ABSTRACT
SARD1 and CBP60g are two master regulators in plant immunity. They are required for the constitutive defense responses in the Arabidopsis snc2-1D mutant, which carries a gain-of-function mutation in a receptor-like protein. Here we report that WRKY54 and WRKY70 are required for activation of SARD1 and CBP60g expression and defense responses in snc2-1D. In addition, the induction of SARD1 and CBP60g by the bacterial pathogen Pseudomonas syringae pv. maculicola is significantly reduced in sid2 wrky54 wrky70 triple mutants compared to the sid2 single mutants, suggesting that WRKY54 and WRKY70 positively regulate the SID2-independent expression of SARD1 and CBP60g during pathogen infection. Our study revealed WRKY54 and WRKY70 as positive regulators of SARD1 and CBP60g expression in plant defense.
KEYWORDS: WRKY54, WRKY70, SARD1, CBP60g, Plant immunity
Arabidopsis SARD1 and CBP60g are two key transcription factors that regulate the expression of a large number of defense-related genes and play broad roles in plant immunity.1 They are required for pathogen-induced biosynthesis of the plant defense hormone salicylic acid (SA),2,3 which is produced mainly from isochorismate.4 Following the conversion of chorismate to isochorismate by ISOCHORISMATE SYNTHASE 1 (ICS1) in the plastids, isochorismate is transported to the cytosol by ENHANCED DISEASE SUSCEPTIBILITY 5 (EDS5), where it is conjugated to glutamate by AVRPPHB SUSCEPTIBLE 3 (PBS3).4–6 The resulting isochorismate-9-Glu subsequently decomposes to produce SA.5,6 The induction of ICS1, EDS5 and PBS3 during bacterial infection is coordinately regulated by SARD1 and CBP60g.1 Similarly, the induction of genes involved in the biosynthesis of N-hydroxypipecolic acid (NHP), a key signaling molecule in systemic acquired resistance (SAR), also relies on SARD1 and CBP60g.1,7 In sard1 cbp60g double mutant plants, accumulation of SA and NHP during pathogen infection is dramatically reduced.2,3,8 Consistent with the reduced SA and NHP levels, sard1 cbp60g exhibit compromised local immunity and complete loss of SAR.1–3
WRKY70 plays complex roles in plant immunity. The expression of WRKY70 is activated by SA and repressed by JA.9 Overexpression of WRKY70 activates the expression of SA-responsive pathogenesis-related (PR) genes, whereas silencing of WRKY70 results in elevated expression of JA-responsive genes, suggesting that WRKY70 acts as an activator of SA-responsive genes and a repressor of JA-responsive genes, which is further supported by the opposite roles of WRKY70 in JA-mediated resistance to the necrotrophic pathogen Alternaria brassicicola and SA-mediated resistance to biotrophic pathogen Erysiphe cichoracearum.9,10 The wrky70 single mutants display enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm) ES4326,11 but the wrky70 wrky46 double mutant is more susceptible to Pseudomonas syringae pv. tomato DC3000 than the wild type and single mutants. 12 In wrky70 and wrky70 wrky54 mutant plants, both basal and Psm ES4326 avrRpt2-induced SA levels are higher than in wild type, suggesting that WRKY70 also negatively regulates SA levels.13 Recently it was reported that the activity of WRKY70 is regulated by phosphorylation and the accumulation of phosphorylated WRKY70 is regulated by the E3 ubiquitin ligase CHYR1.14
Arabidopsis snc2-1D carries a gain-of-function mutation in a receptor-like protein which constitutively activates plant immune responses.15 WRKY70 was previously shown to function downstream of SNC2 to regulate NPR1-independent defense signaling.15 To further analyze the roles of WRKY70 and its close homolog WRKY54 in SNC2-mediated immunity, we generated the snc2-1D wkry70 double mutant and the snc21D wkry54 wrky70 triple mutant. As shown in Figure 1A, the dwarf morphology of snc2-1D is only partially suppressed in snc2-1D wkry70, but the snc2-1D wkry54 wrky70 triple mutant is much bigger and has similar morphology as wkry54 wrky70. Similarly, the increase of PR2 and ICS1 expression in snc2-1D is reduced in snc2-1D wkry70, and blocked in snc2-1D wkry54 wrky70 (Figure 1B and 1C). To test whether WRKY70 and WRKY54 are required for the enhanced disease resistance in snc21D, we challenged the seedlings of snc2-1D wkry70 and snc2-1D wkry70 wrky54 with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) Noco2. As shown in Figure 1D, the resistance to Hpa Noco2 conferred by snc2-1D is completely lost in the snc2-1D wkry54 wrky70 triple mutant. Together, these findings suggest that WRKY70 and WRKY54 function redundantly downstream of SNC2 to activate plant defense responses.
Figure 1.
WRKY54 and WRKY70 are required for the up-regulation of SARD1 and CBP60g expression and the constitutive defense responses in snc2-1D.
(A) Morphology of Col-0 (WT), snc2-1D, snc2-1D wrky70, snc2-1D wrky54 wrky70 and wrky54 wrky70 plants. The pictures were photographed on four-week-old plants grown on soil.(B-C) Expression of PR2 (B) and ICS1(C) in the indicated genotypes. Bars represent means ± SD. Different letters indicate samples with statistical differences (P < .05, Student’s t-test; n = 3).(D) Growth of H.a. Noco2 on the indicated genotypes. Bars represent means ± SD. Different letters indicate samples with statistical differences (P < .05, Student’s t-test; n = 4).(E-F) Expression of SARD1 (E) and CBP60g (F) in the indicated genotypes. Bars represent means ± SD. Different letters indicate samples with statistical differences (P < .05, Student’s t-test; n = 3).Gene expression analysis in (B, C, E, F) was performed on two-week-old plate-grown seedlings.
SARD1 and CBP60g are known to function downstream of SNC2 and be required for SNC2-mediated immunity.1 Loss of function of both SARD1 and CBP60g leads to almost complete suppression of the constitutive defense responses in snc2-1D. WRKY70 has been shown to bind to a GACTTTT sequence motif known as WT-box and a single WT-box is present in the promoter of SARD1.11 To test whether WRKY70 and WRKY54 regulate the expression levels of SARD1 and CBP60g, we compared SARD1 and CBP60g expression levels in two-week-old wild type, snc2-1D, snc2-1D wkry70 and snc2-1D wkry54 wrky70 seedlings (Figure 1E and 1 F). The expression levels of SARD1 and CBP60g are much higher in snc2-1D compared to the wild type. The increase in SARD1 and CBP60g expression is dramatically reduced in snc2-1D wkry70 and completed suppressed in snc2-1D wkry54 wrky70, suggesting that WRKY70 and WRKY54 function redundantly to promote SARD1 and CBP60g expression in snc2-1D.
Previously it was shown that the basal expression level of SARD1 is elevated in wrky70 mutant plants.11 To test whether WRKY70 negatively regulates SARD1 expression through the WT-box, we mutated the WT-box in the SARD1 promoter by site-directed mutagenesis and generated Arabidopsis transgenic plants expressing the luciferase reporter gene under the wild type and mutant SARD1 promoter in wild type Col-0 background. Quantification of the luciferase activities in the transgenic lines showed that there is no significant difference between the pSARD1-Luc and pSARD1mt-Luc transgenic lines (Figure 2), suggesting that the WT-box in the SARD1 promoter is not required for negative regulation of basal expression level.
Figure 2.
Levels of luciferase expressed under the wild type and mutant SARD1 promoters in Arabidopsis transgenic lines
Firefly luciferase activities were determined on two-week-old Arabidopsis seedlings. Bars represent means ± SD (n = 8). Statistical significance was determined using Student’s t-test. The ns represents no significance.
In four-week-old wrky54 wrky70 double mutant plants, the basal expression levels of SARD1 and CBP60g are much higher compared to the wild type (Figure 3A and 3B). To test whether the increased basal SARD1 and CBP60g expression in wrky54 wrky70 is caused by increased SA biosynthesis, we crossed sid2-1 and sid2-3 mutants with wrky54 wrky70 to obtain the sid2-1 wrky54 wrky70 and sid2-3 wrky70 wrky54 triple mutants. As shown in Figure 3C, the dwarf morphology of wrky54 wrky70 is suppressed by the sid2 mutations. In the sid2 wrky54 wrky70 mutants, the basal expression levels of SARD1 and CBP60g are much lower compared to wrky54 wrky70 (Figure 3A and 3B), suggesting that the elevated SARD1 and CBP60g levels in wrky54 wrky70 are at least partially caused by increased SA levels.
Figure 3.
Effects of sid2 mutations on the mutant morphology and expression of SARD1 and CBP60g in wrky54 wrky70.
(A-B) Induction of SARD1 (A) and CBP60g (B) expression in Col-0 (WT), sid2-1, sid2-3, wrky54 wrky70, sid2-1 wrky54 wrky70 and sid2-3 wrky54 wrky70 plants. Leaves of four-week-old plants were infiltrated with Pseudomonas syringae p.v. maculicola ES4326 (OD600 = 0.001) or 10 mM MgCl2 (Mock). Samples were collected 24 hours after infiltration.(C) Morphology of four-week-old plants of the indicated genotypes.(D) A working model of the relationships between WRKY54/WRKY70 and SARD1/CBP60g. Upon pathogen infection, SARD1 and CBP60g activate the expression of WRKY54 and WRKY70, which in turn promote the transcription of SARD1 and CBP60g in a positive feedback loop. WRKY54 and WRKY70 also negatively regulate SA biosynthesis through an unknown mechanism.
To test whether WRKY54 and WRKY70 are required for the induction of SARD1 and CBP60g, we checked the expression levels of SARD1 and CBP60g after infection by Psm ES4326 in sid2, wrky54 wrky70 and sid2 wrky54 wrky70 mutants. As shown in Figure 3A and 3B, induction of both SARD1 and CBP60g by Psm ES4326 is significantly lower in the sid2 single mutants and further reduced in the sid2 wrky54 wrky70 triple mutants, suggesting that WRKY54 and WRKY70 contribute to the SID2-independent expression of SARD1 and CBP60g during pathogen infection.
The requirement of WRKY54 and WRKY70 for the induction of SARD1 and CBP60g in snc2-1D and during Psm ES4326 infection suggests that WRKY54 and WRKY70 positively regulate the expression of SARD1 and CBP60g in plant immunity. As the WT-box WRKY70 binds to is present in the promoter regions of both SARD1 and CBP60g, most likely WRKY70 directly activates the expression of SARD1 and CBP60g through the WT-box. Interestingly, SARD1 and CBP60g also bind to the promoter of WRKY70 and are required for the up-regulation of WRKY70 in snc2-1D and during infection by Psm ES4326 1. These findings suggest that WRKY54/WRKY70 and SARD1/CBP60g form an amplification loop to promote each other’s expression (Figure 3D).
In wrky70 and wrky54 wrky70 mutant plants, the basal levels of ICS1 and SA are significantly higher than in wild type plants.13 How loss of WRKY54/WRKY70 leads to increased ICS1, SARD1 and CBP60g basal expression levels is currently unclear. Because WRKY70 binds to the WT-box in the SARD1 promoter, it was suggested that binding of WRKY70 to the SARD1 promoter results in suppression of its expression.11 However, mutating the WT-box does not lead to increased expression of the luciferase reporter gene under the control of the SARD1 promoter, suggesting that the effect of loss of WRKY54 and WRKY70 on basal SARD1 expression could be indirect. One possibility is that WRKY54/WRKY70 are guarded by an unknown NLR protein and loss of WRKY70/54 triggers partial activation of the NLR protein, leading to increased SARD1 and ICS1 expression. Alternatively, WRKY54 and WRKY70 are required for the expression of a negative regulator of SA biosynthesis. Loss of WRKY54/70 triggers increased SA biosynthesis and elevated SA level, which induces the expression of ICS1, SARD1 and CBP60g.
Supplementary Material
Acknowledgments
This study was financially supported by grants from the Natural Sciences and Engineering Research Council (NSERC) Discovery program of Canada and National Natural Science Foundation of China (31828008), and scholarships to SC and YD from the Chinese Scholarship Council.
Competing financial interests
The authors declare no competing financial interests.
Supplementary material
Supplemental data for this article can be accessed on the publisher’s website
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