Mitogen-activated protein (MAP) kinase signaling cascades play important roles in eukaryotic defense against various pathogens. Activation of the extracellular ATP (eATP) receptor P2K1 triggers MAP kinase 3 and 6 (MPK3/6) phosphorylation, which leads to an elevated plant defense response. However, the mechanism by which P2K1 activates the MAPK cascade is unclear. In this study, we show that in Arabidopsis thaliana, P2K1 phosphorylates the Raf-like MAP kinase kinase kinase (MAPKKK) INTEGRIN-LINKED KINASE 5 (ILK5) on serine 192 in the presence of eATP. The interaction between P2K1 and ILK5 was confirmed both in vitro and in planta and their interaction was enhanced by ATP treatment. Similar to P2K1 expression, ILK5 expression levels were highly induced by treatment with ATP, flg22, Pseudomonas syringae pv. tomato DC3000, and various abiotic stresses. ILK5 interacts with and phosphorylates the MAP kinase MKK5. Moreover, phosphorylation of MPK3/6 was significantly reduced upon ATP treatment in ilk5 mutant plants, relative to wild-type (WT). The ilk5 mutant plants showed higher susceptibility to P. syringae pathogen infection relative to WT plants. Plants expressing only the mutant ILK5S192A protein, with decreased kinase activity, did not activate the MAPK cascade upon ATP addition. These results suggest that eATP activation of P2K1 results in transphosphorylation of the Raf-like MAPKKK ILK5, which subsequently triggers the MAPK cascade, culminating in activation of MPK3/6 associated with an elevated innate immune response.
- Award ID(s):
- 1758434
- NSF-PAR ID:
- 10318495
- Date Published:
- Journal Name:
- Stress Biology
- Volume:
- 1
- Issue:
- 1
- ISSN:
- 2731-0450
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract -
ABSTRACT EseN is an
Edwardsiella ictaluri type III secretion system effector with phosphothreonine lyase activity. In this work, we demonstrate that EseN inactivates p38 and c-Jun-N-terminal kinase (JNK) in infected head-kidney-derived macrophages (HKDMs). We have previously reported inactivation of extracellular-regulated kinase 1/2 (ERK1/2). Also, for the first time, we demonstrated that EseN is involved in the inactivation of 3-phosphoinositide-dependent kinase 1 (PDK1), which has not been previously demonstrated for any of the EseN homologs in other species. We also found that EseN significantly affected mRNA expression ofIL-10 , pro-apoptoticbaxa , andp53 , but had no significant effect on anti-apoptoticbcl2 or pro-apoptotic apoptotic peptidase activating factor 1. EseN is also involved in the inhibition of caspase-8 and caspase-3/7 but does not affect caspase-9 activity. Repression of apoptosis was further confirmed with flow cytometry using Alexa Fluor 647-labeled annexin V and propidium iodide. In addition, we found that theE. ictaluri T3SS is essential for the inhibition of IL-1β maturation, but EseN is not involved in this process. EseN did not affect cell pyroptosis, as indicated by the lack of EseN impact on the release of lactate dehydrogenase from infected HKDM. The transmission electron microscopy data also indicate that HKDM infected with WT or aneseN mutant died by apoptosis, while HKDM infected with the T3SS mutant more likely died by pyroptosis. Collectively, our results indicate thatE. ictaluri EseN is involved in inactivation of ERK1/2, p38, JNK, and PDK1 signaling pathways that lead to modulation of cell death among infected HKDMs.IMPORTANCE This work has global significance in the catfish industry, which provides food for increasing global populations.
E. ictaluri is a leading cause of disease loss, and EseN is an important player inE. ictaluri virulence. TheE. ictaluri T3SS effector EseN plays an essential role in establishing infection, but the specific role EseN plays is not well characterized. EseN belongs to a family of phosphothreonine lyase effectors that specifically target host mitogen activated protein kinase (MAPK) pathways important in regulating host responses to infection. No phosphothreonine lyase equivalents are known in eukaryotes, making this family of effectors an attractive target for indirect narrow-spectrum antibiotics. Targeting of major vault protein and PDK1 kinase by EseN has not been reported in EseN homologs in other pathogens and may indicate unique functions ofE. ictaluri EseN. EseN targeting of PDK1 is particularly interesting in that it is linked to an extraordinarily diverse group of cellular functions. -
The plant cell wall (CW) is an outer cell skeleton that plays an important role in plant growth and protection against both biotic and abiotic stresses. Signals and molecules produced during host–pathogen interactions have been proven to be involved in plant stress responses initiating signal pathways. Based on our previous research findings, the present study explored the possibility of additively or synergistically increasing plant stress resistance by stacking beneficial genes. In order to prove our hypothesis, we generated transgenic Arabidopsis plants constitutively overexpressing three different Aspergillus nidulans CW-modifying enzymes: a xylan acetylesterase, a rhamnogalacturonan acetylesterase and a feruloylesterase. The two acetylesterases were expressed either together or in combination with the feruloylesterase to study the effect of CW polysaccharide deacetylation and deferuloylation on Arabidopsis defense reactions against a fungal pathogen, Botrytis cinerea. The transgenic Arabidopsis plants expressing two acetylesterases together showed higher CW deacetylation and increased resistance to B. cinerea in comparison to wild-type (WT) Col-0 and plants expressing single acetylesterases. While the expression of feruloylesterase alone compromised plant resistance, coexpression of feruloylesterase together with either one of the two acetylesterases restored plant resistance to the pathogen. These CW modifications induced several defense-related genes in uninfected healthy plants, confirming their impact on plant resistance. These results demonstrated that coexpression of complementary CW-modifying enzymes in different combinations have an additive effect on plant stress response by constitutively priming the plant defense pathways. These findings might be useful for generating valuable crops with higher protections against biotic stresses.more » « less
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The amount of benefit microbiome members confer to plants differs among classes of microbes, depending on whether plants are grown in stressful or non‐stressful environments. In the absence of stress, beneficial bacteria tend to confer greater plant benefits than do fungi. However, symbiotic fungi, especially arbuscular mycorrhizal fungi, more strongly ameliorate plant stress than do bacteria. In particular, beneficial microbes ameliorate salinity, foliar herbivory and fungal pathogen stress.
These results highlight the fact that the impacts of beneficial and antagonistic components of the microbiome on plant performance depend on biotic and abiotic environmental contexts. Furthermore, beneficial microbes are especially critical for plant health in stressful environments and thus present opportunities to mitigate negative consequences of global change.
A free
plain language summary can be found within the Supporting Information of this article. -
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