ATR and MKP1 play distinct roles in response to UV‐B stress in Arabidopsis

Ultraviolet‐B (UV‐B) stress activates MAP kinases (MAPKs) MPK3 and MPK6 in Arabidopsis. MAPK activity must be tightly controlled in order to ensure an appropriate cellular outcome. MAPK phosphatases (MKPs) effectively control MAPKs by dephosphorylation of phosphothreonine and phosphotyrosine in their activation loops. Arabidopsis MKP1 is an important regulator of MPK3 and MPK6, and mkp1 knockout mutants are hypersensitive to UV‐B stress, which is associated with reduced inactivation of MPK3 and MPK6. Here, we demonstrate that MPK3 and MPK6 are hyperactivated in response to UV‐B in plants that are deficient in photorepair, suggesting that UV‐damaged DNA is a trigger of MAPK signaling. This is not due to a block in replication, as, in contrast to atr, the mkp1 mutant is not hypersensitive to the replication‐inhibiting drug hydroxyurea, hydroxyurea does not activate MPK3 and MPK6, and atr is not impaired in MPK3 and MPK6 activation in response to UV‐B. We further show that mkp1 leaves and roots are UV‐B hypersensitive, whereas atr is mainly affected at the root level. Tolerance to UV‐B stress has been previously associated with stem cell removal and CYCB1;1 accumulation. Although UV‐B‐induced stem cell death and CYCB1;1 expression are not altered in mkp1 roots, CYCB1;1 expression is reduced in mkp1 leaves. We conclude that the MKP1 and ATR pathways operate in parallel, with primary roles for ATR in roots and MKP1 in leaves.     

Identification and characterization of an ABA‐activated MAP kinase cascade in Arabidopsis thaliana

Abscisic acid (ABA) is a major phytohormone involved in important stress‐related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA‐triggered phosphoproteins as putative mitogen‐activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA‐activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3‐1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA‐dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR‐SnRK2‐PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA‐induced MAPK pathway in plant stress signalling.     

A Förster resonance energy transfer sensor for live‐cell imaging of mitogen‐activated protein kinase activity in Arabidopsis

The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl‐induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22‐induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.     

Signal convergence through the lenses of MAP kinases: paradigms of stress and hormone signaling in plants

Common mechanisms plants use to translate the external stimuli into cellular responses are the activation of mitogen-activated protein kinase (MAPK) cascade. These MAPK cascades are highly conserved in eukaryotes and consist of three subsequently acting protein kinases, MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK) and MAP kinase (MAPK) which are linked in various ways with upstream receptors and downstream targets. Plant MAPK cascades regulate numerous processes, including various environmental stresses, hormones, cell division and developmental processes. The number of MAPKKs in Arabidopsis and rice is almost half the number of MAPKs pointing important role of MAPKKs in integrating signals from several MAPKKKs and transducing signals to various MAPKs. The cross talks between different signal transduction pathways are concentrated at the level of MAPKK in the MAPK cascade. Here we discussed the insights into MAPKK mediated response to environmental stresses and in plant growth and development.     

Overexpression of cotton GhMKK4 enhances disease susceptibility and affects abscisic acid,gibberellin and hydrogen peroxide signalling in transgenic Nicotiana benthamiana

Mitogen‐activated protein kinase (MAPK) cascades are involved in plant development, stress responses and hormonal signal transduction. MAPK kinases (MAPKKs), as the key nodes in these cascades, link MAPKs and MAPKK kinases (MAPKKKs). In this study, GhMKK4, a novel group C MAPKK gene from cotton (Gossypium hirsutum), was isolated and identified. Its expression can be induced by various stresses and signalling molecules. The overexpression of GhMKK4 in Nicotiana benthamiana enhanced its susceptibility to bacterial and fungal pathogens, but had no significant effects on salt or drought tolerance. Notably, the overexpressing plants showed increased sensitivity to abscisic acid (ABA) and gibberellin A3 (GA3), and ABA and gibberellin (GA) signalling were affected on infection with Ralstonia solanacearum bacteria. Furthermore, the overexpressing plants showed more reactive oxygen species (ROS) accumulation and stronger inhibition of catalase (CAT), a ROS‐scavenging enzyme, than control plants after salicylic acid (SA) treatment. Interestingly, two genes encoding ornithine decarboxylase (ODC) and S‐adenosylmethionine decarboxylase (SAMDC), the key enzymes in polyamine synthesis, exhibited reduced R. solanacearum‐induced expression in overexpressing plants. These findings broaden our knowledge about the functions of MAPKKs in diverse signalling pathways and the negative regulation of disease resistance in the cotton crop.     

MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6

Mitogen‐activated protein kinase (MAPK) cascades have important functions in plant stress responses and development and are key players in reactive oxygen species (ROS) signalling and in innate immunity. In Arabidopsis, the transmission of ROS and pathogen signalling by MAPKs involves the coordinated activation of MPK6 and MPK3; however, the specificity of their negative regulation by phosphatases is not fully known. Here, we present genetic analyses showing that MAPK phosphatase 2 (MKP2) regulates oxidative stress and pathogen defence responses and functionally interacts with MPK3 and MPK6. We show that plants lacking a functional MKP2 gene exhibit delayed wilting symptoms in response to Ralstonia solanacearum and, by contrast, acceleration of disease progression during Botrytis cinerea infection, suggesting that this phosphatase plays differential functions in biotrophic versus necrotrophic pathogen‐induced responses. MKP2 function appears to be linked to MPK3 and MPK6 regulation, as indicated by BiFC experiments showing that MKP2 associates with MPK3 and MPK6 in vivo and that in response to fungal elicitors MKP2 exerts differential affinity versus both kinases. We also found that MKP2 interacts with MPK6 in HR‐like responses triggered by fungal elicitors, suggesting that MPK3 and MPK6 are subject to differential regulation by MKP2 in this process. We propose that MKP2 is a key regulator of MPK3 and MPK6 networks controlling both abiotic and specific pathogen responses in plants.     

Activation of members of a MAPK module in β-glucan elicitor-mediated non-host resistance of soybean

Plants recognize microbial pathogens by discriminating pathogen-associated molecular patterns from self-structures. We study the non-host disease resistance of soybean (Glycine max L.) to the oomycete, Phytophthora sojae. Soybean senses a specific molecular pattern consisting of a branched heptaglucoside that is present in the oomycetal cell walls. Recognition of this elicitor may be achieved through a β-glucan-binding protein, which forms part of a proposed receptor complex. Subsequently, soybean mounts a complex defense response, which includes the increase of the cytosolic calcium concentration, the production of reactive oxygen species, and the activation of genes responsible for the synthesis of phytoalexins. We now report the identification of two mitogen-activated protein kinases (MAPKs) and one MAPK kinase (MAPKK) that may function as signaling elements in triggering the resistance response. The use of specific antisera enabled the identification of GmMPKs 3 and 6 whose activity is enhanced within the signaling pathway leading to defense reactions. Elicitor specificity of MAPK activation as well as the sensitivity against inhibitors suggested these kinases as part of the β-glucan signal transduction pathway. An upstream GmMKK1 was identified based on sequence similarity to other plant MAPKKs and its interaction with the MAPKs was analyzed. Recombinant GmMKK1 interacted predominantly with GmMPK6, with concomitant phosphorylation of the MAPK protein. Moreover, a preferential physical interaction between GmMKK1 and GmMPK6 was demonstrated in yeast. These results suggest a role of a MAPK cascade in mediating β-glucan signal transduction in soybean, similar to other triggers that activate MAPKs during innate immune responses in plants. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. The nucleotide sequences encoding the MAPKs and MAPKK1 from soybean can be accessed through the GenBank database under GenBank accession numbers AF104247, AF329506, and AY070230.     

An Arabidopsis kinase cascade influences auxin‐responsive cell expansion

Mitogen‐activated protein kinase (MPK) cascades are conserved mechanisms of signal transduction across eukaryotes. Despite the importance of MPK proteins in signaling events, specific roles for many Arabidopsis MPK proteins remain unknown. Multiple studies have suggested roles for MPK signaling in a variety of auxin‐related processes. To identify MPK proteins with roles in auxin response, we screened mpk insertional alleles and identified mpk1‐1 as a mutant that displays hypersensitivity in auxin‐responsive cell expansion assays. Further, mutants defective in the upstream MAP kinase kinase MKK3 also display hypersensitivity in auxin‐responsive cell expansion assays, suggesting that this MPK cascade affects auxin‐influenced cell expansion. We found that MPK1 interacts with and phosphorylates ROP BINDING PROTEIN KINASE 1 (RBK1), a protein kinase that interacts with members of the Rho‐like GTPases from Plants (ROP) small GTPase family. Similar to mpk1‐1 and mkk3‐1 mutants, rbk1 insertional mutants display auxin hypersensitivity, consistent with a possible role for RBK1 downstream of MPK1 in influencing auxin‐responsive cell expansion. We found that RBK1 directly phosphorylates ROP4 and ROP6, supporting the possibility that RBK1 effects on auxin‐responsive cell expansion are mediated through phosphorylation‐dependent modulation of ROP activity. Our data suggest a MKK3 ? MPK1 ? RBK1 phosphorylation cascade that may provide a dynamic module for altering cell expansion.     

Chemical genetic identification of a lectin receptor kinase that transduces immune responses and interferes with abscisic acid signaling

Insight into how plants simultaneously cope with multiple stresses, for example, when challenged with biotic stress from pathogen infection and abiotic stress from drought, is important both for understanding evolutionary trade‐offs and optimizing crop responses to these stresses. Mechanisms by which initial plant immune signaling antagonizes abscisic acid (ABA) signal transduction require further investigation. Using a chemical genetics approach, the small molecule [5‐(3,4‐dichlorophenyl)furan‐2‐yl]‐piperidine‐1‐ylmethanethione (DFPM) has previously been identified due to its ability to suppress ABA signaling via plant immune signaling components. Here, we have used forward chemical genetics screening to identify DFPM‐insensitive loci by monitoring the activity of ABA‐inducible pRAB18::GFP in the presence of DFPM and ABA. The ability of DFPM to attenuate ABA signaling was reduced in rda mutants (resistant to DFPM inhibition of ABA signaling). One of the mutants, rda2, was mapped and is defective in a gene encoding a lectin receptor kinase. RDA2 functions in DFPM‐mediated inhibition of ABA‐mediated reporter expression. RDA2 is required for DFPM‐mediated activation of immune signaling, including phosphorylation of mitogen‐activated protein kinase (MAPK) 3 (MPK3) and MPK6, and induction of immunity marker genes. Our study identifies a previously uncharacterized receptor kinase gene that is important for DFPM‐mediated immune signaling and inhibition of ABA signaling. We demonstrate that the lectin receptor kinase RDA2 is essential for perceiving the DFPM signal and activating MAPKs, and that MKK4 and MKK5 are required for DFPM interference with ABA signal transduction.     

The Arabidopsis CERK1‐associated kinase PBL27 connects chitin perception to MAPK activation

Perception of microbe‐associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen‐activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho‐signaling transduction pathway from PRR‐mediated pathogen recognition to MAPK activation in plants. We found that the receptor‐like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1‐LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin‐induced MAPK activation and disease resistance to Alternaria brassicicola. PBL27 phosphorylates MAPKKK5 in vitro, which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo. Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin‐induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants.     

Mitogen‐activated protein kinase phosphatase 1 reduces the replication efficiency of Bamboo mosaic virus in Nicotiana benthamiana

In plants, the mitogen‐activated protein kinase (MAPK) cascades are the central signaling pathways of the complicated defense network triggered by the perception of pathogen‐associated molecular patterns to repel pathogens. The Arabidopsis thaliana MAPK phosphatase 1 (AtMKP1) negatively regulates the activation of MAPKs. Recently, the AtMKP1 homolog of Nicotiana benthamiana (NbMKP1) was found in association with the Bamboo mosaic virus (BaMV) replication complex. This study aimed to investigate the role of NbMKP1 in BaMV multiplication in N. benthamiana. Silencing of NbMKP1 increased accumulations of the BaMV‐encoded proteins and the viral genomic RNA, although the same condition reduced the infectivity of Pseudomonas syringae pv. tomato DC3000 in N. benthamiana. On the other hand, overexpression of NbMKP1 decreased the BaMV coat protein accumulation in a phosphatase activity‐dependent manner in protoplasts. NbMKP1 also negatively affected the in vitro RNA polymerase activity of the BaMV replication complex. Collectively, the activity of NbMKP1 seems to reduce BaMV multiplication, inconsistent with the negatively regulatory role of MKP1 in MAPK cascades in terms of warding off fungal and bacterial invasion. In addition, silencing of NbMKP1 increased the accumulation of Foxtail mosaic virus but decreased Potato virus X. The discrepant effects exerted by NbMKP1 on different pathogens foresee the difficulty to develop plants with broad‐spectrum resistance through genetically manipulating a single player in MAPK cascades.     

The Pseudomonas syringae effector HopF2 suppresses Arabidopsis immunity by targeting BAK1

Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. The P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe‐associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms for suppression of two branches of MAMP‐activated MAP kinase (MAPK) cascades. In addition to blocking MKK5 (MAPK kinase 5) activation in the MEKK1 (MAPK kinase kinase 1)/MEKKs–MKK4/5–MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1–MKK1/2–MPK4 cascade and the plasma membrane‐localized receptor‐like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane‐localized receptor‐like kinase that is involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 and between two other P. syringae effectors, AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild‐type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to show that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells.     

In Silico Identification and Evolutionary Analysis of Plant <Emphasis Type="Italic">MAPKK6s</Emphasis>

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in plants. Linking upstream MAPK kinase kinase (MAPKKK) to downstream MAPK, MAPK kinase (MAPKK) plays a crucial role in MAPK cascade. MAPKK6 is one member of the MAPKK family. In this study, we have found that plant MAPKK6 genes are widely distributed in different plant species, including moss, seedless vascular plants, gymnosperms, and angiosperms. However, no MAPKK6 can be found in genomes of algae. Analysis of exon–intron organization and intron phase showed that plant MAPKK6s are highly conserved genes during plant evolution. In Physcomitrella patens, Selaginella moellendorffii, and Picea glauca, MAPKK6s exist as multicopy genes. In most high plants, however, MAPKK6s exist as single-copy. Phylogenetic analysis indicated that the occurrence of single-copy of MAPKK6s in high plants is likely because of genomic copy-number loss.     

Two guard cell mitogen‐activated protein kinases,MPK9 and MPK12, function in methyl jasmonate‐induced stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) and abscisic acid (ABA) signalling cascades share several signalling components in guard cells. We previously showed that two guard cell‐preferential mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signalling in Arabidopsis thaliana. In this study, we examined whether these two MAP kinases function in MeJA signalling using genetic mutants for MPK9 and MPK12 combined with a pharmacological approach. MeJA induced stomatal closure in mpk9‐1 and mpk12‐1 single mutants as well as wild‐type plants, but not in mpk9‐1 mpk12‐1 double mutants. Consistently, the MAPKK inhibitor PD98059 inhibited the MeJA‐induced stomatal closure in wild‐type plants. MeJA elicited reactive oxygen species (ROS) production and cytosolic alkalisation in guard cells of the mpk9‐1, mpk12‐1 and mpk9‐1 mpk12‐1 mutants, as well in wild‐type plants. Furthermore, MeJA triggered elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in the mpk9‐1 mpk12‐1 double mutant as well as wild‐type plants. Activation of S‐type anion channels by MeJA was impaired in mpk9‐1 mpk12‐1. Together, these results indicate that MPK9 and MPK12 function upstream of S‐type anion channel activation and downstream of ROS production, cytosolic alkalisation and [Ca²⁺]_cyt elevation in guard cell MeJA signalling, suggesting that MPK9 and MPK12 are key regulators mediating both ABA and MeJA signalling in guard cells.     

MPK9 and MPK12 function in SA-induced stomatal closure in Arabidopsis thaliana

Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.     

Arabidopsis EXTRA-LARGE G PROTEIN 1 (XLG1) functions together with XLG2 and XLG3 in PAMP-triggered MAPK activation and immunity

Pattern-triggered immunity (PTI) is an essential strategy used by plants to deploy broad-spectrum resistance against pathogen attacks. Heterotrimeric G proteins have been reported to contribute to PTI. Of the three non-canonical EXTRA-LARGE G PROTEINs (XLGs) in Arabidopsis thaliana, XLG2 and XLG3 were shown to positively regulate immunity, but XLG1 was not considered to function in defense, based on the analysis of a weak xlg1 allele. In this study, we characterized the xlg1 xlg2 xlg3 triple knockout mutants generated from an xlg1 knockout allele. The strong xlg1 xlg2 xlg3 triple mutants compromised pathogen-associated molecular pattern (PAMP)-triggered activation of mitogen-activated protein kinases (MAPKs) and resistance to pathogen infection. The three XLGs interacted with MAPK cascade proteins involved in defense signaling, including the MAPK kinase kinases MAPKKK3 and MAPKKK5, the MAPK kinases MKK4 and MKK5, and the MAPKs MPK3 and MPK6. Expressing a constitutively active form of MKK4 restored MAPK activation and partially recovered the compromised disease resistance seen in the strong xlg1 xlg2 xlg3 triple mutant. Furthermore, mutations of all three XLGs largely restored the phenotype of the autoimmunity mutant bak1-interacting receptor-like kinase 1. Our study reveals that all three XLGs function redundantly in PAMP-triggered MAPK activation and plant immunity.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Mitogen-activated protein kinase OsMPK6 negatively regulates rice disease resistance to bacterial pathogens

Mitogen-activated protein kinase (MAPK) cascades play important roles in diverse developmental and physiological processes of plants, including pathogen-induced defense responses. Although at least 17 rice MAPKs have been identified and more than half of these MAPK genes have been shown to be pathogen or elicitor responsive, the exact role of most of the MAPKs in host-pathogen interaction is unknown. Here we report that OsMPK6 is an important regulator in rice disease resistance. Suppressing OsMPK6 or knocking out of OsMPK6 enhanced rice resistance to different races of Xanthomonas oryzae pv. oryzae, causing bacterial blight, one of the most devastating diseases of rice worldwide. The resistant plants showed increased expression of a subset of defense-responsive genes functioning in the NH1 (an Arabidopsis NPR1 orthologue)-involved defense signal transduction pathway. These results suggest that OsMPK6 functions as a repressor to regulate rice defense responses upon bacterial invasion. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Discovery of rice essential genes by characterizing a CRISPR‐edited mutation of closely related rice MAP kinase genes

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 nuclease (Cas9) system depends on a guide RNA (gRNA) to specify its target. By efficiently co‐expressing multiple gRNAs that target different genomic sites, the polycistronic tRNA‐gRNA gene (PTG) strategy enables multiplex gene editing in the family of closely related mitogen‐activated protein kinase (MPK) genes in Oryza sativa (rice). In this study, we identified MPK1 and MPK6 (Arabidopsis AtMPK6 and AtMPK4 orthologs, respectively) as essential genes for rice development by finding the preservation of MPK functional alleles and normal phenotypes in CRISPR‐edited mutants. The true knock‐out mutants of MPK1 were severely dwarfed and sterile, and homozygous mpk1 seeds from heterozygous parents were defective in embryo development. By contrast, heterozygous mpk6 mutant plants completely failed to produce homozygous mpk6 seeds. In addition, the functional importance of specific MPK features could be evaluated by characterizing CRISPR‐induced allelic variation in the conserved kinase domain of MPK6. By simultaneously targeting between two and eight genomic sites in the closely related MPK genes, we demonstrated 45–86% frequency of biallelic mutations and the successful creation of single, double and quadruple gene mutants. Indels and fragment deletion were both stably inherited to the next generations, and transgene‐free mutants of rice MPK genes were readily obtained via genetic segregation, thereby eliminating any positional effects of transgene insertions. Taken together, our study reveals the essentiality of MPK1 and MPK6 in rice development, and enables the functional discovery of previously inaccessible genes or domains with phenotypes masked by lethality or redundancy.