Abstract
Mitogen-activated protein kinase (MAPK) signaling networks represent important means of signal transduction in plants and other eukaryotes, controlling intracellular signaling by linking perception of environmental or developmental cues to downstream targets. In the Arabidopsis MEKK subfamily, the MKKK19, 20, and 21 form a highly supported clade with the Solanaceous Fertilization-Related Kinases. In Arabidopsis, little is known about this group, except for MKKK20, which is involved in osmotic stress. Using a directed MKKK-MKK yeast two-hybrid (Y2H) screen, MKKK20 was found to interact only with MKK3, while a MKKK20 large-scale Y2H screen retrieved MPK18 as a direct interactant. In vitro phosphorylation assays showed that MKKK20 phosphorylates both MKK3 and MPK18. However, when all three kinases are combined, no synergistic effect is observed on MPK18 phosphorylation, suggesting a direct access to MPK18, consistent with the absence of interaction between MKK3 and MPK18 in protein–protein interaction assays. Since mpk18 mutant plants were previously shown to be defective in microtubule-related functions, phenotypes of mkkk20 single and mkkk20/mpk18 double mutants were investigated to determine if MKKK20 acts upstream of MPK18. This was the case, as mkkk20 root length was shorter than WT in media containing microtubule-disrupting drugs as previously observed for mpk18 plants. Surprisingly, mkk3 plants were also similarly affected, suggesting the presence of two non-complementary pathways involved in Arabidopsis cortical microtubule function, the first including MKKK20, MKK3 and an unknown MPK; the second, a non-canonical MAPK cascade made of MKKK20 and MPK18 that bypasses the need for an MKK intermediate.
Highlights
Microtubules are polymers of α- and β-tubulin heterodimers that help form the eukaryotic cytoskeleton
Arabidopsis MAPKKs have been classified into four groups, A–D (Soyano et al, 2003; Teige et al, 2004; Dai et al, 2006), with AtMKK3 belonging to group B, a singlemember clade characterized by the presence of a C-terminal NTF2 domain known to promote nuclear import of cargo proteins (Steggerda and Paschal, 2002)
Diploid yeast cells were selected based on their ability to grow on a -Leu/-Trp media that contained Aureobasidine A (AbA), an antibiotic toxic to yeast (AUR1-C gene reporter) and X-α-Gal (MEL1 gene reporter)
Summary
Microtubules are polymers of α- and β-tubulin heterodimers that help form the eukaryotic cytoskeleton. Close sister clades such as the ZIK and MLK-like kinases are considered as MAPKKKs so far, only the MEKKs are involved in MAPK cascades (Rodriguez et al, 2010).These kinases have been implicated in a wide array of plant biological processes ranging from stress responses to development (Rodriguez et al, 2010). Arabidopsis MAPKKs have been classified into four groups, A–D (Soyano et al, 2003; Teige et al, 2004; Dai et al, 2006), with AtMKK3 belonging to group B, a singlemember clade characterized by the presence of a C-terminal NTF2 domain known to promote nuclear import of cargo proteins (Steggerda and Paschal, 2002) This feature could confer to MKK3 an important and singular rule among all other AtMKKs (Colcombet et al, 2016). This, combined with the analysis of mkkk20/mpk and mkk3/mpk double mutants suggests that MKKK20 modulates cortical microtubule functions through two independent MAPK signaling cascades, including a non-canonical pathway that bypasses the need for an intermediary MKK
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