Self/nonself perception and recognition mechanisms in plants: a comparison of self-incompatibility and innate immunity

Analyses of emerging concepts indicate that parallels exist between self-incompatibility and pathogen recognition. In the case of surveillance of 'nonself', plant immune responses are triggered either by pattern recognition receptors (PRRs) that detect conserved pathogen-associated molecular patterns (PAMPs) or by resistance (R) proteins recognizing isolate-specific pathogen effectors. PAMP detection is an important component of innate immunity in plants and serves as an early warning system for the presence of potential pathogens and activation of plant defense mechanisms. In the Brassicaceae, the recognition of 'self' and self-incompatibility are components of a receptor-ligand based mechanism that utilizes an S receptor kinase (SRK) to perceive and reject 'self'-pollen. SRK is an S-domain receptor-like kinase (RLK), which in turn is part of the RLK family, some members of which represent PRRs involved in the detection of PAMPs. S-domain RLKs also occur in species that do not exhibit self-incompatibility and are up-regulated in response to wounding, PAMPs and pathogen recognition. Although evolution may have driven expansion of certain RLK families to serve roles in particular physiological processes, this may not exclude these receptor types from functioning in different programs. Recent findings on self/nonself recognition are reviewed and conceptual and mechanistic links between microbial recognition and self-incompatibility are discussed.     

Molecular characterization of a new type of receptor-like kinase (wlrk) gene family in wheat

In plants, several types of receptor-like kinases (RLK) have been isolated and characterized based on the sequence of their extracellular domains. Some of these RLKs have been demonstrated to be involved in plant development or in the reaction to environmental signals. Here, we describe a RLK gene family in wheat (wlrk, wheat leaf rust kinase) with a new type of extracellular domain. A member of this new gene family has previously been shown to cosegregate with the leaf rust resistance gene Lr10. The diversity of the wlrk gene family was studied by cloning the extracellular domain of different members of the family. Sequence comparisons demonstrated that the extracellular domain consists of three very conserved regions interrupted by three variable regions. Linkage analysis indicated that the wlrk genes are specifically located on chromosome group 1 in wheat and on the corresponding chromosomes of other members of the Triticeae family. The wlrk genes are constitutively expressed in the aerial parts of the plant whereas no expression was detected in roots. Protein immunoblots demonstrated that the WLRK protein coded by the Lrk10 gene is an intrinsic plasma membrane protein. This is consistent with the hypothesis that WLRK proteins are receptor protein kinases localized to the cell surface. In addition, we present preliminary evidence that other disease resistance loci in wheat contain genes which are related to wlrk.     

Molecular dissection of the response of a rice leucine-rich repeat receptor-like kinase (LRR-RLK) gene to abiotic stresses

Leucine-rich repeat (LRR) receptor-like kinase (RLK) proteins play key roles in a variety of biological pathways. In a previous study, we analyzed the members of the rice LRR-RLK gene family using in silico analysis. A total of 23 LRR-RLK genes were selected based on the expression patterns of a genome-wide dataset of microarrays. The Oryza sativa gamma-ray induced LRR-RLK1 (OsGIRL1) gene was highly induced by gamma irradiation. Therefore, we studied its expression pattern in response to various different abiotic and phytohormone treatments. OsGIRL1 was induced on exposure to abiotic stresses such as salt, osmotic, and heat, salicylic acid (SA), and abscisic acid (ABA), but exhibited downregulation in response to jasmonic acid (JA) treatment. The OsGIRL1 protein was clearly localized at the plasma membrane. The truncated proteins harboring juxtamembrane and kinase domains (or only harboring a kinase domain) exhibited strong autophosphorylation. The biological function of OsGIRL1 was investigated via heterologous overexpression of this gene in Arabidopsis plants subjected to gamma-ray irradiation, salt stress, osmotic stress, and heat stress. A hypersensitive response was observed in response to salt stress and heat stress, whereas a hyposensitive response was observed in response to gamma-ray treatment and osmotic stress. These results provide critical insights into the molecular functions of the rice LRR-RLK genes as receptors of external signals.     

植物凝集素类受体蛋白激酶研究进展

自然界中植物的生长发育受到各种环境变化的影响。为了响应外界各种环境条件,植物演化出一系列识别和传递环境信号的蛋白分子,其中比较典型的是植物细胞质膜上的类受体蛋白激酶(RLKs)。凝集素类受体蛋白激酶(LecRLKs)是类受体蛋白激酶家族中的一个亚族,它主要包含3个结构域:细胞外凝集素结构域、跨膜结构域和细胞内激酶结构域。根据细胞外凝集素结构域的不同,LecRLKs可分为3种不同类型:L、G和C型。近年来,研究表明LecRLKs在植物生物/非生物胁迫和发育调控中发挥非常重要的作用。该文综述了植物凝集素类受体蛋白激酶的研究历史、结构特点、分类以及生物学功能,并重点阐述凝集素类受体蛋白激酶在植物生物/非生物胁迫响应和调控发育方面的功能。对不同类型和不同功能的植物凝集素类受体蛋白激酶进行阐述将有利于对该类蛋白开展功能研究,并为作物改良提供有益借鉴。     

Activation of hypersensitive cell death by pathogen-induced receptor-like protein kinases from <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, there is a family of receptor-like protein kinases (RLKs) containing novel cysteine-rich repeats in their extracellular domains. Genes encoding many of these cysteine-rich RLKs (CRKs) are induced by pathogen infection, suggesting a possible role in plant defense responses. We have previously generated Arabidopsis plants expressing four pathogen-regulated CRK genes (CRK5, 6, 10 and 11) under control of a steroid-inducible promoter and found that induced expression of CRK5, but not the other three CRK genes, triggered hypersensitive response-like cell death in transgenic plants. In the present study, we have analyzed the structural relationship of the CRK family and identified three CRKs (CRK4, 19 and 20) that are structurally closely related to CRK5. Genes encoding these three CRKs are all induced by salicylic acid and pathogen infection. Furthermore, induced expression of CRK4, 19and 20 all activates rapid cell death in transgenic plants. Thus, the activity of inducing rapid cell death is shared by these structurally closely related CRKs. We have also performed yeast two-hybrid screens and identified proteins that interact with the kinase domains of CRKs. One of the identified CRK-interacting proteins is the kinase-associated type 2C protein phospohatase known to interact with a number of other RLKs through its kinase-interacting FHA domain. Other CRK-interacting proteins include a second protein with a FHA domain and another type 2C protein phosphatase. Interactions of CRKs with these three proteins in vivo were demonstrated through co-immunoprecipitation. These CRK-interacting proteins may play roles in the regulation and signaling of CRKs.     

An autophosphorylation site database for leucine‐rich repeat receptor‐like kinases in Arabidopsis thaliana

Leucine‐rich repeat receptor‐like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single‐pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites. In this study we employed high‐throughput cloning and a variety of mass spectrometry approaches to generate an autophosphorylation site database representative of more than 30% of the approximately 223 LRR RLKs in Arabidopsis thaliana. We used His‐tagged constructs of complete cytoplasmic domains to identify a total of 592 phosphorylation events across 73 LRR RLKs, with 497 sites uniquely assigned to specific Ser (268 sites) or Thr (229 sites) residues in 68 LRR RLKs. Multiple autophosphorylation sites per LRR RLK were the norm, with an average of seven sites per cytoplasmic domain, while some proteins showed more than 20 unique autophosphorylation sites. The database was used to analyze trends in the localization of phosphorylation sites across cytoplasmic kinase subdomains and to derive a statistically significant sequence motif for phospho‐Ser autophosphorylation.     

A novel protein elicitor (PeSy1) from Saccharothrix yanglingensis induces plant resistance and interacts with a receptor-like cytoplasmic kinase in Nicotiana benthamiana

Previously, we reported a rare actinomycete Saccharothrix yanglingensis Hhs.015 with strong biocontrol ability, which can colonize plant tissues and induce resistance, but the key elicitor and immune mechanisms were unclear. In this study, a novel protein elicitor screened from the genome of Hhs.015, PeSy1 (protein elicitor of S. yanglingensis 1), could induce a strong hypersensitive response (HR) and resistance in plants. The PeSy1 gene encodes an 11 kDa protein with 109 amino acids that is conserved in Saccharothrix species. PeSy1-His recombinant protein induced early defence events such as a cellular reactive oxygen species burst, callose deposition, and the activation of defence hormone signalling pathways, which enhanced Nicotiana benthamiana resistance to Sclerotinia sclerotiorum and Phytophthora capsici, and Solanum lycopersicum resistance to Pseudomonas syringae pv. tomato DC3000. Through pull-down and mass spectrometry, candidate proteins that interacted with PeSy1 were obtained from N. benthamiana. We confirmed the interaction between receptor-like cytoplasmic kinase RSy1 (Response to PeSy1) and PeSy1 using co-immunoprecipitation, bimolecular fluorescence complementation, and microscale thermophoresis. PeSy1 treatment promoted up-regulation of marker genes in pattern-triggered immunity. The cell death it elicited was dependent on the co-receptors NbBAK1 and NbSOBIR1, suggesting that PeSy1 acts as a microbe-associated molecular pattern from Hhs.015. Additionally, RSy1 positively regulated PeSy1-induced plants resistant to S. sclerotiorum. In conclusion, our results demonstrated a novel receptor-like cytoplasmic kinase in the plant perception of microbe-associated molecular patterns, and the potential of PeSy1 in induced resistance provided a new strategy for biological control of actinomycetes in agricultural diseases.     

蔷薇科果树类受体激酶的研究进展

类受体激酶(receptor like kinase,RLK)参与调控植物几乎所有的生命活动,是植物生长发育和环境适应的“中央处理器”。该文对近年来国内外有关蔷薇科果树RLK基因鉴定、进化特征及其在各器官生长发育、非生物和生物逆境中的作用及调控机制等方面的研究进展进行了综述。蔷薇科果树基因组中存在数目庞大的RLKs,不同树种间的RLK数目和各亚家族成员数目都存在较大差异,而且蔷薇科果树RLK存在极为普遍的部分重复和串联重复现象,是导致家族成员迅速变化的重要原因。有研究发现,一些RLKs调控蔷薇科果树器官发育和对环境的适应性。在器官发育方面,LRR RLK亚家族成员调控根系发育,CrRLK1L、LysM RLK和LRR RLK亚家族部分成员参与调控果实发育,CrRLK1L亚家族成员参与调控花粉管发育,LRR RLK、LysM RLK、L LEC RLK和B Lectin RLK亚家族部分成员调控蔷薇科果树对生物逆境的适应。今后RLK功能研究可侧重于蔷薇科果树特色性状,通过提高目标基因的筛选和验证的效率,加速主效RLKs的筛选进程,并通过筛选主效RLKs诱导方式和加速分子育种进程等途径,将研究成果应用于实际生产。     

植物类受体激酶CrRLK1-L亚家族及其生物学功能

植物CrRLK1-L亚家族类受体激酶的胞外域具有新颖结构基序,但功能大都未知.该家族成员广泛存在于被子植物中,但在动物和微生物中不存在其同源物.CrRLK1-L家族成员相对较少,但组织表达非常广泛.它们定位于细胞质膜上,并且部分成员的定位还具有极性,这与其参与雌雄配子体的识别和受精作用密切相关.该家族成员普遍具有激酶活性,该活性对其功能的发挥至关重要.目前仅报道在拟南芥中参与助细胞与花粉的识别和调控营养组织的细胞伸长,但参与这些生物学过程的作用机制似乎独立于已知的信号通路之外,可能有自身独特的信号传导机制.所以对这一类具特有结构基序的类受体激酶基因的功能研究,将有助于解析植物特有生物学过程的分子作用机制,特别是在植物有性生殖过程中,合理利用这些分子开展育种实践对未来农业生产具有潜在的应用价值.     

A cluster of five cell wall-associated receptor kinase genes,Wak1–5, are expressed in specific organs of Arabidopsis

WAK1 (wall-associated kinase 1) is a cytoplasmic serine/threonine kinase that spans the plasma membrane and extends into the extracellular region to bind tightly to the cell wall. The Wak1 gene was mapped and found to lie in a tight cluster of five highly similar genes (Wak1–5) within a 30 kb region. All of the Wak genes encode a cytoplasmic serine/threonine protein kinase, a transmembrane domain, and an extracytoplasmic region with several epidermal growth factor (EGF) repeats. The extracellular regions also contain limited amino acid identities to the tenascin superfamily, collagen, or the neurexins. RNA blot analysis with gene-specific probes revealed that Wak1, Wak3 and Wak5 are expressed primarily in leaves and stems of Arabidopsis. Wak4 mRNA is only detected in siliques, while Wak2 mRNA is found in high levels in leaves and stems, and in lower levels in flowers and siliques. A trace amount of Wak2 can also be detected in roots. Wak1 is induced by pathogen infection and salicylic acid or its analogue INA and is involved in the plant's response, and Wak2, Wak3 and Wak5 also can be greatly induced by salicylic acid or INA. The WAK proteins have the potential to serve as both linkers of the cell wall to the plasma membrane and as signaling molecules, and since Wak expression is organ-specific and the isoforms vary significantly in the cell wall associated domain this family of proteins may be involved in cell wall-plasma membrane interactions that direct fundamental processes in angiosperms.     

Domain-Specific Positive Selection Contributes to the Evolution of Arabidopsis Leucine-Rich Repeat Receptor-Like Kinase (LRR RLK) Genes

Leucine-rich repeat receptor-like kinases (LRR RLKs) comprise the largest group within the plant receptor-like kinase (RLK) superfamily, and the Arabidopsis genome alone contains over 200 LRR RLK genes. Although there is clear evidence for diverse roles played by individual LRR RLK genes in Arabidopsis growth and development, the evolutionary mechanism for this functional diversification is currently unclear. In this study, we focused on the LRRII RLK subfamily to investigate the molecular mechanisms that might have led to the functional differentiation of Arabidopsis LRR RLK genes. Phylogenetic analysis of 14 genes in this subfamily revealed three well-supported groups (I, II, and III). RT-PCR analysis did not find many qualitative differences in expression among these 14 genes in various Arabidopsis tissues, suggesting that evolution of regulatory sequences did not play a major role in their functional divergence. We analyzed substitution patterns in the predicted ligand-binding regions of these genes to examine if positive selection has acted to produce novel ligand-binding specificities, using the nonsynonymous/synonymous rate ratio (d _N/d _S) as an indicator of selective pressure. Estimates of d _N/d _S ratios from multiple methods indicate that nonsynonymous substitutions accumulated during divergence of the three lineages. Positive selection is likely to have occurred along the lineages ancestral to groups II and III. We suggest that positive selection on the ligand-binding sites of LRRII RLKs promoted diversification of ligand-binding specificities and thus contributed to the functional differentiation of Arabidopsis LRRII RLK genes during evolution. [Reviewing Editor: Dr. Martin Kreitman]     

A lectin receptor-like kinase is required for pollen development in Arabidopsis

Lectin receptor-like kinases (Lectin RLKs) are a large family of receptor-like kinases with an extracellular legume lectin-like domain. There are approximately 45 such receptor kinases in Arabidopsis thaliana. Surprisingly, although receptor-like kinases in general are well investigated in Arabidopsis, relatively little is known about the functions of members of the Lectin RLK family. A number of studies implicated members of this family in various functions, such as disease resistance, stress responses, hormone signaling, and legume-rhizobium symbiosis. Our current work demonstrated that mutation in one Lectin RLK gene led to male sterility in Arabidopsis. The sterility was due to defects in pollen development. Pollen development proceeded normally in the mutant until anther stage 8. After that, all pollen grains deformed and collapsed. Mature pollen grains were much smaller than wild-type pollen grains, glued together, and totally collapsed. Therefore, the mutant was named sgc, standing for small, glued-together, and collapsed pollen mutant. The mutant phenotype appeared to be caused by an unidentified sporophytic defect due to the mutation. As revealed by analysis of the promoter-GUS transgenic plants and the gene expression analysis using RT-PCR, the gene showed an interesting temporal and spatial expression pattern: it had no or a low expression in young flowers (roughly before anther stage 6), reached a maximum level around stages 6-7, and then declined gradually to a very low level in young siliques. No expression was detected in microspores or pollen. Together, our data demonstrated that SGC Lectin RLK plays a critical role in pollen development.     

Autophosphorylation of gatekeeper tyrosine by symbiosis receptor kinase

Plant receptor-like kinases (RLKs) share their evolutionary origin with animal interleukin-1 receptor-associated kinase (IRAK)/Pelle family of soluble kinases and are distinguished by having tyrosine as ‘gatekeeper’. This position is adjacent to the hinge region and is hidden in a hydrophobic pocket of the catalytic cleft of protein kinases and is therefore least probable to be a target for any modification. This communication illustrates the accessibility of the gatekeeper site (Y670) towards both autophosphorylation and dephosphorylation in the recombinant cytoplasmic domain of symbiosis receptor kinase from Arachis hypogaea (AhSYMRK). Autophosphorylation on gatekeeper tyrosine was detected prior to extraction but never under in vitro conditions. We hypothesize gatekeeper phosphorylation to be associated with synthesis/maturation of AhSYMRK and this phenomenon may be prevalent among RLKs.     

GsSRK, a G-type lectin S-receptor-like serine/threonine protein kinase,is a positive regulator of plant tolerance to salt stress

Receptor-like protein kinases (RLKs) play vital roles in sensing outside signals, yet little is known about RLKs functions and roles in stress signal perception and transduction in plants, especially in wild soybean. Through the microarray analysis, GsSRK was identified as an alkaline (NaHCO₃)-responsive gene, and was subsequently isolated from Glycine soja by homologous cloning. GsSRK encodes a 93.22 kDa protein with a highly conserved serine/threonine protein kinase catalytic domain, a G-type lectin region, and an S-locus region. Real-time PCR results showed that the expression levels of GsSRK were largely induced by ABA, salt, and drought stresses. Over expression of GsSRK in Arabidopsis promoted seed germination, as well as primary root and rosette leaf growth during the early stages of salt stress. Compared to the wild type Arabidopsis, GsSRK overexpressors exhibited enhanced salt tolerance and higher yields under salt stress, with higher chlorophyll content, lower ion leakage, higher plant height, and more siliques at the adult developmental stage. Our studies suggest that GsSRK plays a crucial role in plant response to salt stress.     

Identification of three clones which commonly interact with the kinase domains of highly homologous two receptor-like kinases, RLK902 and RKL1

We have previously reported the characterization of highly homologous two leucine-rich repeat (LRR)-receptor-like kinase (RLK) genes, RLK902 and RKL1, which showed 75% identity at the amino acid sequence level. To investigate the RLK902 and RKL1 mediated signal transduction pathways, we performed yeast two-hybrid screening using the kinase domains of RLK902 and RKL1 as baits. Three clones, Y-1, 2 and 3, were found to interact commonly with the kinase domain of RLK902 and RKL1 and not to interact with the kinase domain of BRI1, a member of LRR-RLKs. This result suggests that RLK902 and RKL1 may have common biochemical functions, especially in their downstream signal transduction. Furthermore, the detail analysis of their responsiveness to various conditions suggests their involvement in such stress conditions as mechanical wounding, treatment with salicylic acid, and pathogen infection.     

Functional immobilization of plant receptor-like kinase onto microbeads towards receptor array construction and receptor-based ligand fishing

Despite the large number of receptor-like kinases (RLKs) in plants, few of their specific ligands are known. Ligand fishing is one of the most challenging post-genomic technologies. Here, we report a strategy for functional immobilization of plant RLKs on microbeads via covalent linkage. Because of the high density of immobilized RLKs, ligand-receptor interaction can be visualized at high sensitivity using fluorescent-labeled ligands under the confocal laser scanning microscope. Moreover, using a receptor-based affinity chromatography system with RLK microbeads, the ligand of the receptor was directly retrieved at high purity from complex natural samples. Our RLK microbeads and receptor-based ligand fishing approach is a feasible alternative to conventional forward genetics and bioassay-based biochemical purification for identification of novel ligand-receptor pairs in plants.