MAP kinase cascades in plant development and immune signaling

Abstract

Mitogen‐activated protein kinase (MAPK) cascades are important signaling modules regulating diverse biological processes. During the past 20 years, much progress has been made on the functions of MAPK cascades in plants. This review summarizes the roles of MAPKs, known MAPK substrates, and our current understanding of MAPK cascades in plant development and innate immunity. In addition, recent findings on the molecular links connecting surface receptors to MAPK cascades and the mechanisms underlying MAPK signaling specificity are also discussed.

Mitogen‐activated protein kinase cascades are important signaling modules in plants. This review highlights their roles and known substrates and summarizes our current understanding of MAPK cascades in plant development and innate immunity.

Glossary

ABI4

ABA‐INSENSITIVE 4

ACS2

1‐aminocyclopropane‐1‐carboxylic acid (ACC) Synthase 2

AHL13

AT‐HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 13

AIK1

ABA‐Insensitive protein kinase 1

ANP1/2/3

Arabidopsis thaliana homologs of NPK1 1/2/3

ASR3

Arabidopsis SH4‐RELATED3

BAK1

BRI1‐associated receptor kinase 1

BASL

BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE

BIN2

BRASSINOSTEROID‐INSENSITIVE 2

CA

Constitutively active

CAMTA3

CALMODULIN‐BINDING TRANSCRIPTION ACTIVATOR 3

CC

Coiled‐coil domain

CDKC;1/2

RNA polymerase II C‐terminal domain (CTD) kinase

CERK1

CHITIN ELICITOR RECEPTOR KINASE 1

CKX2

CYTOKININ OXIDASE2

CRCK3

CALMODULIN‐BINDING RECEPTOR‐LIKE CYTOPLASMIC KINASE 3

DST

DROUGHT AND SALT TOLERANCE

EDR1

ENHANCED DISEASE RESISTANCE1

EDS1

ENHANCED DISEASE SUSCEPTIBILITY 1

EFR

EF‐TU RECEPTOR

EPF1

EPIDERMAL PATTERNING FACTOR 1

EPFLs

EPF‐LIKE peptides

ER

ERECTA

ERF6

ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 6

ERL1

ERECTA‐like1

ETI

Effector‐triggered immunity

Exo70B2

EXOCYST SUBUNIT EXO70 FAMILY PROTEIN B2

FLS2

FLAGELLIN‐SENSITIVE 2

GAD1

GRAIN NUMBER, GRAIN LENGTH, AND AWN DEVELOPMENT peptide

HD2B

HISTONE DEACETYLASE 2

HR

Hypersensitive response

IDA

INFLORESCENCE DEFICIENT IN ABSCISSION

IDD4

IMPERIAL EAGLE, INDETERMINATE(ID)‐DOMAIN 4

KEG

KEEP ON GOING

LIP5

HOMOLOG OF MAMMALIAN LYST‐INTERACTING PROTEIN 5

LjSIP2

a MKK from Lotus japonicus

LYK5

LYSM DOMAIN RECEPTOR‐LIKE KINASE 5

MAP65

Microtubule‐associated protein 65

MAPK

mitogen‐activated protein kinase, or MPK

MAPKK

MAPKK Kinases, or MKK, MEK

MAPKKK

MAPK Kinase Kinase, or MEKK, MKKK

MASS

MAPK SUBSTRATES IN THE STOMATAL LINEAGE

MKD1

MAPKKK δ‐1, a Raf‐like MAPKKK

MKP1

MAP KINASE PHOSPHATASE 1

MKS1

MAP KINASE SUBSTRATE 1

MVQ1

MPK3/6‐targeted VQ‐motif‐containing protein 1

NACK1

a kinesin‐like protein

NLR

intercellular nucleotide‐binding leucine‐rich repeat receptor

NPK1

nucleus‐ and phragmoplast‐localized protein kinase 1

NQK1

a tobacco MAPKK, or NtMEK1

NRK1

a tobacco MAPK, or NTF6

NTF4

a tobacco MAPK homolog of SIPK

PAD4

PHYTOALEXIN DEFICIENT 4

PBL19

PBS1‐LIKE 19

PCRK1

PTI COMPROMISED RECEPTOR‐LIKE CYTOPLASMIC KINASE 1

PIN1

Arabidopsis thaliana PIN‐FORMED 1

PIP5K6

Phosphatidylinositol 4‐phosphate 5‐kinase 6

PTI

Pattern‐triggered immunity

PTP1

PROTEIN TYROSINE PHOSPHATASE 1

PUB22

PLANT U‐BOX 22

RGF1

ROOT GROWTH FACTOR 1 peptide

RGIs

RGF1 INSENSITIVEs

RLCK

Receptor‐like cytoplasmic kinase

RLK

Receptor‐like kinase

RLP

Receptor‐like protein

SAR

Systemic acquired resistance

SERK

Somatic embryogenesis receptor‐like kinase

SIPK

Salicylic acid‐induced protein kinase

SOBIR1

SUPPRESSOR OF BIR1 1

SPCH

SPEECHLESS

SPL

SPOROCYTELESS

SUMM2

SUPPRESSOR OF MKK1 MKK2 2

TIR

Toll/interleukin 1‐receptor‐like domain

TMM

TOO MANY MOUTHS

VIP1

VIRE2‐INTERACTING PROTEIN 1

WIPK

Wounding‐induced protein kinase

WRKY33

WRKY DNA‐BINDING PROTEIN 33

Introduction

Mitogen‐activated protein kinase (MAPK) cascades are highly conserved signaling modules consisting of MAPK Kinase Kinases (MAPKKKs/MEKKs), MAPK Kinases (MAPKKs/MKKs/MEKs), and MAPKs/MPKs, which are widely used to relay and amplify signal from plasma membrane receptors to various downstream responses in eukaryotes (Widmann et al, ¹⁹⁹⁹; MAPK‐Group, ²⁰⁰²). A typical signaling pathway mediated by a MAPK cascade is illustrated in Fig 1A, which includes upstream receptors for perceiving ligands, signal transducers, the MAPK cascade, and downstream MAPK substrates. Perception of extracellular stimuli by plasma membrane receptors leads to activation of MAPKKKs, which activate MKKs through phosphorylation. Activated MKKs subsequently phosphorylate the Thr and Tyr residues in the Thr‐X‐Tyr activation motif of MPKs, leading to MPK activation and subsequent phosphorylation of their substrates to regulate downstream responses (Rodriguez et al, ²⁰¹⁰).

Figure 1. MAPK cascades in plant growth and development.

(A) A typical signal transduction pathway involving MAPK cascade includes upstream receptors for perceiving ligands, signal transducers, and a MAPK cascade (MAPKKK, MKK, and MPK) as well as downstream MAPK substrates for modulating specific biological processes. (B) The tobacco MAPK cascade consisting of NPK1, NQK1, and NRK1 targets NtMAP65 during cytokinesis. The tobacco kinesin‐like proteins NACK1 and NACK2 act as upstream activators of NPK1. (C) The Arabidopsis YDA‐MKK4/5‐MPK3/6 cascade acts downstream of multiple ligand–receptor pairs in plant development. EPF1/2, EPFL4/6, IDA, RGF1, and ESF1 are all peptide ligands. SHORT SUSPENSOR (SSP) belongs to the RLCK II subfamily. MPK3/MPK6 negatively regulate the stability of transcription factors SPCH and SCRM to modulate stomatal development. (D) The rice OsMKKK10‐OsMKK4‐OsMPK6 cascade functions downstream of OsER to regulate the trade‐off between grain size and grain number by targeting transcription factors OsWRKY53 and DST.

In plants, plasma membrane receptors in the Receptor‐like kinase (RLK) and Receptor‐like protein (RLP) families function upstream of MAPK cascades. They carry a transmembrane domain and an extracellular domain for sensing different stimuli (Shiu et al, ²⁰⁰⁴). RLKs also have an intracellular kinase domain used for initiating signal transduction. In comparison, RLPs lack the intracellular kinase domain and transduce signals via their interacting RLKs such as SOBIR1 (Liebrand et al, ²⁰¹⁴; Lee et al, ²⁰²¹). Another well‐studied RLK, BAK1 (also known as SERK3), acts as a co‐receptor for a large number of plasma membrane receptors (Liebrand et al, ²⁰¹⁴; Lee et al, ²⁰²¹). Increasing evidence suggests that RLCKs act as central signal transducers that associate with plasma membrane receptor complexes and relay signals to various downstream events including MAPK cascades (Lin et al, ²⁰¹³; Liang & Zhou, ²⁰¹⁸).

Arabidopsis genome encodes approximately 60 putative MAPKKKs, 10 MAPKKs, and 20 MAPKs (MAPK‐Group, ²⁰⁰²). MAPK cascades have been shown to regulate diverse aspects of plant biology, including plant growth and development as well as response to abiotic and biotic stresses (Rodriguez et al, ²⁰¹⁰; Xu & Zhang, ²⁰¹⁵; Zhang et al, ²⁰¹⁸). Remarkable progress has been made in the understanding of regulation of plant development and immunity by MAPK cascades during the past two decades (Table 1). In this review, we summarize the current known MAPK cascades in plant development and immune signaling, the downstream substrates of these MAPK cascades, and regulatory components controlling their activities. In addition, the mechanisms underlying MAPK signaling specificity are also discussed.

Table 1.

Functions of MAPKs (cascades) in plant development and immunity.

Plant species	MAPKs (cascade)	Biological functions	References
Arabidopsis thaliana	ANP1/2/3‐MKK6‐MPK4	Cytokinesis and immune response	Krysan et al (2002), Beck et al (2010), Kosetsu et al (2010), Takahashi et al (2010), Lian et al (2018), Tanaka et al (2004)
	AIK1‐MKK5‐MPK6	ABA‐regulated root growth	Li et al (2017b)
	MAPKKK3/5‐MKK4/5‐MPK3/6	Immune response	Sun et al (2018), Bi et al (2018), Yamada et al (2016), Yan et al (2018)
	MEKK1‐MKK1/2/6‐MPK4	Immune response	Zhang et al (2017b), Roux et al (2015), Bi et al (2018), Qiu et al (2008a), Li et al (2015), Lian et al (2018)
	MKD1‐MKK1/MKK5‐MPK3/MPK6	Immune response	Asano et al (2020)
	YDA‐MKK4/5‐MPK3/6	Stomatal development and patterning	Lampard et al (2008), Nadeau and Sack (2002), Meng et al (2015), Zhang et al (2016), Xue et al (2020), Putarjunan et al (2019), Li et al (2017a), Zhao et al (2017a)
	YDA‐MKK4/5‐MPK3/6	Floral organ abscission	Cho et al (2008), Meng et al (2016), Santiago et al (2016), Taylor et al (2019)
	YDA‐MKK4/5‐MPK3/6	Root meristem growth	Shao et al (2020), Lu et al (2020)
	YDA‐MKK4/5‐MPK6	Suspensor elongation	Costa et al (2014), Zhang et al (2017a)
	MKK1/2/3/7/9‐MPK3/4	Stigma receptivity	Jamshed et al (2020)
	MKK3‐MPK1/2/7/14	Wounding response	Sözen et al (2020)
	MKK4/5‐MPK3/6	Funicular guidance of pollen tubes	Guan et al (2014a)
	MKK4/5‐MPK3/6	Cell separation during lateral root emergence.	Zhu et al (2019)
	MKK4/5‐MPK3/6	Ethylene production	Li et al (2012)
	MKK4/5‐MPK3/6	Pipecolic acid and camalexin accumulation	Mao et al (2011), Wang et al (2018b)
	MKK4/5‐MPK3/6	Immune response	Jiang et al (2020), Li et al (2014a)
	MKK4/5‐MPK3/6	Wounding response	Sözen et al (2020)
	MKK4/5‑MPK1/2	Leaf senescence	Zhang et al (2020)
	MKK6‐MPK13	Lateral root formation	Zeng et al (2011)
	MKK7‐MPK6	Shoot apical meristem activity and branching	Jia et al (2016), Dai et al (2006), Dóczi et al (2019)
	MKK9‐MPK3/6	Ethylene and camalexin production	Xu et al (2008)
	YDA	Immune response	Sopeña‐Torres et al (2018)
	MKK7	Immune response	Zhang et al (2007)
	MPK3/6	Anther development	Zhao et al (2017b)
	MPK3/6	Pollen development and zygote asymmetry	Zheng et al (2018), Guan et al (2014b), Han et al (2019), Ueda et al (2017)
	MPK3/6	Shoot apical meristem maintenance	Lee et al (2019)
	MPK3/6	Immune response	Sheikh et al (2016), Rayapuram et al (2021), Brillada et al (2021), Pecher et al (2014), Djamei et al (2007), Latrasse et al (2017), Furlan et al (2017), Wang et al (2014), Kang et al (2015), Su et al (2018)
	MPK3/6	4MI3G biosynthesis	Meng et al (2013)
	MPK4	PAMP‐induced alternative splicing	Bazin et al (2020)
	MPK4	Anthocyanin accumulation	Wersch et al (2018), Li et al (2016)
	MPK6	Pollen tube growth	Hempel et al (2017)
	MPK6	Leaf senescence	Chai et al (2014)
	MPK6	Ethylene signaling	Bethke et al (2009)
Glycine max (Soybean)	GmMEKK1	Cell death and immune response	Xu et al (2018a)
Gossypium hirsutum (Cotton)	GhMKK6‐GhMPK4	Immune response	Wang et al (2020)
	GhMKK4‐GhMPK20	Immune response	Wang et al (2018a)
Lotus japonicus	LjSIP2‐LjMPK6	Nodule formation	Yin et al (2019), Yan et al (2020)
Medicago sativa	MsSIMKK‐MsSIMK	Nodule formation	Hrbáčková et al (2021)
Medicago truncatula	MtMAPKK4‐MtMAPK3/6	Plant growth and nodule formation	Chen et al (2017)
Nicotiana tabacum (tobacco)	NPK1‐MEK1(NQK1)‐NRK1(NTF6)	Cytokinesis and immune response	Nishihama et al (2001), Jin et al (2002), Takahashi et al (2004), Soyano et al (2003), Nishihama et al (2002), Sasabe et al (2006)
Nicotiana benthamiana	NbMAPKKKα‐MEK2‐SIPK	Hypersensitive response (HR) cell death	Del Pozo et al (2004)
	NbMAPKKKε‐MEK2‐WIPK/SIPK	HR cell death and disease resistance	Melech‐Bonfil and Sessa (2010)
	NbMEK2‐SIPK/WIPK	Immune response	Adachi et al (2015)
	SIPK/NTF4/WIPK	Immune response	Ishihama et al (2011)
Oryza sativa (rice)	OsMKKK10‐OsMKK4‐OsMPK6	Inflorescence architecture	Duan et al (2014), Liu et al (2015), Guo et al (2018), Xu et al (2018b), Guo et al (2020), Jin et al (2016)
	OsMAPKKK18‐OsMKK4/5‐OsMPK3/6	Immune response	Yamada et al (2017)
	OsMAPKKK24‐OsMKK4/5‐OsMPK3/6	Immune response	Wang et al (2017)
	OsMKK3‐OsMPK7	Immune response	Jalmi and Sinha (2016)
	OsMPKK10.2‐OsMPK6	Immune response	Ma et al (2017), Ma et al (2021)
	OsMPK15	Immune response	Hong et al (2019)
Solanum lycopersicum (tomato)	SlMAPKKKε‐MEK2‐WIPK/SIPK	HR cell death and disease resistance	Melech‐Bonfil and Sessa (2010)
	SlYDA	Immune response	Téllez et al (2020)
	SlMKK2/4	Immune response	Li et al (2014b)
	SlMPK20	Pollen development	Chen et al (2018)
Solanum tuberosum (potato)	StMAP3Kβ2‐MEK2‐SIPK;	HR Cell death	Ren et al (2019)
	StMAP3Kε‐MEK2‐SIPK/WIPK	HR Cell death	Ren et al (2019)
	StMKK1‐StMPK7	Immune response	Zhang et al (2021)

MAPK cascades in plant growth and development

Several MAPK cascades have been established for plant growth and development (Fig 1). The NPK1‐NQK1‐NRK1 cascade in tobacco and the analogous ANPs‐MKK6‐MPK4 cascade in Arabidopsis play essential roles in plant cytokinesis, whereas the YODA (YDA)‐MKK4/5‐MPK3/6 cascade is known to regulate many other aspects of plant development in Arabidopsis (Table 1). Recent studies also identified an analogous cascade of YDA‐MKK4/5‐MPK3/6 in rice (Oryza sativa), which consists of OsMKKK10, OsMKK4, and OsMPK6, and regulates panicle architecture (Table 1).

Regulation of plant cytokinesis by MAPK cascades

The tobacco MAPKKK NPK1 was reported as a regulator of cell plate formation during cytokinesis about 20 years ago (Nishihama et al, ²⁰⁰¹). The MKK NQK1 (NtMEK1) and MPK NRK1 (NTF6) were later shown to function downstream of NPK1 to regulate cytokinesis (Soyano et al, ²⁰⁰³; Takahashi et al, ²⁰⁰⁴). NPK1 is activated by two kinesin‐like proteins, NACK1 and NACK2, which promote cell plate formation and cytokinesis in tobacco (Nishihama et al, ²⁰⁰²). NRK1 phosphorylates the microtubule‐associated protein NtMAP65‐1 to reduce its microtubule‐bundling activity and stimulate progression of cytokinesis (Sasabe et al, ²⁰⁰⁶). These findings suggest that the signaling pathway consisting of NACK1/2‐NPK1‐MQK1‐NRK1‐MAP65‐1 regulates plant cytokinesis in tobacco (Fig 1B). An analogous pathway consisting AtNACK1/2‐ANP1/2/3‐MKK6‐MPK4 in Arabidopsis was also reported to regulate cytokinesis by targeting AtMAP65‐1 (Krysan et al, ²⁰⁰²; Beck et al, ²⁰¹⁰; Kosetsu et al, ²⁰¹⁰; Takahashi et al, ²⁰¹⁰).

Regulation of plant development by the YDA‐MKK4/5‐MPK3/6 cascade

The MKK4/5‐MPK3/6 module regulates many aspects of plant development, including stomatal patterning, floral organ abscission, meristem maintenance, and plant inflorescence architecture in Arabidopsis (Xu & Zhang, 2015). Increasing evidence shows that the MAPKKK YDA acts downstream of various peptide ligand–receptor pairs to activate this module and the downstream responses (Fig 1C).

The YDA‐MKK4/5‐MPK3/6 cascade regulates stomatal development by acting downstream of the receptor complex containing the RLK ERECTA (ER)/ER‐Like 1 (ERL1)/ERL2 and the RLP Too Many Mouths (TMM) (Lee et al, ²⁰¹²), which recruit members of SERK family as co‐receptors upon recognition of the EPIDERMAL PATTERNING FACTOR1 (EPF1) and EPF2 peptide ligands (Meng et al, ²⁰¹⁵). Perception of EPF‐LIKE (EPFL) peptides EPFL4 and EPFL6 by ER also activates the YDA‐MKK4/5‐MPK3/6 cascade, which regulates Arabidopsis inflorescence architecture (Meng et al, ²⁰¹²; Uchida et al, ²⁰¹²).

During floral organ abscission, the YDA‐MKK4/5‐MPK3/6 cascade is activated by the Inflorescence Deficient in Abscission (IDA) peptide and its receptor HAE/HSL2 (Cho et al, ²⁰⁰⁸; Taylor et al, ²⁰¹⁹). IDA induces heterodimerization of HAE/HSL2 and SERKs, which leads to activation of their intracellular kinase domains and subsequently the downstream YDA‐MKK4/5‐MPK3/6 cascade (Meng et al, ²⁰¹⁶; Santiago et al, ²⁰¹⁶). A recent study showed that the MKK4/5‐MPK3/6 module also acts downstream of IDA‐HAE/HSL2 to regulate lateral root formation by modulating the expression of cell wall remodeling genes in cells overlaying the lateral root primordia (Zhu et al, ²⁰¹⁹), although the MAPKKK(s) involved in such process is yet to be identified.

The YDA‐MKK4/5‐MPK3/6 cascade was recently shown to also act downstream of the tyrosine‐sulfated peptide ROOT GROWTH FACTOR 1 (RGF1) and its receptors RGF1 INSENSITIVEs (RGIs) to regulate root apical meristem (RAM) maintenance by activating the expression of two downstream transcription factor genes, PLETHORA 1 (PLT1) and PLT2 (Lu et al, ²⁰²⁰; Shao et al, ²⁰²⁰). Loss‐of‐function mutants of this MAPK cascade, including chemical sensitive mpk3 mpk6 double mutants, mkk4 mkk5 and yda, all phenocopy the RAM defect of rgi1/2/3/4/5 quintuple mutant, while expression of constitutively active MKK4DD, MKK5DD, or YDA‐CA driven by the RGI1 promoter partially rescues RAM defect in the rgi1/2/3/4/5 mutant plants. Furthermore, RGF1‐induced MPK3/6 activation depends on RGIs, YDA, and MKK4/5, and RGF1‐induced expression of PLT1/2 requires MKK4/5 and MPK3/6. How MPK3/6 activation leads to the upregulation of PLT1/2 remains to be determined.

Regulation of rice inflorescence architecture by the OsMKKK10‐OsMKK4‐OsMPK6 cascade

The rice OsMKKK10‐OsMKK4‐OsMPK6 MAPK cascade has been shown to regulate rice inflorescence (panicle) architecture. Mutations in OsMKK4 and OsMPK6 result in reduced grain size and dense panicles with increased grain number per panicle (Duan et al, ²⁰¹⁴; Liu et al, ²⁰¹⁵; Guo et al, ²⁰²⁰). Loss‐of‐function mutants of OsMKKK10 also have smaller grains as well as increased number of grains per panicle compared to wild type (Guo et al, ²⁰¹⁸; Xu et al, ^2018b). In contrast, overexpression of constitutively active mutants of OsMKKK10 and OsMPK6, or a gain‐of‐function mutant allele of OsMKK4, results in increased grain size (Xu et al, ^2018b). OsMKKK10 interacts with and phosphorylates OsMKK4 (Xu et al, ^2018b). The phosphorylated OsMKK4 can phosphorylate OsMPK6 at the typical MAPK TEY motif in vitro (Xu et al, ^2018b). Meanwhile, loss‐of‐function mutants of OsMKKK10 and OsMKK4 exhibit greatly decreased OsMPK6 phosphorylation in vivo (Guo et al, ²⁰¹⁸). These genetic and biochemical evidence strongly supports that OsMKKK10, OsMKK4, and OsMPK6 form a MAPK cascade, which regulates the balance between grain size and grain number per panicle (Fig 1D).

Recently, the upstream regulators of this MAPK cascade are emerging. Rice ERECTA1 (OsER1) was shown to regulate panicle morphogenesis. oser1 mutant displayed reduced grain size and increased grain number per panicle, similar to mutants of the OsMKKK10‐OsMKK4‐OsMPK6 cascade (Guo et al, ²⁰²⁰). Expressing CA‐OsMKKK10 or CA‐OsMKK4 leads to the suppression of the increase in grain number per panicle and restored phosphorylation of OsMPK6 in the oser1 mutant, suggesting that OsER1 acts upstream of this MAPK cascade (Guo et al, ²⁰²⁰). Interestingly, GAD1 (GRAIN NUMBER, GRAIN LENGTH, AND AWN DEVELOPMENT1), a peptide in the EPFL family, is also involved in regulating grain number and size in rice (Jin et al, ²⁰¹⁶). Loss‐of‐function mutation of GAD1 confers oser1‐like panicle morphology (Jin et al, ²⁰¹⁶), suggesting that OsER1 may perceive GAD1 peptide to activate the OsMKKK10‐OsMKK4‐OsMPK6 cascade and thus regulate the panicle morphology in rice.

MAPK cascades in plant immunity

Plants deploy cell surface and intercellular receptors to sense pathogen infection and initiate immune signaling (Zhou & Zhang, 2020). Cell surface receptors perceiving immunogenic patterns including Pathogen‐associated molecular patterns (PAMPs) and Damage‐associated molecular patterns (DAMPs) are collectively known as pattern recognition receptors (PRRs). Perception of PAMPs and DAMPs leads to activation of pattern‐triggered immunity (PTI) (Jones & Dangl, 2006). Some well‐studied PAMPs include flg22 and elf18 from bacteria, chitin derived from fungi, and nlp20 from bacteria, fungi, and oomycete, which are recognized by PRR complexes FLS2/BAK1, EFR/BAK1, LYK5/CERK1, and RLP23/BAK1/SOBIR1, respectively (Lee et al, ²⁰²¹). On the other hand, intercellular nucleotide‐binding leucine‐rich repeat receptors (NLRs) recognize pathogen effectors delivered into the plant cells to initiate effector‐triggered immunity (ETI) (Jones & Dangl, 2006). NLRs usually carry a coiled‐coil (CC) or Toll/interleukin1 receptor‐like (TIR) domain at their N‐termini. Activation of PTI and ETI at the infection site leads to induction of systemic acquired resistance (SAR), which confers enhanced resistance against broad‐spectrum pathogens throughout the whole plant. MAPK cascades are used in signal transduction during PTI and ETI as well as SAR (Thulasi Devendrakumar et al, ²⁰¹⁸; Wang et al, ^2018b). There are two major MAPK cascades functioning downstream of PRRs (Fig 2). One consists of MAPKKK3/5, MKK4/5, and MPK3/6 (Bi et al, ²⁰¹⁸; Sun et al, ²⁰¹⁸), and the other comprises MEKK1, MKK1/2, and MPK4 (Gao et al, ²⁰⁰⁸; Qiu et al, ^2008b). Another MAPK cascade consisting of ANP2/3, MKK6, and MPK4, previously known to control cytokinesis, was recently shown to be involved in plant immunity as well (Fig 2) (Takahashi et al, ²⁰¹⁰; Lian et al, ²⁰¹⁸).

Figure 2. MAPK cascades in plant immunity.

(A) The MAPKKK3/5‐MKK4/5‐MPK3/6 cascade acts downstream of PAMP receptors to regulate diverse immune responses by phosphorylating downstream substrates such as WRKY33. (B) The MEKK1‐MKK1/2‐MPK4 cascade functions downstream of PAMP receptors to target various substrates such as MKS1 and CRCK3, which are involved in basal defense and regulation of SUMM2‐mediated immunity, respectively. (C) The ANP2/3‐MKK6‐MPK4 cascade is involved in modulating PAD4/EDS1‐mediated defense responses. (D) The rice MAPK cascade consisting of OsMAPKKK18/24‐OsMKK4/5‐OsMPK3/6 functions downstream of OsCERK1 and OsRLCK185 to activate immune responses upon perception of Chitin.

The MAPKKK3/5‐MKK4/5‐MPK3/6 cascade in plant immunity

Besides its critical roles in regulating plant growth and development, the MAPK module MKK4/5‐MPK3/6 is also essential for plant immunity (Meng & Zhang, 2013; Thulasi Devendrakumar et al, ²⁰¹⁸). MAPKKK3 and MAPKKK5 were recently shown to function upstream of the MKK4/5‐MPK3/6 module in PTI and contribute to resistance against bacterial and fungal pathogens (Yamada et al, ²⁰¹⁶; Bi et al, ²⁰¹⁸; Sun et al, ²⁰¹⁸; Yan et al, ²⁰¹⁸). MPK3/6 activation induced by five different elicitors (PAMPs and DAMPs) including flg22, efl18, nlp20, chitin, and pep1 was significantly compromised in the mapkkk3 mapkkk5 double mutant, but not in the single mutants (Bi et al, ²⁰¹⁸; Sun et al, ²⁰¹⁸), suggesting that MAPKKK3 and MAPKKK5 act redundantly in MPK3/6 activation during PTI (Fig 2A). Interestingly, activated MPK6 phosphorylates MAPKKK5 at Ser‐682/692, which in turn enhances MPK3/6 activation, suggesting a positive feedback regulation (Bi et al, ²⁰¹⁸).

As MAPK activation is not completely blocked in the mapkkk3 mapkkk5 double mutant (Bi et al, ²⁰¹⁸; Sun et al, ²⁰¹⁸), additional MAPKKK(s) is involved in MPK3/6 activation during PTI. YDA is closely related to MAPKKK3/5 and regulates MPK3/6 activation in plant development. It was recently reported to regulate defense response as well (Sopeña‐Torres et al, ²⁰¹⁸; Téllez et al, ²⁰²⁰). However, flg22‐induced MAPK activation is enhanced in a partial loss‐of‐function mutant of YDA and YDA silencing lines (Sopeña‐Torres et al, ²⁰¹⁸; Sun et al, ²⁰¹⁸). MAPKKK δ‐1 (MKD1), a Raf‐like MAPKKK, was recently shown to also contribute to flg22‐triggered activation of MPK3/6 (Asano et al, ²⁰²⁰). The potential functional redundancy between MKD1 and MAPKKK3/5 needs to be tested in future.

The MEKK1‐MKK1/2/6‐MPK4 cascade in plant immunity

The MEKK1‐MKK1/2‐MPK4 cascade is another well‐studied MAPK cascade in plant immunity (Fig 2B; Gao et al, ²⁰⁰⁸; Qiu et al, ^2008b). It was initially suggested to negatively regulate plant immunity based on the autoimmune phenotypes of the mekk1, mkk1 mkk2, and mpk4 mutants (Rodriguez et al, ²⁰¹⁰). Further studies of the suppressor of mkk1 mkk2 (summ) mutants showed that disruption of the MEKK1‐MKK1/2‐MPK4 cascade activates immunity mediated by the CC‐NLR protein SUMM2 (Zhang et al, ²⁰¹²). The MEKK1‐MKK1/2‐MPK4 cascade is activated by various elicitors, suggesting that it may function in disease resistance. Indeed, mekk1 summ2 and mkk1 mkk2 summ2 mutant plants, in which SUMM2‐mediated immunity is blocked, showed enhanced susceptibility to pathogens (Zhang et al, ²⁰¹²), supporting a positive role of this MAPK cascade in basal defense. Thus, this cascade contributes to basal resistance and is guarded by CC‐NLR protein SUMM2. Interestingly, a recent study showed that mutations in RPS6, a TIR‐type NLR protein, can also partially suppress the autoimmune phenotypes of mekk1 and mpk4 plants (Takagi et al, ²⁰¹⁹), suggesting that RPS6 may also monitor the integrity of the MEKK1‐MKK1/2‐MPK4 cascade. However, SUMM2 appears to play a predominant role in surveillance of the MEKK1‐MKK1/2‐MPK4 cascade, as loss of SUMM2 fully blocks the autoimmunity in mekk1 and mkk1 mkk2 mutants and largely suppresses the autoimmunity of mpk4 (Zhang et al, ²⁰¹²).

MKK6 was recently shown to function in parallel with MKK1/2 (Lian et al, ²⁰¹⁸), consistent with the observation that mekk1 mutant plants display a much more severe dwarf phenotype than mkk1 mkk2 (Rodriguez et al, ²⁰¹⁰). summ4‐1D, a gain‐of‐function mutation in the MKK6 promoter region that results in elevated expression of MKK6, can almost fully suppress the autoimmunity of mkk1 mkk2, but has no effect on the autoimmune phenotypes of mekk1 and mpk4 (Lian et al, ²⁰¹⁸). In addition, MKK6 associates with MEKK1 and MPK4, and overexpression of MKK6 rescues flg22‐induced MPK4 activation in mkk1 mkk2, but not in mekk1. These data suggest that MEKK1, MKK6, and MPK4 function together in a MAPK cascade (Lian et al, ²⁰¹⁸). Contribution of MKK6 to MPK4 activation is minor in wild type and mkk1 mkk2 plants due to its low expression levels. In summ4‐1D, the elevated expression of MKK6 is sufficient to compensate for the loss of MKK1 and MKK2.

The ANP2/3‐MKK6‐MPK4 cascade in plant immunity

In addition to their roles in regulating cytokinesis, ANP2/3 and MKK6 are involved in plant immunity, as mkk6 and anp2 anp3 mutant plants display elevated expression of defense genes and enhanced disease resistance (Lian et al, ²⁰¹⁸). The autoimmunity in mkk6 and anp2 anp3 mutants was largely suppressed by expression of a constitutively active version of MPK4 (CA‐MPK4), suggesting that ANP2/3 and MKK6 form a MAPK cascade together with MPK4 in plant immunity (Lian et al, ²⁰¹⁸). Interestingly, the constitutive defense response in anp2 anp3 is not suppressed by loss of SUMM2, but depends on PHYTOALEXIN DEFICIENT 4 (PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), two positive regulators of immunity mediated by TIR‐type NLR proteins (Wiermer et al, ²⁰⁰⁵). Therefore, MPK4 participates in two separate MAPK cascades, namely, the ANP2/3‐MKK6‐MPK4 cascade and the MEKK1‐MKK1/2/6‐MPK4 cascade, and blocking these pathways triggers activation of EDS1/PAD4‐dependent defense pathway and SUMM2‐mediated immunity, respectively (Fig 2B and C). This is in line with the observation that the mutant phenotypes of mekk1 and mkk1 mkk2 rely on SUMM2 fully, whereas the autoimmune phenotypes of mpk4 can only be partially suppressed by summ2 mutations (Zhang et al, ²⁰¹²). How the ANP2/3‐MKK6‐MPK4 cascade regulates plant immunity remains to be determined.

The molecular links connecting surface receptors to MAPK cascades

Increasing evidence suggests that RLCKs function downstream of PRRs in activation of MAPK cascades during PTI. An early study on Xanthomonas campestris pv campestris effector AvrAC, a uridylyltransferase inactivating multiple RLCK VII subfamily members, showed that AvrAC can block flg22‐triggered MAPK activation, but MAPK activation was not affected in individual RLCK knockout mutants (Feng et al, ²⁰¹²). Analysis of various combined mutants of the six members in RLCK VII‐4 subgroup (PBL19, PBL20, PBL37, PBL38, PCRK1, and PCRK2) showed that these RLCKs play crucial roles in activation of MAPKs in PTI (Rao et al, ²⁰¹⁸; Tian et al, ²⁰²¹). In the pcrk1 pcrk2 double mutant, a modest reduction of flg22‐induced MAPK activation was observed (Kong et al, ²⁰¹⁶). Analysis of the pcrk1/2 pbl19/20 quadruple mutant showed that both flg22‐ and nlp20‐induced MAPK activation is significantly lower than in wild type (Tian et al, ²⁰²¹). In addition, chitin‐triggered MAPK activation was greatly compromised in a sextuple mutant of all the RLCK VII‐4 members (Rao et al, ²⁰¹⁸). As MAPK activation induced by the elicitors is not completely blocked in the rlck vii‐4 mutant, additional RLCKs likely contribute to the activation of MAPKs during PTI.

RLCK VII‐4 subgroup members were further shown to connect the chitin receptor CERK1 to MAPKKK5 and MEKK1 (Bi et al, ²⁰¹⁸). MAPKKK5 interacts with PBL19 and can be phosphorylated by CERK1‐activated PBL19 at Ser‐599 in vitro. Phosphorylation of MAPKKK5 at Ser‐599 is required for MPK3/6 activation and disease resistance. On the other hand, MEKK1 is phosphorylated predominantly at Ser‐603 after treatment with chitin, which is essential for its function in plant immunity. In the rlck vii‐4 sextuple mutant, chitin‐triggered phosphorylation at Ser‐603 of MEKK1 was compromised, and CERK1‐activated PBL19 can phosphorylate MEKK1 at Ser‐603 in vitro, suggesting that members of RLCK VII‐4 subgroup directly phosphorylate MEKK1 at Ser‐603 to link the chitin receptor to MEKK1 (Bi et al, ²⁰¹⁸).

In addition to members in the Arabidopsis RLCK VII‐4 subgroup, several other RLCKs were also shown to function upstream of MAPKKKs. BSK1 in the Arabidopsis RCLK II subfamily interacts and phosphorylates MAPKKK5 (Yan et al, ²⁰¹⁸). In rice, OsRLCK185 functions downstream of OsCERK1 to activate the OsMAPKKK18/24‐OsMKK4/5‐OsMPK3/6 cascades upon perception of chitin (Fig 2D) (Wang et al, ²⁰¹⁷; Yamada et al, ²⁰¹⁷). SlMai1, a tomato RLCK closely related to Arabidopsis BSK1, functions upstream of MAPKKKα to regulate NLR‐induced cell death (Roberts et al, ²⁰¹⁹).

How the upstream surface receptors are connected to the YDA‐MKK4/5‐MPK3/6 cascade in plant development is largely unknown. SHORT SUSPENSOR (SSP), an RLCK II subfamily member that functions downstream of the EMBRYO SURROUNDING FACTOR 1 (ESF1) peptide to controls embryonic patterning, interacts with and activates YDA‐dependent signaling during embryogenesis (Bayer et al, ²⁰⁰⁹; Costa et al, ²⁰¹⁴; Ueda et al, ²⁰¹⁷; Yuan et al, ²⁰¹⁷), suggesting that RLCKs could also serve as molecular links between membrane receptors and YDA in developmental signaling. However, the specific RLCKs involved in different developmental processes regulated by YDA‐MKK4/5‐MPK3/6 need to be identified in future.

MAPK substrates in plants

MAPK cascades regulate diverse biological processes by phosphorylating different downstream substrates. MAPK substrates include proteins with diverse biochemical functions, such as transcription factors, enzymes, and other proteins with specific functions (Table 2). Phosphorylation of MAPK substrates could affect their functions by altering their protein stability, subcellular localization, interactions with other proteins, or the activities of the proteins. For example, SIPK‐mediated phosphorylation of NbWRKY8 enhances its DNA binding activity and transcriptional activation activity, thus promoting disease resistance through activation of downstream genes (Ishihama et al, ²⁰¹¹). Arabidopsis CAMTA3, a transcriptional factor known as a negative regulator of plant immunity, is phosphorylated by MPK3/6 upon flg22 treatment, which has dual impact on CAMTA3 including destabilizing CAMTA3 protein and promoting its nuclear‐to‐cytoplasmic trafficking (Jiang et al, ²⁰²⁰).

Table 2.

Reported MAPK substrates in plant development and immunity.

Substrates	MAPKs	Effect of phosphorylation	Biological processes	References
ABI4	MPK3/6	Increased DNA binding activity	Chloroplast retrograde signaling	Guo et al (2016)
ACS2/6	MPK3/6	Stabilization	Ethylene production	Li et al (2012)
AHL13	MPK6	Stabilization	Immune response	Rayapuram et al (2021)
ASR3	MPK4	Increased DNA binding activity	Immune response	Li et al (2015)
BASL	MPK3/6	BASL polarization	Stomatal development	Zhang et al (2016)
BES1	MPK6	Unknown	Immune response	Kang et al (2015)
CAMTA3	MPK3/6	Destabilization and nuclear export	Immune response	Jiang et al (2020)
CDKC;1/2	MPK3/6	Kinase activity	Immune response	Li et al (2014a)
CRCK3	MPK4	Unknown	SUMM2‐mediated ETI	Zhang et al (2017b)
ERF6/104	MPK3/6	Stabilization	Defensin expression	Meng et al (2013), Bethke et al (2009)
Exo70B2	MPK3	Localization and interaction with ATG8	Immune response and autophagy	Brillada et al (2021)
HD2B	MPK3	Intra‐nuclear localization	Immune response	Latrasse et al (2017)
IDD4	MPK6	Increased DNA binding activity	Immune response	Völz et al (2019)
LIP5	MPK3/6	Destabilization	Immune response	Wang et al (2014)
MAP65	NRK1/MPK4	Microtubule‐bundling activity	Cytokinesis	Sasabe et al (2006), Beck et al (2010)
MAPKKK5	MPK6	MPK3/6 activation	Immune response	Bi et al (2018)
MASS	MPK3/6	Subcellular localization	Stomatal development	Xue et al (2020)
MEKK1	MPK4	MPK4 activation	Immune response	Bi et al (2018)
MKS1	MPK4	Localization	Immune response	Qiu et al (2008a)
MVQ1	MPK3/6	Destabilization	Immune response	Pecher et al (2014)
MYB75	MPK4	Stabilization	Anthocyanin biosynthesis	Li et al (2016)
NPR1	MPK1/2	Monomerization	Leaf senescence	Zhang et al (2020)
PAT1	MPK4	Localization	SUMM2‐mediated ETI	Roux et al (2015)
PIN1	MPK6	Subcellular localization, polar localization	Shoot branching	Jia et al (2016), Dai et al (2006)
PIP5K6	MPK6	Decreased phosphoinositide kinase activity	Pollen tube growth and ROS production	Hempel et al (2017), Menzel et al (2019)
PUB22	MPK3	Stabilization	Immune response	Furlan et al (2017)
SCRM	MPK3/6	Destabilization	Stomatal development	Putarjunan et al (2019)
SGT1a/b	MPK3/6	Interaction with RPS2	Immune response	Yu et al (2020)
SPCH	MPK3/6	Destabilization	Stomatal development	Lampard et al (2008)
SPL	MPK3/6	Stabilization	Anther development	Zhao et al (2017b)
VIP1	MPK3	Nuclear import	Immune response	Djamei et al (2007)
WRKY2/34	MPK3/6	Unknown	Pollen development and zygote asymmetry	Zheng et al (2018), Guan et al (2014b)
WRKY33	MPK3/6	Increased transcriptional activity	Biosynthesis of camalexin, ethylene and pipecolic acid	Mao et al (2011), Wang et al (2018b), Li et al (2012), Zhou et al (2020)
WRKY46	MPK3	Destabilization	Immune response	Sheikh et al (2016)
GhWRKY40	GhMPK20	Unknown	Disease resistance	Wang et al (2018a)
NbWRKY7/8/9/11	SIPK/WIPK	Increased transcriptional activity	HR‐like cell death	Adachi et al (2015)
NbWRKY8	SIPK/NTF4/WIPK	Increased DNA binding and transactivation activities	Immune response	Ishihama et al (2011)
MdWRKY17	MdMPK3	Increased DNA binding activity	Immune response	Shan et al (2021)
OsDST	OsMPK6	Increased transcriptional activity	Cytokinin homeostasis Grain number per panicle	Guo et al (2020)
OsEDR1	OsMPK6	Destabilization	Immune response	Ma et al (2021)
OsWRKY30	OsMPK7	Unknown	Disease resistance	Jalmi and Sinha (2016)
OsWRKY53	OsMPK3/6	Increased DNA binding and transactivation activities	Grain size and BR signaling Immune response	Chujo et al (2014), Tian et al (2017)

A large number of candidate MAPK substrates have been identified in Arabidopsis by comparing the phosphoproteomes of wild type, mpk3, mpk4, and mpk6 (Rayapuram et al, ²⁰¹⁸). Their roles in plant development and immunity remain to be determined. In Table 2, we summarized the currently known MAPK substrates involved in plant development and immunity, and the effects of phosphorylation on them. Some of the well characterized MAPK substrates are discussed below.

During plant development, several substrate proteins of Arabidopsis MPK3/6 including SPEECHLESS (SPCH), SCREAM (SCRM), and BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) have been shown to regulate stomatal development and pattering (Lampard et al, ²⁰⁰⁸; Putarjunan et al, ²⁰¹⁹; Xue et al, ²⁰²⁰). SPCH is a bHLH transcription factor that is required for stomatal initiation (Lampard et al, ²⁰⁰⁸). SCRM also known as ICE1 and its paralogue SCRM2 act as partner bHLH proteins of SPCH (Kanaoka et al, ²⁰⁰⁸). The polarity protein BASL is required for stomatal asymmetric division (Dong et al, ²⁰⁰⁹). The WRKY transcription factor OsWRKY53 is a substrate of OsMPK6, which function downstream of the OsMKKK10‐OsMKK4‐OsMPK6 cascade to positively regulates brassinosteroid (BR) signaling and grain size (Tian et al, ²⁰¹⁷). Phosphorylation of OsWRKY53 by OsMPK6 enhances its DNA‐binding and transactivation activity, which is critical for its function in regulating grain size and BR signaling (Chujo et al, ²⁰¹⁴; Hu et al, ²⁰¹⁵; Tian et al, ²⁰¹⁷). The zinc finger transcription factor DROUGHT AND SALT TOLERANCE (DST) is another substrate of OsMPK6 (Guo et al, ²⁰²⁰). DST negatively regulates grain number by directly activating the expression of CYTOKININ OXIDASE2 (OsCKX2) (Li et al, ²⁰¹³). OsMPK6‐mediated phosphorylation of DST enhances its transcriptional activity and stimulates the expression of OsCKX2, leading to decreased cytokinin accumulation in the reproductive meristem and reduced grain number per panicle (Guo et al, ²⁰²⁰).

Arabidopsis MPK3 and MPK6 have also been shown to target many substrates involved in diverse immune responses (Table 2). For example, MPK3/6 promote ethylene biosynthesis through phosphorylation and stabilization of 1‐aminocyclopropane‐1‐carboxylic acid (ACC) Synthase 2 (ACS2) /ACS6 proteins (Han et al, ²⁰¹⁰). MPK3/6 also promote the expression of ACS2 and ACS6 as well as camalexin biosynthetic genes by phosphorylating WRKY33 (Mao et al, ²⁰¹¹; Li et al, ²⁰¹²). MPK3/6‐mediated phosphorylation of WRKY33 was recently shown to enhance its transactivation activity (Zhou et al, ²⁰²⁰). In addition, MPK3/6 are required for accumulation of pipecolic acid (Pip), a precursor of the SAR signaling molecule N‐hydroxypipecolic acid, induced by Pseudomonas syringae pv tomato DC3000 AvrRpt2, which triggers immunity mediated by the resistance protein RPS2 (Wang et al, ^2018b). Stimulation of Pip biosynthesis by MPK3/6 is probably facilitated by WRKY33, which directly up‐regulates the expression of the Pip biosynthetic gene ALD1. Furthermore, MPK3/6 also contribute to ETI by promoting reactive oxygen species (ROS) production and cell death (Su et al, ²⁰¹⁸).

MPK4 also has multiple substrates and plays complex roles in plant immunity. CALMODULIN‐BINDING RECEPTOR‐LIKE CYTOPLASMIC KINASE 3 (CRCK3), a substrate of MEKK1‐MKK1/2‐MPK4 cascade, is required for the autoimmune phenotypes in the mekk1, mkk1 mkk2, and mpk4 mutants (Zhang et al, ^2017b). CRCK3 associates with SUMM2 in planta and MPK4‐mediated phosphorylation of CRCK3 prevents activation of SUMM2‐mediated immunity, suggesting that SUMM2 monitors the integrity of the MEKK1‐MKK1/2‐MPK4 cascade through CRCK3 (Zhang et al, ^2017b). The mRNA decay factor PAT1 is another substrate of MPK4 (Roux et al, ²⁰¹⁵). Knockout mutants of PAT1 activate SUMM2‐dependent defense responses (Roux et al, ²⁰¹⁵). How loss of PAT1 leads to activation of SUMM2‐mediated immunity requires further investigation. In addition, MPK4 targets the positive defense regulator MKS1 and the negative immune regulator ASR3 (Qiu et al, ^2008a; Li et al, ²⁰¹⁵). MPK4 is required for the expression of approximately 50% of flg22‐induced gene (Frei dit Frey et al, ²⁰¹⁴), whereas overexpression of a constitutively active version of MPK4 (CA‐MPK4) compromises PTI as well as ETI‐mediated by TIR‐type NLRs (Berriri et al, ²⁰¹²). MPK4 likely executes its diverse functions through participating in multiple MAPK pathways and targeting different components of plant immunity in a spatial and temporal manner.

Specificity of MAPK cascade signaling in plants

Different MAPK cascades often share the same MKKs and/or MPKs. One given MAPK can also target multiple substrates involved in different biological processes. Uncovering how plants achieve the specificity of MAPK signaling is essential to our understanding of MAPK functions. The specificity of MAPK signaling can be regulated at different stages, including signal perception by upstream regulators, signal relay through independent MAPK cascades, and downstream responses mediated by specific MAPK substrates.

Various studies have shown that spatial and temporal expression of upstream receptors, and/or their ligands is one of the major mechanisms that govern the specificity of a MAPK signaling pathway during development. For example, the YDA‐MKK4/5‐MPK3/6 cascade functions downstream of different ligand–receptor pairs in distinct cell types to regulate stomatal development, floral organ abscission, and root growth, respectively (Cho et al, ²⁰⁰⁸; Lee et al, ²⁰¹²; Lu et al, ²⁰²⁰; Shao et al, ²⁰²⁰). The specific expression of EPF1/2 peptides in stomatal lineage cells and the presence of EPFL4/6 peptides in stem endodermis determine the roles of this cascade in stomatal patterning and inflorescence architecture, respectively (Lee et al, ²⁰¹²; Uchida et al, ²⁰¹²).

The spatial and temporal expression of MAPK substrates also contributes to the specificity of MAPK signaling. For instance, several WRKY transcription factors identified as MPK3/6 substrates have different expression patterns. WRKY34 is expressed specifically in pollen and MPK3/6 regulate pollen development by phosphorylating WRKY34 (Guan et al, ^2014b), whereas WRKY2 is expressed in the basal embryo and acts as a substrate of MPK3/6 to regulate zygote asymmetry and embryo patterning (Ueda et al, ²⁰¹⁷). WRKY33 is induced during pathogen infection, allowing it to regulate immune responses upon MAPK activation (Mao et al, ²⁰¹¹; Birkenbihl et al, ²⁰¹⁷; Wang et al, ^2018b).

Interaction among components of MAPK cascades mediated by scaffold proteins is another well‐known mechanism determining the specificity of MAPK cascades in signal transduction. Several scaffold proteins for MAPK cascades have been reported in plants, including tomato 14‐3‐3 protein 7 (TFT7), receptor for activated C kinase 1 (RACK1), and the polarity protein BASL in Arabidopsis (Oh & Martin, 2011; Cheng et al, ²⁰¹⁵; Zhang et al, ²⁰¹⁵). Upon phosphorylation by MPK3/6, BASL acts as a scaffold and recruits YDA and MPK3/6 to spatially organize at the cell cortex, leading to polar localization of BASL and the YDA cascade in the cell and the asymmetric cell division of stomatal lineage cells (Zhang et al, ²⁰¹⁵). Both TFT7 and RACK1 function as scaffolds for MAPK cascades in plant immunity. TFT7 interacts with SlMAPKKKα and SlMKK2 and is required for programmed cell death mediated by various disease resistance proteins (Oh & Martin, 2011). RACK1 interacts with all three tiers of the Arabidopsis MEKK1‐MKK4/5‐MPK3/6 cascade and contributes to MAPK activation triggered by a protease from P. aeruginosa (Cheng et al, ²⁰¹⁵). Interestingly, MEKK1 was also suggested to function as a scaffold protein for the MEKK1‐MKK1/2‐MPK4 cascade, because it associates with both MKK1/2 and MPK4, and the kinase activity of MEKK1 is dispensable for its function in MPK4 activation (Suarez‐Rodriguez et al, ²⁰⁰⁷; Gao et al, ²⁰⁰⁸; Qiu et al, ^2008b). A recent study reported that the cotton WD40 domain repeat protein GhMORG1 serves as a scaffold protein for the GhMKK6‐GhMPK4 cascade and enhances disease resistance to the fungal pathogen Fusarium oxysporum (Wang et al, ²⁰²⁰).

MAPK signaling specificity can also be facilitated by interactions between MAPKs and their substrates. Many MAPK substrates carry a MAPK docking domain as the direct interaction interface with their cognate MAPKs (Sharrocks et al, ²⁰⁰⁰). For example, the docking domain of NbWRKY8 mediates the interaction with SIPK, NTF4, and WIPK, and this interaction is required for effective phosphorylation of NbWRKY8 in plants (Ishihama et al, ²⁰¹¹). Interestingly, the MPK3/6 substrate SCRM also acts as a scaffold that recruits MPK3/MPK6 to SPCH, a long known MAPK substrate with no direct interaction with MAPKs detected (Putarjunan et al, ²⁰¹⁹). SCRM interacts with MPK3/MPK6 and SPCH through the KiDoK motif and C‐terminal region, respectively, and thus brings MPK3/MPK6 and SPCH to close proximity, allowing effective phosphorylation of SPCH by MAPKs in regulation of stomatal patterning (Putarjunan et al, ²⁰¹⁹).

Post‐translational modifications of substrates are also involved in regulating their interactions with MAPKs and affect the specificity of MAPK signaling. For example, WRKY33 undergoes rapid SUMOylation in response to Botrytis cinerea infection or flg22 treatment, which enables its association with MPK3/6 during plant immunity (Verma et al, ²⁰²¹). The SUMO interaction motif (SIM) present in MPK3/6 is required for their interactions with WRKY33 and phosphorylation of WRKY33, but is dispensable for the association with the non‐SUMOylation form of another substrate, SPCH, supporting a role for SUMOylation in substrate selectivity by MAPKs (Verma et al, ²⁰²¹).

Modulation of MAPK signaling by post‐translational regulations

MAPK signaling can be regulated by post‐translational modifications of MAPK cascade components, such as phosphorylation by other protein kinases, de‐phosphorylation by protein phosphatases, and E3 ligase‐mediated ubiquitination (Fig 3). In addition, de‐phosphorylation of MAPK substrates by protein phosphatases could antagonize the effect of MAPK‐mediated phosphorylation. For example, the conserved Protein Phosphatase 2A (PP2A) interacts with and dephosphorylates SPCH and thus antagonizes MPK3/6‐mediated phosphorylation and destabilization of SPCH (Bian et al, ²⁰²⁰).

Figure 3. Examples of modulation of MAPK signaling by post‐translational modifications.

(A) De‐phosphorylation of MPK3/6 by protein phosphatases including AP2C1/3, PTP1, and MKP1/2 inactivates their kinase activity. (B) BIN2‐mediated phosphorylation of YDA and MKK4/5 attenuates their kinase activity and regulates stomatal development. (C) Ubiquitination of MKK4/5 mediated by the E3 ligase KEG leads to their degradation and dampens immune responses.

Since MAPK activation requires phosphorylation of residues in the conserved activation loop, MAPKs can be inactivated via de‐phosphorylation of these residues by protein phosphatases. In line with the diverse roles of MAPKs in plant development and immunity, several protein phosphatases were shown to regulate plant development and immunity by modulating activities of MAPKs (Fig 3A). Two members of protein phosphatase 2C subfamily, AP2C1 and PP2C5 (also known as AP2C3), directly interact with and inactivate MPK3, MPK4, and MPK6 in Arabidopsis (Schweighofer et al, ²⁰⁰⁷; Brock et al, ²⁰¹⁰; Shubchynskyy et al, ²⁰¹⁷). Loss‐of‐function mutants of AP2C1 or PP2C5 exhibited enhanced activation of MPK3/6, and transgenic lines overexpressing AP2C1 or PP2C5 showed reduced MPK3/6 activation in response to flg22 treatment or pathogen infection (Galletti et al, ²⁰¹¹; Shubchynskyy et al, ²⁰¹⁷). LjPP2C, a type 2C protein phosphatase of Lotus japonicas, was recently shown to fine‐tune nodule development via dephosphorylating LjMPK6 (Yan et al, ²⁰²⁰).

Arabidopsis PROTEIN TYROSINE PHOSPHATASE 1 (PTP1) and two dual‐specificity phosphatases, MAP KINASE PHOSPHATASE1 (MKP1) and MKP2, are also involved in the downregulation of MAPKs. mkp1 mutant plants exhibit elevated basal levels of active MPK3/6 and constitutive defense responses, which are further enhanced in the mkp1 ptp1 double mutant, suggesting that PTP1 and MKP1 have partially overlapping functions in negative regulation of plant immunity through inactivating MPK3/6 (Bartels et al, ²⁰⁰⁹; Anderson et al, ²⁰¹¹). MKP2 also interacts with MPK3/6 and contributes to negative regulation of immune responses (Lumbreras et al, ²⁰¹⁰). Several recent studies showed that MKP1 and its rice homolog OsMKP1 are also involved in regulation of plant development. In Arabidopsis, MKP1‐mediated inactivation of MAPKs in early stomatal precursor determines cell fate transition leading to stomatal differentiation (Tamnanloo et al, ²⁰¹⁸). OsMKP1, also known as GRAIN SIZE AND NUMBER1 (GSN1)/GRAIN LENGTH AND AWN 1 (GLA1)/LARGE8, modulates rice panicle architecture via negative regulation of the OsER1‐OsMKKK10‐OsMKK4‐OsMPK6 pathway (Guo et al, ^{2018, 2020}; Xu et al, ^2018c; Wang et al, ²⁰¹⁹).

The kinase activities of YDA and MKK4/5 were shown to be regulated through phosphorylation by the GSK3/Shaggy‐like kinase BRASSINOSTEROID‐INSENSITIVE 2 (BIN2) and its homologs (Fig 3B; Kim et al, ²⁰¹²; Khan et al, ²⁰¹³). Phosphorylation of YDA by BIN2 inhibits the phosphorylation of MKK4 by YDA (Kim et al, ²⁰¹²). BIN2 also interacts with and phosphorylate MKK4/5, and BIN2‐mediated phosphorylation of MKK4 inhibits its activity to phosphorylate MPK6 in vitro (Khan et al, ²⁰¹³). These findings suggest that BR inhibits stomatal development by BIN2‐mediated suppression of the YDA‐MKK4/5‐MPK3/6 cascade.

Protein levels of MKK4/5 are also regulated by 26S proteasome‐mediated degradation (Gao et al, ²⁰²¹). ENHANCED DISEASE RESISTANCE1 (EDR1) is a Raf‐like MAPKKK involved in negative regulation of cell death and defense responses in Arabidopsis (Frye et al, ²⁰⁰¹). EDR1 directly interacts with MKK4/5 and negatively affects their protein levels (Zhao et al, ²⁰¹⁴). KEEP ON GOING (KEG), an E3 ligase required for edr1‐mediated disease resistance (Wawrzynska et al, ²⁰⁰⁸; Gu & Innes, ²⁰¹¹), was recently shown to associate with MKK4/5 and mediate the ubiquitination and degradation of MKK4/5 (Fig 3C; Gao et al, ²⁰²¹).

In addition to regulation of MAPK signaling through post‐translational modifications, a recent study showed that MEKK2, a paralog of MEKK1, interacts with MPK4 and related MPK11/MPK13 to inhibit their activation by the upstream MKK1/2 (Nitta et al, ²⁰²⁰). The inhibition of MPK4/11/13 is required for the amplification of SUMM2‐mediated immunity, as loss‐of‐function mutants of MEKK2 can suppress the autoimmune phenotypes in the mekk1, mkk1, mkk2, and mpk4 mutants (Kong et al, ²⁰¹²; Su et al, ²⁰¹³).

Concluding remarks

MAPK cascades play essential roles in relaying and amplifying signals from membrane receptors to various downstream responses. Considerable progress has recently been made in MAPK signaling in plant development and immune signaling. However, some key questions remain to be addressed (see BOX 1). Several ligand–receptor pairs have been shown to act upstream of the YDA cascade to regulate plant development, but the molecular links between the receptor complexes to the YDA cascade remain unclear. In plant immunity, RLCK VII‐4 subfamily members are only partially required for elicitor‐induced MAPK activation. Other RLCKs involved in MAPK activation downstream of PRRs need to be uncovered. In addition, MPK3/6 were also shown to be activated during ETI specified by the CC‐NLR RPS2, RPS5, and RPM1 (Tsuda et al, ²⁰¹³; Ngou et al, ²⁰²¹), but the mechanism of how these CC‐NLR activates MAPK signaling is unclear. Activation of MPK3/6 is known to contribute to ETI‐induced cell death (Su et al, ²⁰¹⁸). How activation of MPK3/6 induces cell death is currently unknown. Identification of additional MPK3/6 substrates using technologies suitable for detecting transient protein–protein interactions such as TurboID‐based proximity labeling may help understand the mechanism of MPK3/6‐induced cell death.

Box 1: In need of answers.

1. What are the molecular links between the surface receptors to the YDA cascades in plant development?

2. To what extend do different subgroups of RLCK VII contributes to MAPK activation in response to different elicitors?

3. Beside the well‐studied MPK3/4/6 and MKK1/2/4/5/6, what are the roles of other MPKs and MKKs in Arabidopsis development and growth?

4. What’s underlying mechanism of MPK3/6 activation during ETI specified by the CC‐NLRs including RPS2, RPS5, and RPM1?

5. How does constitutive activation of MPK3/6 lead to cell death?

EMBO Reports (2022) 23: e53817.