strain with the host plant as previously suggested in studies with the plant and animal pathogens A. fumigatus, A. brassicicola, and U. maydis (Joubert et al., 2011; Richie et al., 2011; Heimel et al., 2013).

It is possible that the plant is able to activate defense responses and suppress fungal colonization of the HAC1 deletion strain due to a lack of fungal effector gene expression and secretion, which are required to modulate the plants immune system. Examples for regulation of fungal effector genes by the corresponding UPR transcription factor in the maize colonizer U. maydis are given (Hampel et al., 2016). Further, it is possible that V. dahliae Hac1 regulates virulence factors independent of ER stress induction, similar to the signal peptide peptidase Ssp1 in U. maydis (Pinter et al., 2019). Here, deletion of the

Cib1-regulated gene ssp1 resulted in an avirulent in planta phenotype without impacts of ssp1 on vegetative growth or ER stress response (Pinter et al., 2019).

Figure 32: V. dahliae HAC1 regulates differentiation and virulence. The HAC1 mRNA occurs in two splice variants in the cell. The HAC1^u mRNA contains an unconventionally spliced intron, which is presumably targeted by the ER transmembrane endoribonuclease Ire1 (indicated by an arrow located at the ER membrane). Splicing of the unconventional intron results in the mRNA splice variant HAC1ⁱ, which is translated into the protein Hac1.

The transcription factor Hac1 presumably regulates transcription of UPR target genes by binding to UPR elements (UPRE) in their promoters and is essential for the development of microsclerotia as resting structures. Hac1 regulates vegetative growth under non-stress and ER stress conditions. Furthermore, HAC1 or Hac1-regulated genes are important for sporulation, fungal propagation within host plants, and induction of disease symptoms.

In this study, the impact of the constitutive presence of unconventionally spliced HAC1ⁱ mRNA on growth, differentiation, and virulence of V. dahliae was tested by construction of HAC1ⁱ-HA. Ectopic integration into the HAC1 deletion strain resulted in decreased gene expression in comparison to wild type similar to the strain containing an ectopic integration of the HAC1^u mRNA, HAC1^u-HA (Figure 18). The decreased expression levels were sufficient to complement the microsclerotia and growth phenotype of the HAC1 deletion strain in HAC1^u-HA. For HAC1ⁱ-HA even increased microsclerotia formation could be observed. The decreased growth in HAC1ⁱ-HA is assumed to be caused by a differential gene expression of metabolic genes in response to the perception of ER stress similar to A. niger (Carvalho et al., 2012) and not due to an incomplete complementation. However, tomato plants treated with HAC1^u-HA or HAC1ⁱ-HA strains displayed a similar induction of disease symptoms with intermediate severity between wild type and HAC1 deletion strain infected plants, suggesting an impact of the decreased expression levels of HAC1 on fungal virulence. Since the differences in HAC1 gene expression to wild type are similar in both strains and both

strains induce similar severities of disease symptoms, an impact of the constitutive presence of unconventionally spliced HAC1 mRNA on virulence cannot be assumed.

In conclusion, the UPR regulator Hac1 plays a major impact on the fungal ability to colonize host plants, either due to decreased growth and fungal propagation on the root surface as well as decreased conidiation and spreading within the plant, but likely as well due to the inability to evade the plant immune response, since it is not required for penetration, but for propagation inside the host (Figure 32).

4.4 The V. dahliae oleate ∆12-fatty acid desaturase Ode1 has