3.4 B. cereus AR156 triggers the accumulation of camalexin dependent on WRKY33 upon pathogen infection
There are reported that the transcription factor WRKY33 act as a key positive regulator of pathogen-induced camalexin biosynthesis. Therefore, we investigated whether WRKY33 was required for enhancing camalexin accumulation by AR156-induced ISR under P. capsici ,Pst DC3000, and B. cinerea infection. As we expected, the expression level of WRKY33 increased at 6 h and 12 h afterP. capsici , Pst DC3000, and B. cinerea infection with or without AR156 pretreated, while the expression level ofWRKY33 was significantly increased under the AR156 pretreatment, compared with the Mock (Figure 4 A, D, and G). These results suggested that AR156-induced ISR increased the expression of WRKY33 . To further explore whether AR156 regulates camalexin synthesis via wrky33 , we examined the accumulation levels of camalexin in wild-type Arabidopsis and wrky33mutants inoculated with pathogenic bacteria 5 d after pretreatment with AR156 or 0.85% NaCl. AR156 enhanced camalexin accumulation in Col-0, while the induction of camalexin by P. capsici , PstDC3000, and B. cinerea was blocked. Meanwhile, the induction of camalexin accumulation in wrky33 by AR156 was also impaired under pathogen inoculation (Figure 4 B, E, and H). The compromised camalexin induction in wrky33 mutant with or without AR156 pretreatment was correlated with the significantly attenuated activation of camalexin biosynthetic genes CYP71A13 and PAD3 after P. capsici , Pst DC3000, and B. cinerea infection (Figure 4 C, D, G, H, K, and L). Therefore, these data suggest that WRKY33plays a crucial role in AR156-induced ISR-regulated camalexin biosynthesis.
3.5 B. cereusAR156-triggered ISR against different pathogens is impaired in thewrky33 mutant
The above results suggested that WRKY33 acts as a positive regulator of camalexin synthesis by AR156-triggered ISR, and it remains unclear whether WRKY33 functions in the process of AR156-triggered ISR against P. capsici , Pst DC3000, andB. cinerea . To further investigate the function of WRKY33in the AR156-triggered ISR defense against pathogens, A. thalianaCol-0 and wrky33 were inoculated with B. cinerea ,Pst DC3000, and P. capsici 5 d after pretreated withB. cereus AR156 or 0.85% NaCl. The P. capsici biomass in the leaves of AR156-treated Col-0 plants were significantly lower than that in the mock plants inoculated only with P. capsici ; by contrast, the P. capsici biomass in the leaves of AR156-treatedwrky33 mutant plants was similar to that in the mock plants only inoculated with P. capsici (Figure 5 A and B), indicating that AR156-triggered ISR against P. capsici was abolished inwrky33 mutant plants. The B. cinerea biomass in the leaves of AR156-treated Col-0 plants were significantly lower than that in the control plants inoculated only with B. cinerea . However, theB. cinerea biomass in the leaves of AR156-treated wrky33mutant plants was similar to that in the control plants only inoculated with B. cinerea (Figure 5 C and D), indicating that AR156-triggered ISR against B. cinerea was abolished inwrky33 mutant plants. The pathogen density in the leaves of AR156-treated Col-0 plants was significantly lower than that in the control plants inoculated only with Pst DC3000. At the same time, the AR156-triggered ISR against Pst DC3000 was attenuated inwrky33 mutant plants (Figure 5 E and F). Taken together,WRKY33 functions as a positive regulator in the AR156-triggered ISR process against P. capsici , Pst DC3000, and B. cinerea .
3.6 PEN3 and PDR12 serve as positive regulators involved in AR156-triggered ISR against P. capsici, Pst DC3000, and B. cinerea
By analyzing the RNA-seq results, we found that the AR156-triggered ISR induced the accumulation of Pleiotropic Drug Resistance Transporters PEN3 and PDR12 (Figure 2 A, H and I), which were reported to function in immunity in Arabidopsis through the transport of camalexin and other Trp metabolites (He et al., 2019). Moreover, WRKY33 could bind to the promoters of PEN3 and PDR12 . Therefore, we speculate that AR156-triggered ISR resists P. capsici , Pst DC3000, and B. cinerea by upregulating PEN3 and PDR12 . To reveal the mechanism, pen3 , pdr12-2 single mutant, andpen3-3/pdr12-2 double mutant were used. A. thalianaplants, wild-type Col-0, pen3 , pdr12-2 single mutant, andpen3-3/pdr12-2 double mutant were inoculated with B. cinerea , Pst DC3000 and P. capsici 5 d after being pretreated with B. cereus AR156 or 0.85% NaCl. The P. capsici biomass in the leaves of pen3-3 mutant plants treated with AR156 was slightly reduced compared to the control inoculated withP. capsici only. The P. capsici biomass in the leaves ofpdr12-2 mutant plants treated with AR156 was similar to the control inoculated with P. capsici only. The P. capsicibiomass in leaves of pen3-3/pdr12-2 double mutant plants treated with AR156 had shown no significant difference compared to the control inoculated with P. capsici only (Figure 6 A). Similarly, we found that The B. cinerea biomass in the leaves of pen3-3 mutant plants treated with AR156 was slightly reduced compared to the control inoculated with B. cinerea only. The B. cinerea biomass in the leaves of pdr12-2 mutant plants treated with AR156 was similar to the control inoculated with B. cinerea only. TheB. cinerea biomass in leaves of pen3-3/pdr12-2 double mutant plants treated with AR156 had shown no significant difference compared to the control inoculated with B. cinerea only. These results suggest that PEN3 and PDR12 function as positive regulators of AR156-triggered ISR resistance to P. capsici andB. cinerea. However, the density of AR156-treated pen3-3was similar compared to the control inoculated with Pst DC3000 only, similar in pdr12-2 and pen3-3/pdr12-2 mutant leaves. The results suggested that it is PEN3 but not PDR12 acting as a positive regulator of AR156 triggering ISR resistance to Pst DC300. In summary, PEN3 and PDR12 serve as positive regulators involved in AR156-triggered ISR against pathogens. Specifically, PEN3 and PDR12 were jointly involved in AR156-triggered ISR against fungi and oomycetes, while PEN3 was involved in AR156-triggered ISR against Pst DC3000 (Zheng et al., 2006).