Abstract 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.
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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. YDA, also known as 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 MKK4/MKK5–MPK3/MPK6 module. However, the residual activation of MPK3/MPK6 in the mapkkk3 mapkkk5 double mutant in response to flg22 PAMP treatment suggests the presence of additional MAPKKK(s) in this MAPK cascade in signaling plant immunity. To investigate whether YDA is also involved in plant immunity, we attempted to generate mapkkk3 mapkkk5 yda triple mutants. However, it was not possible to recover one of the double mutant combinations (mapkkk5 yda) or the triple mutant (mapkkk3 mapkkk5 yda) due to a failure of embryogenesis. Using the CRISPR-Cas9 approach, we generated weak, N-terminal deletion alleles of YDA, yda-del, in a mapkkk3 mapkkk5 background. PAMP-triggered MPK3/MPK6 activation was further reduced in the mapkkk3 mapkkk5 yda-del mutant, and the triple mutant was more susceptible to pathogen infection, suggesting YDA also plays an important role in plant immune signaling. In addition, MAPKKK5 and, to a lesser extent, MAPKKK3 were found to contribute to gamete function and embryogenesis, together with YDA. While the double homozygous mapkkk3 yda mutant showed the same growth and development defects as the yda single mutant, mapkkk5 yda double mutant and mapkkk3 mapkkk5 yda triple mutants were embryo lethal, similar to the mpk3 mpk6 double mutants. These results demonstrate that YDA, MAPKKK3, and MAPKKK5 have overlapping functions upstream of the MKK4/MKK5–MPK3/MPK6 module in both plant immunity and growth/development.
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- Award ID(s):
- 1856093
- PAR ID:
- 10340593
- Date Published:
- Journal Name:
- Journal of Integrative Plant Biology
- ISSN:
- 1672-9072
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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