A novel receptor‐like kinase involved in fungal pathogen defence in Arabidopsis thaliana

Plants are under constant attack from a variety of disease‐causing organisms. Lacking an adaptive immune system, plants repel pathogen attack via an array of pathogen recognition machinery. Receptor‐like kinases (RLKs) are involved in the recognition of pathogen‐associated molecular patterns (PAMPs) and activate resistance pathways against broad classes of pathogens. We have identified powdery mildew‐resistant kinase 1, an Arabidopsis gene encoding an RLK that is highly induced by chitin at early time points and localizes to the plasma membrane. Knockout mutants in pmrk1 are more susceptible to both Golovinomyces cichoracearum and Plectosphaerella cucumerina. Our data show that PMRK1 is essential in early stages of defence against fungi and provide evidence that PMRK1 may be unique to chitin‐induced signalling pathways. The results of this study indicate that PMRK1 is a critical component of plant innate immunity against fungal pathogens.     

OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity

Microbe‐associated molecular pattern (MAMP)‐triggered immunity plays critical roles in the basal resistance defense response in plants. Chitin and peptidoglycan (PGN) are major molecular patterns for fungi and bacteria, respectively. Two rice (Oryza sativa) lysin motif‐containing proteins, OsLYP4 and OsLYP6, function as receptors that sense bacterial PGN and fungal chitin. These membrane receptors, which lack intracellular kinase domains, likely contain another component for transmembrane immune signal transduction. Here, we demonstrate that the rice LysM receptor‐like kinase OsCERK1, a key component of the chitin elicitor signaling pathway, also plays an important role in PGN‐triggered immunity in rice. Silencing of OsCERK1 suppressed PGN‐induced (and chitin‐induced) immunity responses, including reactive oxygen species generation, defense gene expression, and callose deposition, indicating that OsCERK1 is essential for both PGN and chitin signaling initiated by OsLYP4 and OsLYP6. OsLYP4 associated with OsLYP6 and the rice chitin receptor chitin oligosaccharide elicitor‐binding protein (CEBiP) in the absence of PGN or chitin, and treatment with PGN or chitin led to their disassociation in vivo. OsCERK1 associated with OsLYP4 or OsLYP6 when induced by PGN but it associated with OsLYP4, OsLYP6, or CEBiP under chitin treatment, suggesting the presence of different patterns of ligand‐induced heterooligomeric receptor complexes. Furthermore, the receptor‐like cytoplasmic kinase OsRLCK176 functions downstream of OsCERK1 in the PGN and chitin signaling pathways, suggesting that these MAMPs share overlapping intracellular signaling components. Therefore, OsCERK1 plays dual roles in PGN and chitin signaling in rice innate immunity and as an adaptor involved in signal transduction at the plasma membrane in conjunction with OsLYP4 and OsLYP6.     

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1‐1 and VvLYK1‐2 mediate chitooligosaccharide‐triggered immunity

Chitin, a major component of fungal cell walls, is a well‐known pathogen‐associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM‐RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM‐RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin‐induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1‐1, ‐2, or ‐3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1‐1 and VvLYK1‐2, but not VvLYK1‐3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide‐induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1‐1 in Atcerk1 restored penetration resistance to the non‐adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1‐1 and VvLYK1‐2 participate in chitin‐ and chitosan‐triggered immunity and that VvLYK1‐1 plays an important role in basal resistance against E. necator.     

Role of ethylene signalling in growth and systemic resistance induction by the plant growth‐promoting fungus Penicillium viridicatum in Arabidopsis

The plant growth‐promoting fungi (PGPF) have long been known to improve plant growth and suppress plant diseases. The PGPF Penicillium viridicatum GP15‐1 elicited plant growth and induced systemic resistance (ISR) in Arabidopsis thaliana against Pseudomonas syringae pv. tomato DC3000 (Pst), leading to a restriction of pathogen growth and disease development. Examination of local and systemic genes indicated that GP15‐1 did not modulate the expression of any of the tested defence‐related marker genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene signalling pathways. Subsequent challenge of GP15‐1‐colonized plants with Pst bacterium primed Arabidopsis plants for enhanced activation of the JA‐inducible Atvsp (vegetative storage protein) gene at a later stage of infection. To assess the contribution of different signalling pathways in GP15‐1‐elicited plant growth and ISR, Arabidopsis genotypes implicated in SA signalling expressing the nahG transgene (NahG) or carrying disruption in NPR1 (npr1), JA signalling (jar1) and ethylene signalling (ein2) were tested. The GP15‐1‐induced plant growth and ISR were fully compromised in an ein2 mutation. Root colonization assay revealed that the inability of the ein2 mutant to express GP15‐1‐induced plant growth and ISR was not associated with reduced root colonization by GP15‐1. In conclusion, our results demonstrate the ethylene signalling pathway is involved in plant growth promotion and ISR elicitation by the PGPF P. viridicatum GP15‐1 in Arabidopsis. These results provide evidence that ethylene signalling has a substantial role in plant growth and disease resistance.     

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14–3–3 isoforms to suppress effector‐triggered immunity

Effector‐triggered immunity (ETI) to host‐adapted pathogens is associated with rapid cell death at the infection site. The plant‐pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI‐associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI‐associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein–protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14–3–3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14–3–3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI‐associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity‐associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ.     

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.     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

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.     

Functional analysis of chimeric lysin motif domain receptors mediating Nod factor‐induced defense signaling in Arabidopsis thaliana and chitin‐induced nodulation signaling in Lotus japonicus

The expression of chimeric receptors in plants is a way to activate specific signaling pathways by corresponding signal molecules. Defense signaling induced by chitin from pathogens and nodulation signaling of legumes induced by rhizobial Nod factors (NFs) depend on receptors with extracellular lysin motif (LysM) domains. Here, we constructed chimeras by replacing the ectodomain of chitin elicitor receptor kinase 1 (AtCERK1) of Arabidopsis thaliana with ectodomains of NF receptors of Lotus japonicus (LjNFR1 and LjNFR5). The hybrid constructs, named LjNFR1–AtCERK1 and LjNFR5–AtCERK1, were expressed in cerk1‐2, an A. thaliana CERK1 mutant lacking chitin‐induced defense signaling. When treated with NFs from Rhizobium sp. NGR234, cerk1‐2 expressing both chimeras accumulated reactive oxygen species, expressed chitin‐responsive defense genes and showed increased resistance to Fusarium oxysporum. In contrast, expression of a single chimera showed no effects. Likewise, the ectodomains of LjNFR1 and LjNFR5 were replaced by those of OsCERK1 (Oryza sativa chitin elicitor receptor kinase 1) and OsCEBiP (O. sativa chitin elicitor‐binding protein), respectively. The chimeras, named OsCERK1–LjNFR1 and OsCEBiP–LjNFR5, were expressed in L. japonicus NF receptor mutants (nfr1‐1; nfr5‐2) carrying a GUS (β‐glucuronidase) gene under the control of the NIN (nodule inception) promoter. Upon chitin treatment, GUS activation reflecting nodulation signaling was observed in the roots of NF receptor mutants expressing both chimeras, whereas a single construct was not sufficient for activation. Hence, replacement of ectodomains in LysM domain receptors provides a way to specifically trigger NF‐induced defense signaling in non‐legumes and chitin‐induced nodulation signaling in legumes.     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

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.     

Calcium acetate‐induced reduction of cadmium accumulation in Oryza sativa: Expression of auto‐inhibited calcium‐ATPase and cadmium transporters

• Calcium (Ca) signalling has an essential role in regulating plant responses to various abiotic stresses.
• This study applied Ca in various forms (Ca acetate and CaCl₂) and concentrations to reduce cadmium (Cd) concentration in rice and propose a possible mechanism through which Ca acts to control the Cd concentration in rice.
• The results showed that supplementation of Cd‐contaminated soil with Ca acetate reduced the Cd concentration in rice after exposure for 7 days in both hydroponic and soil conditions. The possible involvement of the auto‐inhibited Ca²⁺‐ATPase gene (ACA) might act to control the primary signal of the Cd stress response. The messages from ACA3 and ACA13 tended to up‐regulate the low‐affinity cation transporter (OsLCT1) and down‐regulate Cd uptake and the Cd translocation transporter, including the genes, natural resistance‐associated macrophage protein 5 (Nramp5) and Zn/Cd‐transporting ATPase 2 (HMA2), which resulted in a reduction in the Cd concentration in rice. After cultivation for 120 days, the application of Ca acetate into Cd‐contaminated soil inhibited Cd uptake of rice.
• Increasing the Ca acetate concentration in the soil lowered the Cd concentration in rice shoots and grains. Moreover, Ca acetate maintained rice productivity and quality whereas both aspects decreased under Cd stress.

     

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE‐receptor kinase‐WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant‐parasitic cyst nematodes secrete CLE‐like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR‐LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode‐induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock‐down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants.     

Brassinosteroids are involved in ethylene‐induced Pst DC3000 resistance in Nicotiana benthamiana

• Plant immunity is regulated by a huge phytohormone regulation network. Ethylene(ET) and brassinosteroids (BRs) play critical roles in plant response to biotic stress; however, the relationship between BR and ET in plant immunity is unclear.
• We used chemical treatments, genetic approaches and inoculation experiments to investigate the relationship between ET and BR in plant defense against Pst DC3000 in Nicotiana benthamiana.
• Foliar applications of ET and BR enhanced plant resistance to Pst DC3000 inoculation, while treatment with brassinazole (BRZ, a specific BR biosynthesis inhibitor) eliminated the ET induced plant resistance to Pst DC3000. Silencing of DWARF 4(DWF4, a key BR biosynthetic gene), BRASSINOSTEROID INSENSITIVE 1 (BRI1, aBR receptor) and BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1, downstream of BRI1) also neutralised the ET‐induced plant resistance to Pst DC3000. ET can induce callose deposition and reactive oxygen species (ROS) accumulation to resistPst DC3000, BRZ‐treated and gene‐silenced were completely eliminate this response.
• Our results suggest BR is involved in ET‐induced plant resistance, the involvement of ET in plant resistance is possibly by the induction of callose deposition and ROS accumulation, in a BR‐dependent manner.

     

The rice FISH BONE gene encodes a tryptophan aminotransferase,which affects pleiotropic auxin‐related processes

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole‐3‐pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole‐3‐acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin‐related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.