Nobuaki Ishihama

Phosphorylation of the Nicotiana benthamiana WRKY8 Transcription Factor by MAPK Functions in the Defense Response

This study identified WRKY8 as a downstream target of three mitogen-activated protein kinases in Nicotiana benthamiana. Phosphorylation of WRKY8 increased its DNA binding activity, and ectopic expression of WRKY8 induced the expression of various defense-related genes. Mitogen-activated protein kinase (MAPK) cascades have pivotal roles in plant innate immunity. However, downstream signaling of plant defense-related MAPKs is not well understood. Here, we provide evidence that the Nicotiana benthamiana WRKY8 transcription factor is a physiological substrate of SIPK, NTF4, and WIPK. Clustered Pro-directed Ser residues (SP cluster), which are conserved in group I WRKY proteins, in the N-terminal region of WRKY8 were phosphorylated by these MAPKs in vitro. Antiphosphopeptide antibodies indicated that Ser residues in the SP cluster of WRKY8 are phosphorylated by SIPK, NTF4, and WIPK in vivo. The interaction of WRKY8 with MAPKs depended on its D domain, which is a MAPK-interacting motif, and this interaction was required for effective phosphorylation of WRKY8 in plants. Phosphorylation of WRKY8 increased its DNA binding activity to the cognate W-box sequence. The phospho-mimicking mutant of WRKY8 showed higher transactivation activity, and its ectopic expression induced defense-related genes, such as 3-hydroxy-3-methylglutaryl CoA reductase 2 and NADP-malic enzyme. By contrast, silencing of WRKY8 decreased the expression of defense-related genes and increased disease susceptibility to the pathogens Phytophthora infestans and Colletotrichum orbiculare. Thus, MAPK-mediated phosphorylation of WRKY8 has an important role in the defense response through activation of downstream genes.

Post-translational regulation of WRKY transcription factors in plant immunity

Plants have evolved immune system to protect themselves against invading pathogens. Recent research has illustrated that signaling networks, after perception of diverse pathogen-derived signals, facilitate transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. WRKY proteins, which comprise a large family of plant transcription factors, are key players in plant immune responses. WRKY transcription factors participate in the control of defense-related genes either as positive or as negative regulators, and essentially are regulated at the transcriptional level. Emerging evidence emphasizes that group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are also activated by MAPK-dependent phosphorylation, underlining their importance in plant immunity.

Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling

BackgroundPlant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. However, until now this prediction was not testable.ResultsTo investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca2+ levels after herbivory and wounding damage. In vitro kinase assays of CPK3 protein with a suite of substrates demonstrated that the protein phosphorylates transcription factors (including ERF1, HsfB2a and CZF1/ZFAR1) in the presence of Ca2+. CPK13 strongly phosphorylated only HsfB2a, irrespective of the presence of Ca2+. Furthermore, in vivo agroinfiltration assays showed that CPK3-or CPK13-derived phosphorylation of a heat shock factor (HsfB2a) promotes PDF1.2 transcriptional activation in the defense response.ConclusionsThese results reveal the involvement of two Arabidopsis CPKs (CPK3 and CPK13) in the herbivory-induced signaling network via HsfB2a-mediated regulation of the defense-related transcriptional machinery. This cascade is not involved in the phytohormone-related signaling pathways, but rather directly impacts transcription factors for defense responses.

WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana

Phosphorylation of WRKYs by MAPK regulates an NADPH oxidase, demonstrating that the MAPK-WRKY pathway is required for the ROS burst in effector-triggered immunity via upregulation of RBOHB. Pathogen attack sequentially confers pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) after sensing of pathogen patterns and effectors by plant immune receptors, respectively. Reactive oxygen species (ROS) play pivotal roles in PTI and ETI as signaling molecules. Nicotiana benthamiana RBOHB, an NADPH oxidase, is responsible for both the transient PTI ROS burst and the robust ETI ROS burst. Here, we show that RBOHB transactivation mediated by MAPK contributes to R3a/AVR3a-triggered ETI (AVR3a-ETI) ROS burst. RBOHB is markedly induced during the ETI and INF1-triggered PTI (INF1-PTI), but not flg22-tiggered PTI (flg22-PTI). We found that the RBOHB promoter contains a functional W-box in the R3a/AVR3a and INF1 signal-responsive cis-element. Ectopic expression of four phospho-mimicking mutants of WRKY transcription factors, which are MAPK substrates, induced RBOHB, and yeast one-hybrid analysis indicated that these mutants bind to the cis-element. Chromatin immunoprecipitation assays indicated direct binding of the WRKY to the cis-element in plants. Silencing of multiple WRKY genes compromised the upregulation of RBOHB, resulting in impairment of AVR3a-ETI and INF1-PTI ROS bursts, but not the flg22-PTI ROS burst. These results suggest that the MAPK-WRKY pathway is required for AVR3a-ETI and INF1-PTI ROS bursts by activation of RBOHB.

The Colletotrichum orbiculare ssd1 Mutant Enhances Nicotiana benthamiana Basal Resistance by Activating a Mitogen-Activated Protein Kinase Pathway

Plant basal resistance is activated by virulent pathogens in susceptible host plants. A Colletotrichum orbiculare fungal mutant defective in the SSD1 gene, which regulates cell wall composition, is restricted by host basal resistance responses. Here, we identified the Nicotiana benthamiana signaling pathway involved in basal resistance by silencing the defense-related genes required for restricting the growth of the C. orbiculare mutant. Only silencing of MAP Kinase Kinase2 or of both Salicylic Acid Induced Protein Kinase (SIPK) and Wound Induced Protein Kinase (WIPK), two mitogen-activated protein (MAP) kinases, allowed the mutant to infect and produce necrotic lesions similar to those of the wild type on inoculated leaves. The fungal mutant penetrated host cells to produce infection hyphae at a higher frequency in SIPK WIPK-silenced plants than in nonsilenced plants, without inducing host cellular defense responses. Immunocomplex kinase assays revealed that SIPK and WIPK were more active in leaves inoculated with mutant fungus than with the wild type, suggesting that induced resistance correlates with MAP kinase activity. Infiltration of heat-inactivated mutant conidia induced both SIPK and WIPK more strongly than did those of the wild type, while conidial exudates of the wild type did not suppress MAP kinase induction by mutant conidia. Therefore, activation of a specific MAP kinase pathway by fungal cell surface components determines the effective level of basal plant resistance.

Ethylene-responsive AP2/ERF transcription factor MACD1 participates in phytotoxin-triggered programmed cell death

To investigate plant programmed cell death (PCD), we developed the model system using phytotoxin AAL, which is produced by necrotrophic pathogen Alternaria alternata f. sp. lycopersici, and AAL-sensitive Nicotiana umbratica. We previously reported that ethylene (ET) signaling plays a pivotal role in AAL-triggered cell death (ACD). However, downstream signaling of ET to ACD remains unclear. Here, we show that the modulator of AAL cell death 1 (MACD1), which is an APETALA2/ET response factor (ERF) transcription factor, participates in ACD and acts downstream of ET signaling during ACD. MACD1 is a transcriptional activator and MACD1 overexpression plants showed earlier ACD induction than control plants, suggesting that MACD1 positively regulates factors affecting cell death. To investigate the role of MACD1 in PCD, we used Arabidopsis thaliana and a structural analog of AAL, fumonisin B1 (FB1). FB1-triggered cell death was compromised in ET signaling and erf102 mutants. The loh2 mutants showed sensitivity to AAL, and the loh2-1/erf102 double mutant compromised ACD, indicating that ERF102 also participates in ACD. To investigate the PCD-associated genes regulated by ERF102, we compared our microarray data using ERF102 overexpression plants with the database of upregulated genes by AAL treatment in loh2 mutants, and found genes under the control of ERF102 in ACD.

Ethylene Signaling Pathway and MAPK Cascades Are Required for AAL Toxin–Induced Programmed Cell Death

Programmed cell death (PCD), known as hypersensitive response cell death, has an important role in plant defense response. The signaling pathway of PCD remains unknown. We employed AAL toxin and Nicotiana umbratica to analysis plant PCD. AAL toxin is a pathogenicity factor of the necrotrophic pathogen Alternaria alternata f. sp. lycopersici. N. umbratica is sensitive to AAL toxin, susceptible to pathogens, and effective in Tobacco rattle virus-based virus-induced gene silencing (VIGS). VIGS analyses indicated that AAL toxin-triggered cell death (ACD) is dependent upon the mitogen-activated protein (MAP) kinase kinase MEK2, which is upstream of both salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) responsible for ethylene (ET) synthesis. ET treatment of MEK2-silenced N. umbratica re-established ACD. In SIPK- and WIPK-silenced N. umbratica, ACD was compromised and ET accumulation was not observed. However, in contrast to the case of MEK2-silenced plants, ET treatment did not induce cell death in SIPK- and WIPK-silenced plants. This work showed that ET-dependent pathway and MAP kinase cascades are required in ACD. Our results suggested that MEK2-SIPK/WIPK cascades have roles in ET biosynthesis; however, SIPK and WIPK have other roles in ET signaling or another pathway leading to cell death by AAL toxin.

Nicotiana benthamiana MAPK-WRKY pathway confers resistance to a necrotrophic pathogen Botrytis cinerea

ABSTRACT MEK2-SIPK/WIPK cascade, a Nicotiana benthamiana mitogen-activated protein kinase (MAPK) cascade, is an essential signaling pathway for plant immunity and involved in hypersensitive response (HR) accompanied by cell death. WRKY transcription factors as substrates of SIPK and WIPK have been isolated and implicated in HR cell death. Here, we show virus-induced gene silencing of WRKY genes compromised constitutively active MEK2-triggered cell death in N. benthamiana leaves. In general, HR cell death enhances susceptibility to necrotrophic pathogens such as Botrytis cinerea. However, the WRKY gene silencing elevated susceptibility to B. cinerea. These findings suggest that downstream WRKYs of MEK2-SIPK/WIPK cascade are required for cell death-dependent and -independent immunities in N. benthamiana.

In Vivo Phosphorylation of WRKY Transcription Factor by MAPK

Plants activate signaling networks in response to diverse pathogen-derived signals, facilitating transcriptional reprogramming through mitogen-activated protein kinase (MAPK) cascades. Identification of phosphorylation targets of MAPK and in vivo detection of the phosphorylated substrates are important processes to elucidate the signaling pathway in plant immune responses. We have identified a WRKY transcription factor, which is phosphorylated by defense-related MAPKs, SIPK and WIPK. Recent evidence demonstrated that some group I WRKY transcription factors, which contain a conserved motif in the N-terminal region, are activated by MAPK-dependent phosphorylation. In this chapter, we describe protocols for preparation of anti-phosphopeptide antibodies, detection of activated MAPKs using anti-phospho-MAPK antibody, and activated WRKY using anti-phospho-WRKY antibody, respectively.