Spp1 represses defense responses in planta

Deletion of spp1 led to a highly reduced proliferation in planta (Fig 3.22 and 3.23), with chlorosis as the strongest symptom in the plant infection assay (Fig 3.15 and 3.25A and B). As this might indicate an activation of the plant defense response, the strains SG200 (WT), SG200∆spp1 (∆spp1) and SG200∆spp1 Pspp1:spp1^D279A-mC (∆spp1 spp1^D279A) were grown in YEPSlight to an OD600 of 1 and injected into 7-day-old maize seedlings and analyzed for reactive oxygen species (ROS) production. Infected leaf tissue was collected 24 hours post inoculation and stained with 3,3'-Diaminobenzidine (DAB) to visualize the formation of ROS in planta (Molina and Kahmann, 2007). Plants infected with the spp1 deletion mutant and the catalytically inactive Spp1^D279A mutant showed a strong accumulation of DAB when compared to the wildtype strain (Fig 3.26).

Figure 3.26: spp1 mutants elicited plant defense responses. 3,3'-Diaminobenzidine (DAB) staining of leaf tissue infected with U. maydis SG200 (WT), SG200∆spp1 (∆spp1) and SG200∆spp1 Pspp1:spp1^D279A-mC (∆spp1 spp1^D279A) was performed 24 h post inoculation. Brown precipitates reflect the presence of reactive oxygen species (ROS). Scale bar = 100 µm. Data generated in (Hach, 2018).

ROS in plant cells are mainly derived from hydrogen peroxide (Bolwell and Wojtaszek, 1997). To test if the loss of virulence might be connected to hypersensitivity against ROS an oxidative stress assay with H2O2 was performed. The strains SG200 (WT), SG200∆spp1 (∆spp1) and SG200∆spp1 Pspp1 :spp1-mC (∆spp1 spp1-:spp1-mC) were grown in YEPS^light to an OD600 of 1 and spotted on YNBG solid medium containing different concentrations of H2O2. After incubation for 48 hours at 28°C, no differences could be observed between WT, ∆spp1 and ∆spp1 spp1-mC (Fig 3.27). Moreover, the strains SG200 (WT), SG200∆cib1 (∆cib1) and SG200∆spp1 (∆spp1) were used in a cell wall stress assay, which was performed on solid medium containing either Congo Red or Calcufluor for cell wall stress. However, no differences in growth could be observed for the ∆cib1 or the ∆spp1 strain compared to the wildtype (Appendix Fig 7.4). The increased ROS formation in plant infections with U. maydis indicates a strongly activated plant defense response. However, ∆spp1 strains are not susceptible to cell wall stress as well as H2O2 stress, which indicates that spp1 is not necessary for cell wall integrity or detoxification of H2O2, respectively.

Figure 3.27: Spp1 is not crucial for H2O2 detoxification. H2O2 resistance of U. maydis strain SG200 (WT) and the ∆spp1 derivative was tested by serial 10-fold dilutions of strains, spotted on YNBG solid medium supplemented with the indicated concentration of H2O2. Plates were incubated for 48 h at 28°C.

ROS are mainly derived from H2O2, which is produced by the NADPH oxidase complex in plants.

(Bolwell and Wojtaszek, 1997). Therefore, the spp1 deletion strain was used in a plant infection assay with Diphenyleneiodonium (DPI), an inhibitor of ROS production mediated by the plant’s NADPH oxidase. (Molina and Kahmann, 2007; Fernández-Alvarez et al., 2009). However, treatment with DPI did not complement the ∆spp1 phenotype (Fig 3.28A). Furthermore, in Chlorazol Black E stainings of infected leaf tissue, no difference between the treated (Fig 3.28B, ∆spp1, DPI) and untreated (Fig 3.28B,

∆spp1, control) condition could be observed.

48 Figure 3.28: Inhibition of ROS production in planta could not recover the virulence of the ∆spp1 mutant.

(A) U. maydis strain SG200 (WT) and the ∆spp1 derivative were inoculated into 7-day-old maize seedlings.

Cultures used for infection experiments were supplemented with 0.5 µM (f.c.) DPI or an equivalent volume of solvent (DMSO). Disease symptoms were rated 8 dpi and grouped into categories depicted on the right. n represents the total number of inoculated plants from three independent experiments. (B) Fungal morphology of SG200 (WT) and the ∆spp1 was investigated by Chlorazol Black E staining of DPI or control (DMSO) treated infected leaf samples at 3 dpi. Scale bar = 20 μm.

Since infection of maize plants with deletion strain of spp1 as well as the catalytic inactive spp1^D279A mutant led to an increased plant defense response by increased ROS formation (Fig 3.26), gene expression of several plant defense response genes was tested in infected leaf tissue. To determine the gene expression of defense-related plant genes, the strains SG200 (WT), SG200∆spp1 (∆spp1) and SG200∆spp1 Pspp1:spp1^D279A-mC (spp1^D279A) and the complementation strain, SG200∆spp1 Pspp1 :spp1-mC (∆spp1 spp1), were cultivated in YEPS^light to an OD600 of 1 and injected into 7-day-old maize

seedlings. Infected leaf tissue was collected 2 dpi and expression plant genes was analyzed by qRT-PCR. The tested genes can be divided into two groups: PR1, PR3, PR4, PR5, ATFP4 and POX12, which can be conflated in the group of salicylic acid (SA)-related defense response genes (Fig 3.29 and Appendix Fig 7.3, dark gray bars) and CC9 as well as BBI, which are allocated in the group of jasmonic acid (JA)-related response genes (Fig 3.29, light gray bars). Suppression of the plant defense responses by biotrophic pathogens is maintained by counteracting the SA response pathway and induce JA-related defense responses to prevent programmed plant cell death (Glazebrook, 2005). Consistently, all SA marker genes were highly induced in the ∆spp1 and the spp1^D279A strain compared to wildtype or the complementation strain (Fig 3.29 and Appendix Fig 7.3).

Figure 3.29: Strains with loss plants infected with the WT and represent the mean of three biological replicates with two technical duplicates each.

GAPDH was used for normalization. Dark gray and light gray color of the bars indicate SA responsive genes

50 In comparison to plants infected with the WT strain, PR1 revealed the highest induction of all tested plant defense response genes. Gene expression of plants infected with the ∆spp1 mutant or the spp1^D279A mutant, had 78 mean fold changes (mfc) and 151 mfc, respectively, compared to plants infected with the wildtype strain. Differences in expression levels for PR5 compared with plants infected with the wildtype were 15 mfc and 27 mfc in plants infected with the spp1 deletion mutant or the catalytically inactive spp1 mutant, respectively. For ATFP4, a mean fold change of 9 for plants infected with the spp1 deletion mutant, as well as 10 for plants infected with the catalytically inactive spp1 was measured compared to plants infected with the wildtype. For the POX12 gene, 2 mfc and 5 mfc in expression levels compared to plants infected with the wildtype strain were measured for the spp1 deletion mutant and the catalytically inactive spp1 mutant, respectively (Fig 3.29).

For the additionally tested pathogenesis-related gene PR3, a mean fold change of 2 mfc and 4 mfc, for

∆spp1 and spp1^D279A were measured compared to the wildtype strain, and for PR4, mean fold changes of 14 mfc and 24 mfc for ∆spp1 and spp1^D279A were observed compared to the wildtype strain (Appendix Fig 7.3).

In contrast to the group of tested SA-responsive genes, the JA-responsive genes had an overall lower expression in planta. A decreased expression compared to the wildtype strain for CC9 could be observed with 3 mfc for both, the spp1 deletion mutant as well as the catalytically inactive spp1 mutant. A decrease in the expression could also be observed for BBI with 2 mfc in the spp1 deletion mutant and the catalytically inactive spp1 mutant compared to the wildtype strain (Fig 3.29). Taken together, this indicates that the loss of the Spp1 function may be involved in the suppression of the plant defense responses.

3.3.7 Deletion mutants of ER-associated degradation pathway (ERAD) and