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).