Effect of B. cinerea infection after priming with frass or insect skin residue on defense gene

Expression of several defense related genes were also investigated in plants infected with Botrytis cinerea (BC) after they had been exposed to frass and insect skin residue (ISR) for a longer time period, potentially showcasing a priming effect triggered by the frass and ISR treatments.

Arabidopsis Col-0 were grown for 3 weeks in C-soil before transplanted to a frass, ISR or control fertilizer treatment for 2 weeks.

Leaves were inoculated with B. cinerea spores and gene expression was analyzed 8, 24 and 48 hours post inoculation (hpi). In addition to ZAT10, ERF5, and PER4 used for the elicitor treatment described above, the expression of three WRKY transcription factors (WRKY33, WRKY53 and WRKY75) and a myeloblastosis transcription factor (MYB51), two cytochrome P450 monooxygenases (CYP71A13 and CYP71B15), CML37 (calmodulin-like protein 37), and an uncharacterized Chitinase were monitored (Figures 4.11 to 4.16; Appendix 1.8).

The WRKY gene family are transcription factors that are important in the modulation of defense responses as well as many other functions in the plant. Both WRKY33, WRKY53 and WRKY75 are involved in the Arabidopsis defense against fungal pathogens, especially Botrytis cinerea (Aranega-Bou et al., 2014). MYB51 is a transcription factor regulating the production of camalexin and glucosinolate, compounds involved in plant immunity (Frerigmann et al., 2015), while CYP71A13 and CYP71B15 encode proteins important in camalexin biosynthesis (Birkenbihl et al., 2012). CML37 is a Ca²⁺ sensitive defense regulator in plants, connecting Ca²⁺

signaling with the jasmonate response pathway (Scholz et al., 2014). Chitinases are enzymes that hydrolyses chitin and can be synthesized by plants as a defense response against fungal pathogens (Kumar et al., 2018).

Expression of ZAT10 was elevated in frass treatments compared to the ISR and control soil treatments, particularly in the BC infected plants (Figure 4.11). ISR treatments with BC infection also led to a significantly induced ZAT10 expression compared to the control after 24 and 48 hours. After 48 hpi, BC infected plants treated with frass or ISR displayed considerably higher expression values than mock treated plants.

Both ERF5 (Figure 4.12) and WRKY33 (Figure 4.13) were overall higher expressed in frass and ISR treatments than in the control treatment. However, BC inoculation seemed to have little effect on the expression levels of these genes, except after 48 hours, where the BC infestation led to higher expression levels in both genes compared to mock (only significant for ISR). PER4 (presented in Appendix 1.8) showed quite similar results as ERF5 and WRKY33, with an inducing effect by frass and ISR, but the treatment that led to the highest expression varied considerably depending on the time point.

Figure 4.11: Gene expression analysis of ZAT10 target gene after B. cinerea infection. 5-week-old Arabidopsis Col-0 leaves grown for 2 weeks in control (Ctrl), frass or ISR soil treatments. Leaves were inoculated with Botrytis cinerea spores (BC, red bars) or Vogel solution (Mock, blue bars), and harvested at 8 hours, 24 hours or 48 hours post inoculation (hpi). All values are relative to ZAT10 expression in plants grown on Ctrl soil, mock-inoculated and subsequently incubated for 8 h (set to 1). Error bars show 95% confidence intervals (CI), but the upper CI for BC Frass 8 h (32.0) and for BC Frass 48 h (17.9) are capped in the figure for presentation purposes. Different letters denote significant differences within the same time point (no comparisons between time points), Tukey-Kramer multiple comparison test, p < 0.05.

Figure 4.12: Gene expression analysis of ERF5 target gene after B. cinerea infection. 5-week-old Arabidopsis Col-0 leaves grown for 2 weeks in control, frass or ISR soil treatments. Leaves were inoculated with Botrytis cinerea spores (BC, red bars) or Vogel solution (Mock, blue bars), and harvested at 8 hours, 24 hours or 48 hours post inoculation (hpi). All values are relative to ERF5 expression in plants grown on Ctrl soil, mock-inoculated and subsequently incubated for 8 h (set to 1). Error bars show 95% confidence intervals. Letters denote significance within the same time group (no comparisons between time groups), Tukey-Kramer multiple comparison test, p <

0.05.

ISR

ISR

ISR ISR

Figure 4.13: Gene expression analysis of WRKY33 target gene after B. cinerea infection. 5-week-old Arabidopsis Col-0 leaves grown for 2 weeks in control, frass or ISR soil treatments. Leaves were inoculated with Botrytis cinerea spores (BC, red bars) or Vogel solution (Mock, blue bars), and harvested at 8 hours, 24 hours or 48 hours post inoculation (hpi). All values are relative to WRKY33 expression in plants grown on Ctrl soil, mock-inoculated and subsequently incubated for 8 h (set to 1). Error bars show 95% confidence intervals. Letters denote significance within the same time group (no comparisons between time groups), Tukey-Kramer multiple comparison test, p < 0.05.

Figur 4.14: Gene expression analysis of CML37 target gene after B. cinerea infection. 5-week-old Arabidopsis Col-0 leaves grown for 2 weeks in control, frass or ISR soil treatments. Leaves were inoculated with Botrytis cinerea spores (BC, red bars) or Vogel solution (Mock, blue bars), and harvested at 8 hours, 24 hours or 48 hours post inoculation (hpi). All values are relative to CML37 expression in plants grown on Ctrl soil, mock-inoculated and subsequently incubated for 8 h (set to 1). Error bars show 95% confidence intervals (CI), but the upper CI for BC Frass 8 h (76.6), BC Frass 48 h (21.4), and BC IS 48 h (35.4) are capped in the figure for presentation purposes.

Letters denote significance within the same time group (no comparisons between time groups), Tukey-Kramer multiple comparison test, p < 0.05.

ISR ISR

ISR ISR

As to the expression of CML37, a positive effect from frass was seen after 8 hours in both BC and mock inoculation, although non-significant to the control (Figure 4.14). After 24 hours, frass and ISR have a clear effect on CML37 expression levels when infested with BC, while this effect is less clear in mock treatments. After 48 hours, plants exposed to BC show a lot higher expression of CML37 compared to mock-inoculated ones, and frass and ISR treated plants are higher expressed but not significantly different compared to BC control. Considerable variations within treatments led to large confidence intervals and consequently less significant results.

WRKY53 displayed similar expression levels in mock and BC treated plants in all time points (Figure 4.15). Frass and ISR seemed to lead to increased induction also for this gene, especially at 8 hpi. However, after 48 hpi, all treatments exhibited the same low expression values.

Figure 4.15: Gene expression analysis of WRKY53 target gene after B. cinerea infection. 5-week-old Arabidopsis Col-0 leaves grown for 2 weeks in control, frass or ISR soil treatments. Leaves were inoculated with Botrytis cinerea spores (BC, red bars) or Vogel solution (Mock, blue bars), and harvested at 8 hours, 24 hours or 48 hours post inoculation (hpi). All values are relative to WRKY53 expression in plants grown on Ctrl soil, mock-inoculated and subsequently incubated for 8 h (set to 1). Error bars show 95% confidence intervals. Letters denote significance within the same time group (no comparisons between time groups), Tukey-Kramer multiple comparison test, p < 0.05.

In contrast to WRKY33 and WRKY53, WRKY75 (Figure 4.16) showed very low expression values and no significant differences between treatments until 48 hours post inoculation, where the BC infestation led to a substantial increase in expression compared to the mock treatment.

Although this was especially prominent in plants grown on soil supplemented with frass and IS, the levels were not significantly different from plants grown on control soil. The same trend was seen for the expression of CYP71A13 (Figure 4.17) and CYP71B15 (Appendix 1.8).

Chitinase also showed increased expression after 48 hours in BC-infested plants, but only 3-5 times more than in mock-inoculated ones, and without any significant differences between control, frass and IS treatments (results in Appendix 1.8).

ISR

ISR

Figure 4.16: Gene expression analysis of WRKY75 target gene after B. cinerea infection. 5-week-old Arabidopsis Col-0 leaves grown for 2 weeks in control, frass or ISR soil treatments. Leaves were inoculated with Botrytis cinerea spores (BC, red bars) or Vogel solution (Mock, blue bars), and harvested at 8 hours, 24 hours or 48 hours post inoculation (hpi). All values are relative to WRKY75 expression in plants grown on Ctrl soil, mock-inoculated and subsequently incubated for 8 h (set to 1). Error bars show 95% confidence intervals (CI), but the upper CI for BC Ctrl 8 h (133.8), BC Ctrl 48 h (481.2), BC Frass 48 h (42940.0), and BC IS 48 h (635.6) are capped in the figure for presentation purposes. Letters denote significance within the same time group (no comparisons between time groups), Tukey-Kramer multiple comparison test, p < 0.05.

Figure 4.17: Gene expression analysis of CYP71A13 target gene after B. cinerea infection. 5-week-old Arabidopsis Col-0 leaves grown for 2 weeks in control, frass or ISR soil treatments. Leaves were inoculated with Botrytis cinerea spores (BC, red bars) or Vogel solution (Mock, blue bars), and harvested at 8 hours, 24 hours or 48 hours post inoculation (hpi). All values are relative to CYP71A13 expression in plants grown on Ctrl soil, mock-inoculated and subsequently incubated for 8 h (set to 1). Error bars show 95% confidence intervals (CI), but the upper CI for BC Ctrl 48 h (734.), BC Frass 48 h (28682.6), and BC IS 48 h (548.5) are capped in the figure for presentation purposes. Letters denote significance within the same time group (no comparisons between time groups), Tukey-Kramer multiple comparison test, p < 0.05.

ISR ISR

ISR ISR

4.2.5 Pathogen assays with B. cinerea and P. syringae on Arabidopsis thaliana