Abstract
A wide variety of intrinsic and extrinsic cues lead to cell death with unclear mechanisms. The infertility of some death mutants often hurdles the classical suppressor screens for death regulators. We have developed a transient RNA interference (RNAi)-based screen using a virus-induced gene silencing approach to understand diverse cell death pathways in Arabidopsis (Arabidopsis thaliana). One death pathway is due to the depletion of a MAP kinase (MAPK) cascade, consisting of MAPK kinase kinase 1 (MEKK1), MKK1/2, and MPK4, which depends on a nucleotide-binding site Leu-rich repeat (NLR) protein SUMM2. Silencing of MEKK1 by virus-induced gene silencing resembles the mekk1 mutant with autoimmunity and defense activation. The RNAi-based screen toward Arabidopsis T-DNA insertion lines identified SUMM2, MEKK2, and Calmodulin-binding receptor-like cytoplasmic kinase 3 (CRCK3) to be vital regulators of RNAi MEKK1-induced cell death, consistent with the reports of their requirement in the mekk1-mkk1/2-mpk4 death pathway. Similar with MEKK2, overexpression of CRCK3 caused dosage- and SUMM2-dependent cell death, and the transcripts of CRCK3 were up-regulated in mekk1, mkk1/2, and mpk4 MEKK2-induced cell death depends on CRCK3. Interestingly, CRCK3-induced cell death also depends on MEKK2, consistent with the biochemical data that MEKK2 complexes with CRCK3. Furthermore, the kinase activity of CRCK3 is essential, whereas the kinase activity of MEKK2 is dispensable, for triggering cell death. Our studies suggest that MEKK2 and CRCK3 exert concerted functions in the control of NLR SUMM2 activation and MEKK2 may play a structural role, rather than function as a kinase, in regulating CRCK3 protein stability.
Highlights
Plants and microbes are engaged in a continuous battle for survival
Plants have evolved a second layer of defenses whereby a plethora of intracellular nucleotide-binding site leucine-rich repeat (NBS-LRR) receptors (NLRs) detect those effectors directly or indirectly and initiate effector-triggered immunity (ETI) (Cui et al, 2015), which sometimes results in localized programmed cell death (PCD) called hypersensitive response (Coll et al, 2011)
We investigated whether cell death caused by overexpression of Calmodulin-binding receptor-like cytoplasmic kinase 3 (CRCK3) requires SUPPRESSOR OF MKK1 MKK2 2 (SUMM2) by transforming 35S::CRCK3-GFP into the summ2-8 mutant, which is morphologically similar to WT plants
Summary
Plants and microbes are engaged in a continuous battle for survival. Plants have evolved sophisticated defense mechanisms to detect the telltale molecules produced by pathogens and to initiate defense responses to ward off potential infections (Peng et al, 2018). Adapted pathogens deliver an arsenal of effectors into host cells to damp host immune responses and promote parasitism (Dou and Zhou, 2012; Macho and Zipfel, 2015). Facing these challenges, plants have evolved a second layer of defenses whereby a plethora of intracellular nucleotide-binding site leucine-rich repeat (NBS-LRR) receptors (NLRs) detect those effectors directly or indirectly and initiate effector-triggered immunity (ETI) (Cui et al, 2015), which sometimes results in localized programmed cell death (PCD) called hypersensitive response (Coll et al, 2011). A conventional MAPK cascade is assembled by three interlinked kinases: MAPKs (MPKs) are activated by MAPK kinases (MAPKKs, or MKKs), which are further phosphorylated and activated by MAPK kinase kinases (MAPKKKs, or MEKKs) (Zhang et al, 2018). Activated MPK6 phosphorylates and stabilizes 1-AMINOCYCLOPROPANE-1-AARBOXYLIC ACID (ACC)
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