Antagonistic interactions between two MAP kinase cascades in plant development and immune signaling

Mitogen‐activated protein kinase (MAPK) signaling plays important roles in diverse biological processes. In Arabidopsis, MPK3/MPK6, MKK4/MKK5, and the MAPKKK YODA (YDA) form a MAPK pathway that negatively regulates stomatal development. Brassinosteroid (BR) stimulates this pathway to inhibit stomata production. In addition, MPK3/MPK6 and MKK4/MKK5 also serve as critical signaling components in plant immunity. Here, we report that MAPKKK3/MAPKKK5 form a kinase cascade with MKK4/MKK5 and MPK3/MPK6 to transduce defense signals downstream of multiple plant receptor kinases. Loss of MAPKKK3/MAPKKK5 leads to reduced activation of MPK3/MPK6 in response to different pathogen‐associated molecular patterns (PAMPs) and increased susceptibility to pathogens. Surprisingly, developmental defects caused by silencing of YDA are suppressed in the mapkkk3 mapkkk5 double mutant. On the other hand, loss of YDA or blocking BR signaling leads to increased PAMP‐induced activation of MPK3/MPK6. These results reveal antagonistic interactions between a developmental MAPK pathway and an immune signaling MAPK pathway.     

The Arabidopsis mitogen-activated protein kinase kinase MKK3 is upstream of group C mitogen-activated protein kinases and participates in pathogen signaling

Although the Arabidopsis thaliana genome contains genes encoding 20 mitogen-activated protein kinases (MAPKs) and 10 MAPK kinases (MAPKKs), most of them are still functionally uncharacterized. In this work, we analyzed the function of the group B MAPK kinase, MKK3. Transgenic ProMKK3:GUS lines showed basal expression in vascular tissues that was strongly induced by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) infection but not by abiotic stresses. The growth of virulent Pst DC3000 was increased in mkk3 knockout plants and decreased in MKK3-overexpressing plants. Moreover, MKK3 overexpression lines showed increased expression of several PR genes. By yeast two-hybrid analysis, coimmunoprecipitation, and protein kinase assays, MKK3 was revealed to be an upstream activator of the group C MAPKs MPK1, MPK2, MPK7, and MPK14. Flagellin-derived flg22 peptide strongly activated MPK6 but resulted in poor activation of MPK7. By contrast, MPK6 and MPK7 were both activated by H(2)O(2), but only MPK7 activation was enhanced by MKK3. In agreement with the notion that MKK3 regulates the expression of PR genes, ProPR1:GUS expression was strongly enhanced by coexpression of MKK3-MPK7. Our results reveal that the MKK3 pathway plays a role in pathogen defense and further underscore the importance and complexity of MAPK signaling in plant stress responses.     

MEKK1 is required for MPK4 activation and regulates tissue-specific and temperature-dependent cell death in Arabidopsis

Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. An Arabidopsis MAPK cascade (MEKK1, MKK4/MKK5, and MPK3/MPK6) has been proposed to function downstream of the flagellin receptor FLS2 based on biochemical assays using transient overexpression of candidate components. To genetically test this model, we characterized two mekk1 mutants. We show here that MEKK1 is not required for flagellin-triggered activation of MPK3 and MPK6. Instead, MEKK1 is essential for activation of MPK4, a MAPK that negatively regulates systemic acquired resistance. We also showed that MEKK1 negatively regulates temperature-sensitive and tissue-specific cell death and H(2)O(2) accumulation that are partly dependent on both RAR1, a key component in resistance protein function, and SID2, an isochorismate synthase required for salicylic acid production upon pathogen infection.     

Disruption of PAMP-induced MAP kinase cascade by a Pseudomonas syringae effector activates plant immunity mediated by the NB-LRR protein SUMM2

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) serves as a primary plant defense response against microbial pathogens, with MEKK1, MKK1/MKK2, and MPK4 functioning as a MAP kinase cascade downstream of PAMP receptors. Plant Resistance (R) proteins sense specific pathogen effectors to initiate a second defense mechanism, termed effector-triggered immunity (ETI). In a screen for suppressors of the mkk1 mkk2 autoimmune phenotype, we identify the nucleotide-binding leucine-rich repeat (NB-LRR) protein SUMM2 and find that the MEKK1-MKK1/MKK2-MPK4 cascade negatively regulates SUMM2-mediated immunity. Further, the MEKK1-MKK1/MKK2-MPK4 cascade positively regulates basal defense targeted by the Pseudomonas syringae pathogenic effector HopAI1, which inhibits MPK4 kinase activity. Inactivation of MPK4 by HopAI1 results in activation of SUMM2-mediated defense responses. Our data suggest that SUMM2 is an R protein that becomes active when the MEKK1-MKK1/MKK2-MPK4 cascade is disrupted by pathogens, supporting the hypothesis that R proteins evolved to protect plants when microbial effectors suppress basal resistance.     

The MI-1-mediated pest resistance requires Hsp90 and Sgt1

The tomato (Solanum lycopersicum) Mi-1 gene encodes a protein with putative coiled-coil nucleotide-binding site and leucine-rich repeat motifs. Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum euphorbiae), and sweet potato whitefly (Bemisia tabaci). To identify genes required in the Mi-1-mediated resistance to nematodes and aphids, we used tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to repress candidate genes and assay for nematode and aphid resistance. We targeted Sgt1 (suppressor of G-two allele of Skp1), Rar1 (required for Mla12 resistance), and Hsp90 (heat shock protein 90), which are known to participate early in resistance gene signaling pathways. Two Arabidopsis (Arabidopsis thaliana) Sgt1 genes exist and one has been implicated in disease resistance. Thus far the sequence of only one Sgt1 ortholog is known in tomato. To design gene-specific VIGS constructs, we cloned a second tomato Sgt1 gene, Sgt1-2. The gene-specific VIGS construct TRV-SlSgt1-1 resulted in lethality, while silencing Sgt1-2 using TRV-SlSgt1-2 did not result in lethal phenotype. Aphid and root-knot nematode assays of Sgt1-2-silenced plants indicated no role for Sgt1-2 in Mi-1-mediated resistance. A Nicotiana benthamiana Sgt1 VIGS construct silencing both Sgt1-1 and Sgt1-2 yielded live plants and identified a role for Sgt1 in Mi-1-mediated aphid resistance. Silencing of Rar1 did not affect Mi-1-mediated nematode and aphid resistance and demonstrated that Rar1 is not required for Mi-1 resistance. Silencing Hsp90-1 resulted in attenuation of Mi-1-mediated aphid and nematode resistance and indicated a role for Hsp90-1. The requirement for Sgt1 and Hsp90-1 in Mi-1-mediated resistance provides further evidence for common components in early resistance gene defense signaling against diverse pathogens and pests.     

MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants

Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekk1 that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complementation (BiFC) analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases. mkk1 and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkk1 mkk2 double mutants were generated. The mkk1 mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekk1 mutants, the mkk1 mkk2 double mutant seedlings accumulate high levels of H2O2, display spontaneous cell death, constitutively express Pathogenesis Related (PR) genes and exhibit pathogen resistance. In addition, activation of MPK4 by flg22 is impaired in the mkk1 mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants.     

EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity.     

Arabidopsis EXTRA-LARGE G PROTEIN 1 (XLG1) functions together with XLG2 and XLG3 in PAMP-triggered MAPK activation and immunity

Pattern-triggered immunity (PTI) is an essential strategy used by plants to deploy broad-spectrum resistance against pathogen attacks. Heterotrimeric G proteins have been reported to contribute to PTI. Of the three non-canonical EXTRA-LARGE G PROTEINs (XLGs) in Arabidopsis thaliana, XLG2 and XLG3 were shown to positively regulate immunity, but XLG1 was not considered to function in defense, based on the analysis of a weak xlg1 allele. In this study, we characterized the xlg1 xlg2 xlg3 triple knockout mutants generated from an xlg1 knockout allele. The strong xlg1 xlg2 xlg3 triple mutants compromised pathogen-associated molecular pattern (PAMP)-triggered activation of mitogen-activated protein kinases (MAPKs) and resistance to pathogen infection. The three XLGs interacted with MAPK cascade proteins involved in defense signaling, including the MAPK kinase kinases MAPKKK3 and MAPKKK5, the MAPK kinases MKK4 and MKK5, and the MAPKs MPK3 and MPK6. Expressing a constitutively active form of MKK4 restored MAPK activation and partially recovered the compromised disease resistance seen in the strong xlg1 xlg2 xlg3 triple mutant. Furthermore, mutations of all three XLGs largely restored the phenotype of the autoimmunity mutant bak1-interacting receptor-like kinase 1. Our study reveals that all three XLGs function redundantly in PAMP-triggered MAPK activation and plant immunity.     

Induction of γ-aminobutyric acid plays a positive role to Arabidopsis resistance against Pseudomonas syringae

Gamma‐aminobutyric acid (GABA) is an important metabolite which functions in plant growth, development, and stress responses. However, its role in plant defense and how it is regulated are largely unknown. Here, we report a detailed analysis of GABA induction during the resistance response to Pseudomonas syringae in Arabidopsis thaliana. While searching for the mechanism underlying the pathogen‐responsive mitogen‐activated protein kinase (MPK)3/MPK6 signaling cascade in plant immunity, we found that activation of MPK3/MPK6 greatly induced GABA biosynthesis, which is dependent on the glutamate decarboxylase genes GAD1 and GAD4. Inoculation with Pseudomonas syringae pv tomato DC3000 (Pst) and Pst‐avrRpt2 expressing the avrRpt2 effector gene induced GAD1 and GAD4 gene expression and increased the levels of GABA. Genetic evidence revealed that GAD1, GAD2, and GAD4 play important roles in both GABA biosynthesis and plant resistance in response to Pst‐avrRpt2 infection. The gad1/2/4 triple and gad1/2/4/5 quadruple mutants, in which the GABA levels were extremely low, were more susceptible to both Pst and Pst‐avrRpt2. Functional loss of MPK3/MPK6, or their upstream MKK4/MKK5, or their downstream substrate WRKY33 suppressed the induction of GAD1 and GAD4 expression after Pst‐avrRpt2 treatment. Our findings shed light on both the regulation and role of GABA in the plant immunity to a bacterial pathogen.     

CITRX thioredoxin interacts with the tomato Cf-9 resistance protein and negatively regulates defence

To identify proteins involved in tomato Cf-9 resistance protein function, a yeast two-hybrid screen was undertaken using the cytoplasmic C-terminus of Cf-9 as bait. A thioredoxin-homologous clone, interacting specifically with Cf-9, was identified and called CITRX (Cf-9-interacting thioredoxin). Virus-induced gene silencing (VIGS) of CITRX resulted in an accelerated Cf-9/Avr9-triggered hypersensitive response in both tomato and Nicotiana benthamiana, accompanied by enhanced accumulation of reactive oxygen species, alteration of protein kinase activity and induction of defence-related genes. VIGS of CITRX also conferred increased resistance to the fungal pathogen Cladosporium fulvum in the otherwise susceptible Cf0 tomato. CITRX acts as a negative regulator of the cell death and defence responses induced through Cf-9, but not Cf-2. Recognition of the Cf-9 C-terminus by CITRX is necessary and sufficient for this negative regulation. This is the first study that implicates thioredoxin activity in the regulation of plant disease resistance.     

Virus-induced gene silencing: A versatile tool for discovery of gene functions in plants

Virus-induced gene silencing (VIGS) is a technology that exploits an antiviral defense mechanism in plants as a tool for plant reverse genetics. VIGS circumvents the need for plant transformation, is methodologically simple and yields rapid results. Various VIGS vectors have been developed and have helped to unravel the functions of genes involved in processes such as disease resistance, abiotic stress, cellular signaling and secondary metabolite biosynthesis.     

Overlapping functions of YDA and MAPKKK3/MAPKKK5 upstream of MPK3/MPK6 in plant immunity and growth/development

Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3(MAPK3 or MPK3) and MPK6 play important signaling roles in plant immunity and growth/development. MAPK KINASE4(MKK4)and MKK5 function redundantly upstream of MPK3 and MPK6 in these processes. YODA(YDA), also known as MAPK KINASE KINASE4(MAPKKK4), is upstream of MKK4/MKK5 and forms a complete MAPK cascade(YDA–MKK4/MKK5–MPK3/MPK6) in regulating plant growth and development. In plant immunity, MAPKKK3 and MAPKKK5 function redundantly upstream of the same...     

Interfamily transfer of tomato Ve1 mediates Verticillium resistance in Arabidopsis

Vascular wilts caused by soil-borne fungal species of the Verticillium genus are devastating plant diseases. The most common species, Verticillium dahliae and Verticillium albo-atrum, have broad host ranges and are notoriously difficult to control. Therefore, genetic resistance is the preferred method for disease control. Only from tomato (Solanum lycopersicum) has a Verticillium resistance locus been cloned, comprising the Ve1 gene that encodes a receptor-like protein-type cell surface receptor. Due to lack of a suitable model for receptor-like protein (RLP)-mediated resistance signaling in Arabidopsis (Arabidopsis thaliana), so far relatively little is known about RLP signaling in pathogen resistance. Here, we show that Ve1 remains fully functional after interfamily transfer to Arabidopsis and that Ve1-transgenic Arabidopsis is resistant to race 1 but not to race 2 strains of V. dahliae and V. albo-atrum, nor to the Brassicaceae-specific pathogen Verticillium longisporum. Furthermore, we show that signaling components utilized by Ve1 in Arabidopsis to establish Verticillium resistance overlap with those required in tomato and include SERK3/BAK1, EDS1, and NDR1, which strongly suggests that critical components for resistance signaling are conserved. We subsequently investigated the requirement of SERK family members for Ve1 resistance in Arabidopsis, revealing that SERK1 is required in addition to SERK3/BAK1. Using virus-induced gene silencing, the requirement of SERK1 for Ve1-mediated resistance was confirmed in tomato. Moreover, we show the requirement of SERK1 for resistance against the foliar fungal pathogen Cladosporium fulvum mediated by the RLP Cf-4. Our results demonstrate that Arabidopsis can be used as model to unravel the genetics of Ve1-mediated resistance.