Direct regulation of WRKY70 by AtMYB44 in plant defense responses

Abstract

Cross-talk between hormones is required for plant response to developmental cues and environmental stresses. This cross-talk is achieved through several regulators located in convergence point of distinct hormonal signaling. In plant defense responses, salicylic acid and jasmonic acid affect each other in antagonistic manner. In a recent study we showed that AtMYB44 transcription factor positively regulates SA-mediated defense expression and enhanced resistance to Pst DC3000. On the other hand, AtMYB44 negatively regulates expression of JA-mediated defense gene expression and downregulated resistance to Alternaria brassicicola. Effects of AtMYB44 in SA- and JA-mediated defense responses were achieved through direct regulation of WRKY70 expression which acts as an integrator of cross-talk between SA and JA in plant defense responses. Here we provide further evidence that AtMYB44 regulates defense responses by transcriptional activation of downstream gene, WRKY70. This result shows that AtMYB44 is an integrator of cross-talk between SA and JA in plant defense responses.

Plants compete with pathogens for survival throughout their life cycle. To cope with attack from diverse pathogens, plants produce several phytohormones including salicylic acid (SA) and jasmonic acid (JA).^{1, 2} SA and JA have well defined function in the regulation of plant defense responses. SA is associated in defense response against biotrophic pathogen, whereas JA has a function in defense responses against herbivore and necrotrophic pathogen.^3,4 There is a significant cross-talk between SA- and JA-mediated defense signaling.^1,5,6 SA signaling mutants resulted in upregulation of JA-mediated defense response. On the contrary, JA signaling mutants resulted in upregulation of SA-mediated defense. Mutual antagonism between SA and JA is regarded as an effective mechanism for developing specialized defense responses.^2,6 In spite of significance of antagonistic interaction between SA and JA, its network was still not elucidated.

AtMYB44 Directly Regulates Expression of WRKY70

AtMYB44, a R2R3 MYB transcription factor, have been involved in development and abiotic stresses.^7,8 It is also reported that AtMYB44 is induced by defense signals.^8-10 Previously, we reported that AtMYB44 negatively regulated expression of JA-mediated genes such as VSP1 and PDF1.2.^11,12 To understand molecular mechanism of antagonistic interaction between SA and JA, we monitored expression of JA- and SA-responsive gene expression through microarray analysis.⁸ Overexpression of AtMYB44 leads to activation of PR genes and T-DNA insertional knockout mutation resulted in attenuated expression of them.¹² The role of AtMYB44 in positive modulation of SA-mediated defense responses was also reported. AtMYB44 overexpression plants showed enhanced cell death and hydrogen peroxide accumulation against Pst DC3000 infection.¹³

We showed that WRKY70, an integrator in cross-talk between SA and JA, was constitutively expressed in AtMYB44 overexpression plants.^12,14 Furthermore, induced expression of AtMYB44 by the β-esradiol inducible promoter in transgenic plants resulted in epistatic expression of WRKY70 and PR1 in downstream. Overexpression effects of AtMYB44 were abolished by wrky70 mutation. AtMYB44 directly regulates expression of WRKY70 independently of NPR1, a signal transducer of SA.

Transactivation of WRKY70 by AtMYB44

From SELEX, EMSA and ChIP experiments we demonstrated that AtMYB44 directly binds to CNGTTA element in the promoter region of WRKY70.¹² There are three MYB binding elements (REs) in the promoter region of WRKY70. To test their function in transcriptional activation of WRKY70 by AtMYB44, we performed trans-activation analysis by using transient expression system in Nicotiana benthamiana (Fig. 1). The WRKY70 promoter region of 2,380 bp containing three MYB binding elements was successfully activated reporter gene expression by the effector AtMYB44. To define the contribution of each MYB binding sequence to AtMYB44 dependent activation of WRKY70, we co-infiltrated various constructs of truncated WRKY70 promoter sequences with AtMYB44 effector plasmid. Presence of RE1 or RE2 in the reporter plasmid was sufficient to activate GFP gene by AtMYB44. However, GFP gene fused under WRKY70 promoter sequence containing RE3 alone was not activated by AtMYB44. It means that AtMYB44 binds to RE1 and RE2, and directly activates expression of WRKY70. Previously, we showed that WRKY70 promoter fragment containing RE1 was sufficient for AtMYB44 dependent activation and this activation was abolished by mutation in RE1.¹² Those findings demonstrate that AtMYB44 binds to at least two regions of WRKY70 promoter for target gene activation.

Figure 1. Transactivation of WRKY70 by AtMYB44. (A) Structure of AtMYB44 binding sequences, REs, in WRKY70 promoter. (B) A cDNA encoding AtMYB44 was fused to the CaMV 35S promoter as an effector and various constructs of truncated WRKY70 promoter sequences was fused to the GFP reporter gene.(C) The reporter and effector constructs were infiltrated into Nicotiana benthamiana. Transactivation activity was detected by GFP fluorescence.

AtMYB44 Contributes to Plant Innate Immunity

In Arabidopsis, the MPK3 and its upstream regulator MKK4 initiate signal cascade to abiotic and biotic stress responses. In pathogen molecular pattern (PAMP)-induced resistance, VirE2 interacting protein 1 (VIP1) is phosphorylated by MPK3 for nuclear transportation in response to bacterial invasion. In the nucleus, phosphorylated VIP1 activates expression of stress inducible genes including PR1.¹⁵ AtMYB44 is reported to be one of the targets of VIP1.¹⁰ We showed that AtMYB44 directly activates expression of WRKY70 thus PR1.¹² From those data, AtMYB44 could mediate PAMP induced defense responses through WRKY70 and PR1. Even though its effect in biotic stress response is not known, there are reports that AtMYB44 is phosphorylated by MPK3 and its phosphorylation is required for abiotic stress tolerances.^16,17

According to our previous report, AtMYB44 is also induced by abiotic stresses such as drought, cold temperature and salt.⁸ Its overexpression showed tolerance to those ABA-mediated abiotic stresses by modulating stomatal closure. In fact, AtMYB44 is highly expressed in guard cell and its overexpression resulted in rapid closure of stomata. There are reports that ABA and SA are required for stomatal closure to biotrophic pathogen and PAMPs.^18,19 Stomatal closure triggered by bacterial invasion is considered to be a key part in the beginning of plant innate immunity. It is therefore possible that regulation of stomatal movement and defense gene expression by AtMYB44 together could contribute to plant innate immunity against biotrophic pathogens. Further studies on the function and molecular mechanism of AtMYB44 in plant innate immunity are required.

Glossary

Abbreviation:

SA

salicylic acid

JA

jasmonic acid

Pst DC3000

Pseudomonas syringae pv. tomato DC3000

MPK

mitogen-activated protein kinase

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.