HAC1 is essential for microsclerotia formation of V. dahliae

The impact of V. dahliae HAC1 on growth, microsclerotia formation, and fungal conidiation was investigated by comparison of the ex planta phenotypes of the ∆HAC1, HAC1-C, HAC1^u-HA and HAC1ⁱ-HA strains.

Vegetative growth of the HAC1 deletion strain was severely impaired under all tested growth conditions (Figure 19). Colonies formed by the deletion strain ten days after spot inoculation formed less aerial mycelium, appeared less dense, and more transparent in comparison to wild type colonies grown on complete medium (PDM), minimal medium, or minimal medium supplemented with tunicamycin (Figure 19). Melanization of the ∆HAC1 colony centers was not observed under any tested growth condition. Furthermore, the deletion strain formed smaller colonies compared to wild type for every tested condition.

Quantification of vegetative growth showed a significant decrease in the colony diameter about 12% for ∆HAC1 ten days after spot inoculation on CDM (Figure 19B).

Vegetative growth on minimal medium supplemented with tunicamycin was tested to examine the ability to cope with ER stress. Tunicamycin inhibits the glycosylation of proteins and, thereby, prevents folding in the ER und subsequent protein secretion (Guillemette et al., 2011). The mean colony diameter of ∆HAC1 grown on CDM supplemented with tunicamycin was 11% smaller than wild type colonies ten days after spot inoculation (Figure 19B). The supplementation of tunicamycin induced no additional decrease in the colony size of ∆HAC1 relative to wild type and the relative difference was unaltered (Figure 19B).

Figure 19: V. dahliae requires the expression of HAC1 for growth with or without tunicamycin-induced ER stress and melanization. Spot inoculation of 50 000 spores from the wild type JR2 (WT) or ∆HAC1, HAC1-C, HAC1^u-HA, and HAC1ⁱ-HA strains and incubation at 25 °C for ten days. ∆HAC1 displays reduced vegetative growth compared to wild type. HAC1-C and HAC1^u-HA strains display wild type-like growth, whereas colony diameters of HAC1ⁱ-HA are decreased under non-stress conditions and relatively increased upon supplementation of tunicamycin. (A) Ex planta phenotypes on PDM, SXM, CDM, and CDM supplemented with 1 µg/ml tunicamycin (TM). ∆HAC1 forms less aerial mycelium, appears less dense, and more transparent on PDM, CDM, and CDM+TM. ∆HAC1 displays no melanization in the colony centers. (B) Quantification of vegetative growth. Growth was quantified by measurement of two perpendicular colony diameters for three colonies per transformant and medium (n=1). Two independent transformants were tested for ∆HAC1 (VGB371, VGB372), HAC1^u-HA (VGB439, VGB440), and HAC1ⁱ-HA (VGB437, VGB438), as well as one HAC1-C (VGB382) transformant. Bars represent mean values with standard deviations from three independent experiments for wild type and ∆HAC1, and two independent experiment for HAC1-C, HAC1^u-HA and HAC1ⁱ-HA grown on CDM. Mean values with standard deviations are shown from three independent experiments for wild type,

∆HAC1, and HAC1-C, and two independent experiments for HAC1^u-HA and HAC1ⁱ-HA grown on CDM+TM. Significant differences to wild type determined with one-way Anova and Student´s t-test are shown with *p<0.05; **p<0.01; ***p<0.001, ****p=0, ns= non-significant.

The HAC1-C and HAC1^u-HA strains displayed wild type-like growth. Colony diameters of HAC1ⁱ-HA were decreased on all non-stress inducing media, but increased on CDM

supplemented with tunicamycin (Figure 19). A decrease about 14% in the colony diameter was determined for the HAC1ⁱ-HA strain relative to wild type on CDM, whereas supplementation of tunicamycin resulted in a ~15% increase of the colony diameters relative to wild type (Figure 19B). This observations suggests that the presence of the unconventionally spliced HAC1 mRNA in the HAC1ⁱ-HA strain and constitutive activation of the UPR enable a more efficient response to the induced ER stress.

Comparison of the ex planta phenotypes of the HAC1 deletion and the HAC1-C, HAC1^u-HA, and HAC1ⁱ-HA strains to wild type revealed the absence of melanized colony centers for ∆HAC1 on all tested media, whereas the HAC1-C and HAC1ⁱ-HA strains displayed increased melanization. The ability of ∆HAC1 to develop microsclerotia as resting structures, maybe in an unmelanized form, was further analyzed in comparison to the complementation strains. The differences in melanization are most apparent in the colony centers and in colony cross sections (Figure 20). During microscopy of fungal material from colony centers of ∆HAC1 no melanized or unmelanized microsclerotia were observed for any medium, as exemplified for CDM in Figure 20.

Figure 20: V. dahliae HAC1 is essential for microsclerotia formation. Microsclerotia formation was examined in ∆HAC1, HAC1-C, HAC1^u-HA, and HAC1ⁱ-HA strains by spot inoculation of 50 000 spores on CDM and incubation at 25 °C for ten days. Two independent transformants were tested for ∆HAC1 (VGB371, VGB372), HAC1^u-HA (VGB439, VGB440), and HAC1ⁱ-HA (VGB437, VGB438) strains and a single transformant for the HAC1-C (VGB382) strain. Close up pictures were taken from colony centers (indicated as red boxes) and cross sections were cut to visualize the appearance of microsclerotia as melanized structures in the agar. Fungal material from colony centers was scraped off for microscopy to observe microsclerotia morphology. Neither melanized nor unmelanized microsclerotia were observed for the ∆HAC1 strain, whereas formation of wild type-like microsclerotia was observed for all other strains. Microsclerotia formation was qualitatively increased in the HAC1-C and the HAC1ⁱ-HA strains in comparison to wild type. Black scale bar = 1 mm, blue scale bar = 20 µm.

HAC1-C

∆HAC1

WT HAC1^u-HA HAC1ⁱ-HA

For all complementation strains, wild type-like shaped and melanized microsclerotia could be observed, however, in qualitatively increased occurrence for the HAC1-C and HAC1ⁱ-HA strains and wild type-like frequencies in the HAC1^u-HA strain (Figure 20). The absence of microsclerotia in ∆HAC1 and the increase in melanization in HAC1ⁱ-HA regardless of the decreased HAC1 expression levels in HAC1ⁱ-HA (Figure 18A) corroborate the role of HAC1 in regulation of microsclerotia formation.

Within the plants vascular system V. dahliae forms conidiospores for spreading and systemic colonization of the host. The role of the UPR regulator Hac1 in conidiation was characterized by examination of the ability to form spores in the HAC1 deletion strain compared to wild type and the HAC1-C complementation strain. Therefore, the same number of spores was inoculated in liquid simulated xylem medium and quantified after five days. The HAC1 deletion strain displayed significantly reduced conidiospore numbers to 15% of the wild type level (Figure 21). Conidia levels of the HAC1-C strain were more similar to wild type with 86%.

In summary, HAC1 is essential for microsclerotia formation and has important impacts on conidiation. Constant activation of the UPR in the HAC1ⁱ-HA strain is correlated with increased growth in response to ER stress and increased microsclerotia formation in V. dahliae.

Figure 21: V. dahliae HAC1 positively regulates conidiospore formation. Quantification of conidiation was performed by inoculation of 4000 spores/ml in liquid SXM and incubation at 25 °C under constant agitation for five days in triplicates. The number of spores was determined for the wild type JR2, two independent transformants of ∆HAC1 (VGB371, VGB372), and the HAC1-C (VGB382) complementation strain in three independent experiments. The numbers of conidia relative to wild type are shown. The values determined for two independent ∆HAC1 transformants are represented in one bar. Significant differences to wild type determined by one-way Anova and Student´s t-test are shown with ****p=0,

*p<0.05 with n(WT)=3, n(∆HAC1)=6, n(HAC1-C)=3. The HAC1 deletion strain shows reduced conidia formation. Conidia formation of HAC1-C was more similar to the wild type-level.

3.3.5 V. dahliae HAC1 is dispensable for penetration of the A. thaliana root cortex