Big Roles of Small Kinases： The Complex Functions of Receptor-Like Cytoplasmic Kinases in Plant Immunity and Development

Plants have evolved a large number of receptor-like cytoplasmic kinases （RLCKs） that often functionally and physically associate with receptor-like kinases （RLKs） to modulate plant growth, development and immune responses. Without any apparent extracellular domain, RLCKs relay intracellular signaling often via RLK complex-mediated transphosphorylation events. Recent advances have suggested essential roles of diverse RLCKs in concert with RLKs in regulating various cellular and physiological responses. We summarize here the complex roles of RLCKs in mediating plant immune responses and growth regulation, and discuss specific and overlapping functions of RLCKs in transducing diverse signaling pathways.     

植物类受体蛋白参与植物生长与发育的研究进展

类受体蛋白(receptor-like protein,RLP)广泛存在于高等植物中,其家族在拟南芥中有57个成员。研究表明RLP蛋白参与了植物茎尖分生组织(shoot apical meristem,SAM)、根尖分生组织(root apical meristem,RAM)和维管分生组织等器官的生长与发育过程,例如CLV2(CLAVATA2)和FEA2(FASCINATED EAR2)参与了分生组织的维持与分化和其他器官的发育、TMM(TOO MANY MOUTHS)则介导了植物表皮细胞的分裂和气孔分布。另一方面,RLP蛋白在植物抗病中发挥着重要作用,包括番茄抗叶霉菌Cf类蛋白、番茄抗黑白轮枝菌Ve1和Ve2以及番茄应答非致病绿色木霉菌激发子的抗病蛋白LeEix等。同时,研究表明RLP蛋白在细胞间信号的接受与传递过程中起着极为重要的作用。本文综述了近年来对植物类受体蛋白参与植物生长与发育的功能、信号识别、传输与转导等方面的研究进展。     

The Receptor-Like Cytoplasmic Kinase OsRLCK102 Regulates XA21-Mediated Immunity and Plant Development in Rice

Receptor-like cytoplasmic kinases (RLCKs) belong to a large subgroup of kinases that play pivotal roles in plant development and in protecting plants from various stresses. Here, we report the isolation and characterization of rice OsRLCK102, from the OsRLCK VII subgroup. Silencing of OsRLCK102 compromised receptor kinase XA21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) but did not affect plant basal resistance to Xoo or Magnaporthe oryzae (M. oryzae). Plants with silenced OsRLCK102 exhibit architecture alterations, including reduced plant height, enlarged angle of the lamina joint, decreased rates of seed setting and enhanced sensitivity to hormone brassinolide (BR). Collectively, our study reveals that OsRLCK102 positively regulates XA21-mediated immunity and negatively regulates rice development through BR signaling in rice.     

LEA蛋白与植物的抗旱性

文章对LEA蛋白的性质、功能与植物抗旱性的关系,以及lea基因表达与调控的研究进展作了简要述评。     

The DEAD-box Protein Dbp2 Functions with the RNA-Binding Protein Yra1 to Promote mRNP Assembly

Eukaryotic gene expression involves numerous biochemical steps that are dependent on RNA structure and ribonucleoprotein (RNP) complex formation. The DEAD-box class of RNA helicases plays fundamental roles in formation of RNA and RNP structure in every aspect of RNA metabolism. In an effort to explore the diversity of biological roles for DEAD-box proteins, our laboratory recently demonstrated that the DEAD-box protein Dbp2 associates with actively transcribing genes and is required for normal gene expression in Saccharomyces cerevisiae. We now provide evidence that Dbp2 interacts genetically and physically with the mRNA export factor Yra1. In addition, we find that Dbp2 is required for in vivo assembly of mRNA-binding proteins Yra1, Nab2, and Mex67 onto poly(A)+ RNA. Strikingly, we also show that Dbp2 is an efficient RNA helicase in vitro and that Yra1 decreases the efficiency of ATP-dependent duplex unwinding. We provide a model whereby messenger ribonucleoprotein (mRNP) assembly requires Dbp2 unwinding activity and once the mRNP is properly assembled, inhibition by Yra1 prevents further rearrangements. Both Yra1 and Dbp2 are conserved in multicellular eukaryotes, suggesting that this constitutes a broadly conserved mechanism for stepwise assembly of mature mRNPs in the nucleus.     

Auxin Functions Downstream of Ethylene to Regulate Iron Absorption Promoted by Phomopsis liquidambaris in Arachis hypogaea L.

Plant iron (Fe) deficiency is widely present in alkaline calcium soils worldwide, and endophytes show great potential for promoting plant nutrient absorption. However, the underlying mechanisms remain unclear. To clarify the mechanisms by which the endophytic fungus Phomopsis liquidambaris promotes peanut Fe absorption, we designed this study to detect the physiological changes in peanut with P. liquidambaris infection. We measured ethylene and auxin in peanuts under Fe deficiency and found that fungal colonization promoted their accumulation (50% and 20%, respectively, at the top point). Moreover, plant Fe absorption ability and transfer were enhanced according to qPCR and enzyme results; the Fe content in the leaf increased (29.52%) as the symptoms of leaf chlorosis were ameliorated. Finally, the chlorophyll content increased (29%), and plant growth was enhanced (13.3%). We also proved that during Fe insufficiency, auxin functions downstream of ethylene to induce the upregulation of Fe absorption-related gene and enzyme activity including that of AHA4, IRT1, H⁺-ATPase, and FCR. We conclude that the addition of P. liquidambaris activates the auxin signaling pathway downstream of ethylene and improves peanut Fe absorption by promoting rhizosphere acidification, increasing FCR and IRT1 expression in peanut roots, leading to plant Fe absorption and growth.

     

APH1 Polar Transmembrane Residues Regulate the Assembly and Activity of Presenilin Complexes

Complexes involved in the γ/ϵ-secretase-regulated intramembranous proteolysis of substrates such as the amyloid-β precursor protein are composed primarily of presenilin (PS1 or PS2), nicastrin, anterior pharynx defective-1 (APH1), and PEN2. The presenilin aspartyl residues form the catalytic site, and similar potentially functional polar transmembrane residues in APH1 have been identified. Substitution of charged (E84A, R87A) or polar (Q83A) residues in TM3 had no effect on complex assembly or activity. In contrast, changes to either of two highly conserved histidines (H171A, H197A) located in TM5 and TM6 negatively affected PS1 cleavage and altered binding to other secretase components, resulting in decreased amyloid generating activity. Charge replacement with His-to-Lys substitutions rescued nicastrin maturation and PS1 endoproteolysis leading to assembly of the formation of structurally normal but proteolytically inactive γ-secretase complexes. Substitution with a negatively charged side chain (His-to-Asp) or altering the structural location of the histidines also disrupted γ-secretase binding and abolished functionality of APH1. These results suggest that the conserved transmembrane histidine residues contribute to APH1 function and can affect presenilin catalytic activity.The anterior pharynx defective-1 (APH1)⁵ protein is an essential component of presenilin-dependent complexes required for the γ/ϵ-secretase activity (1). The multicomponent γ-secretase is responsible for the intramembrane proteolysis of a variety of substrates including the amyloid-β precursor protein (APP) and Notch receptor. Notch signaling is involved in a variety of important cell fate decisions during embryogenesis and adulthood (2). The γ/ϵ-secretase cleavage of APP protein is related to the pathogenesis of Alzheimer disease by releasing the 4-kDa amyloid β-peptide (Aβ) which accumulates as senile plaques in patients with Alzheimer disease (3, 4).The γ-complexes are composed of multispanning transmembrane proteins that include APH1 (5, 6), presenilin (PS1 or PS2) (7–10), PEN2 (5), and the type 1 transmembrane nicastrin (NCT) (11). All four components are essential for proteolytic activity, and loss of any single component destabilizes the complex, resulting in the loss of substrate cleavage. Conversely, co-expression of all four components increases γ-secretase activity (12–14). During the maturation of the complexes, presenilins undergo an endoproteolytic cleavage to generate amino- and carboxyl-terminal fragments which remain associated as heterodimers in the active high molecular weight complexes (15–18). Although the exact function of presenilins has been debated (19, 20), it has been proposed that the presenilins are aspartyl proteases with two transmembrane residues constituting the catalytic subunit (21). Analogous aspartyl catalytic dyads are found in the signal peptide peptidases (21, 22). Contributions from the other components are under investigation, and it has been shown, for example, that the large ectodomain of NCT plays a key role in substrate recognition (23, 24). It has also been shown that other proteins can regulate activity such as TMP21, a member of p24 cargo protein, which binds to the presenilin complexes and selectively modulates γ but not ϵ cleavage (25, 26).APH1 is a seven-transmembrane protein with a topology such that the amino terminus is oriented with the endoplasmic reticulum and the carboxyl terminus resides in the cytoplasm (6, 27). It is also expressed as different isoforms encoded by two genes in humans (APH1a on chromosome 1; APH1b on chromosome 15) or three genes in rodents (APH1a on chromosome 3; APH1b and APH1c on chromosome 9). APH1a has 55% sequence similarity with APH1b/APH1c, whereas APH1b and APH1c share 95% similarity. In addition to these different genes, APH1a is alternatively spliced to generate a short (APH1aS) and a long isoform (APH1aL). These two isoforms differ by the addition of 18 residues on the carboxyl-terminal part of APH1aL (28, 29). Deletion of APH1a in mice is embryonically lethal and is associated with developmental and patterning defects similar to those found in Notch, NCT, or PS1 null embryos (30, 31). In contrast to the essential nature of APH1a, the combined APH1b/c-deficient mice survive into adulthood (31). This suggests that APH1a is the major homologue involved in presenilin-dependent function during embryonic development. In addition, these different APH1 variants are constituents of distinct, proteolytically active presenilin-containing complexes and may, therefore, make unique contributions to γ-secretase activity (30–32).Despite their importance to complex formation and function, the exact role of the APH1 isoforms in presenilin-dependent γ/ϵ-secretase activity remains under investigation. In the current study, several highly conserved polar and charged residues located within the transmembrane domains of APH1 were identified. Mutagenesis of two conserved histidine residues embedded in TM5 and TM6 (His-171 and His-197) lead to alterations in γ-secretase complex maturation and activity. The histidine residues contribute to APH1 function and are involved in stabilizing interactions with other γ-secretase components. These key histidines may also be physically localized near the presenilin active site and involved in the γ-secretase activity as shown by the decreased activity of γ-secretase complexes that are assembled with the His-mutants.     

The FLS2-Associated Kinase BIK1 Directly Phosphorylates the NADPH Oxidase RbohD to Control Plant Immunity

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Zinc Finger Protein SALL4 Functions through an AT-Rich Motif to Regulate Gene Expression

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Huntingtin-Associated Protein 1 Interacts with Breakpoint Cluster Region Protein to Regulate Neuronal Differentiation

Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.     

The proteasome regulator PTRE1 contributes to the turnover of SNC1 immune receptor

Plants have evolved a sophisticated immune system in order to recognize and respond to microbes in their environments. Nucleotide-binding leucine-rich repeat (NLR) proteins detect the presence of specific effector molecules delivered into host cells by pathogens and activate strong defence responses. However, as excessive accumulation of NLRs can result in inappropriate immune responses, their abundance must be tightly regulated. Targeted degradation of NLRs through the ubiquitin proteasome pathway is an important mechanism to limit NLR accumulation. Mutations that perturb NLR degradation can cause autoimmune phenotypes. In this study, we show that the proteasome regulator PTRE1 also contributes to NLR degradation. ptre1 mutant plants exhibit increased defence marker gene expression and enhanced disease resistance against virulent pathogens. The stability of the NLR, SUPPRESSOR OF npr1-1 CONSTITUTIVE 1 (SNC1) is also increased in the ptre1 mutant. Although the mouse homologue of PTRE1 was reported to interact with a Cell Division Control protein 48 (CDC48) homologue in vitro (Clemen et al., 2015), we only observed interaction between PTRE1 and AtCDC48A in a split luciferase assay, but not in co-immunoprecipitation. In addition, a related Arabidopsis protein PTRE1h shares partial redundancy with PTRE1. Together, PTRE1 acts as a negative regulator of plant immunity partly by facilitating the degradation of immune receptors such as SNC1.     

The Leucine-Rich Repeat Receptor-Like Kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 and the Cytochrome P450 PHYTOALEXIN DEFICIENT3 Contribute to Innate Immunity to Aphids in Arabidopsis

The importance of pathogen-associated molecular pattern-triggered immunity (PTI) against microbial pathogens has been recently demonstrated. However, it is currently unclear if this layer of immunity mediated by surface-localized pattern recognition receptors (PRRs) also plays a role in basal resistance to insects, such as aphids. Here, we show that PTI is an important component of plant innate immunity to insects. Extract of the green peach aphid (GPA; Myzus persicae) triggers responses characteristic of PTI in Arabidopsis (Arabidopsis thaliana). Two separate eliciting GPA-derived fractions trigger induced resistance to GPA that is dependent on the leucine-rich repeat receptor-like kinase BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)/SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3, which is a key regulator of several leucine-rich repeat-containing PRRs. BAK1 is required for GPA elicitor-mediated induction of reactive oxygen species and callose deposition. Arabidopsis bak1 mutant plants are also compromised in immunity to the pea aphid (Acyrthosiphon pisum), for which Arabidopsis is normally a nonhost. Aphid-derived elicitors induce expression of PHYTOALEXIN DEFICIENT3 (PAD3), a key cytochrome P450 involved in the biosynthesis of camalexin, which is a major Arabidopsis phytoalexin that is toxic to GPA. PAD3 is also required for induced resistance to GPA, independently of BAK1 and reactive oxygen species production. Our results reveal that plant innate immunity to insects may involve early perception of elicitors by cell surface-localized PRRs, leading to subsequent downstream immune signaling.Close to a million insect species have so far been described, and nearly one-half of them feed on plants (Wu and Baldwin, 2010). Within these plant-feeding insects, most feed on a few related plant species, with only 10% feeding upon multiple plant families (Schoonhoven et al., 2005). Plant defense to insects include several layers (Bos and Hogenhout, 2011; Hogenhout and Bos, 2011). Physical barriers, volatile cues, and composition of secondary metabolites of plants are important components that determine insect host choice (Howe and Jander, 2008; Bruce and Pickett, 2011). In addition, plants induce a variety of plant defense responses upon perception of herbivore oral secretions (OS), saliva, and eggs (De Vos and Jander, 2009; Bruessow et al., 2010; Ma et al., 2010; Wu and Baldwin, 2010). These responses may provide full protection against the majority of insect herbivores, and insects that are able to colonize specific plant species likely produce effectors in their saliva or during egg laying that suppress these induced defense responses (Bos and Hogenhout, 2011; Hogenhout and Bos, 2011; Pitino and Hogenhout, 2013).Aphids are sap-feeding insects of the order Hemiptera and are among the most destructive pests in agriculture, particularly in temperate regions (Blackman and Eastop, 2000). More than 4,000 aphid species in 10 families are known (Dixon, 1998). Most aphid species are specialists and use one or a few closely related plant species within one family as host for feeding and reproduction. Examples are pea aphid (Acyrthosiphon pisum), cabbage aphid (Brevicoryne brassicae), and English grain aphid (Sitobion avenae) that colonize plant species within the legumes (family Fabaceae), brassicas (Brassicaceae), and grasses (Gramineae), respectively. The green peach aphid (GPA; Myzus persicae) is one of few aphid species with a broad host range and can colonize hundreds of plants species in over 40 plant families, including brassicas (Blackman and Eastop, 2000). Aphids possess mouthparts composed of stylets that navigate to the plant vascular system, predominantly the phloem, for long-term feeding. However, before establishing a long-term feeding site, these insects display a host selection behavior by probing the upper leaf cell layers with their stylets, a behavior seen on host and nonhost plants of the aphid (Nam and Hardie, 2012). When the plant is judged unsuitable, the aphid takes off to find an alternative plant host. It is not yet clear what happens in the initial stages of insect interactions with plants.Plants sense microbial organisms (including bacteria, fungi, and oomycetes) through perception of conserved molecules, named microbe-associated molecular patterns and pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) to induce the first stage of plant immunity, termed PAMP-triggered immunity (PTI). PTI is effective against the majority of plant pathogens. Bacterial and fungal PAMPs characterized so far include bacterial flagellin (or its derived peptide flg22), bacterial elongation factor (EF)-Tu (or its derived peptide elf18), bacterial lipopolysaccharides and bacterial cold shock protein, chitin oligosaccharides, and the oomycete elicitin INF1 (Boller and Felix, 2009)Plant PRRs are either receptor-like kinases (RLKs) or receptor-like proteins. Most leucine-rich repeat (LRR)-type PRRs associate with and rely for their function on the small regulatory LRR-RLK BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1)/SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3 (SERK3; Monaghan and Zipfel, 2012). For example, in Arabidopsis (Arabidopsis thaliana), flg22 and elf18 bind to the LRR-RLKs FLAGELLIN SENSITIVE2 (FLS2) and EF-TU RECEPTOR (EFR), respectively, leading to a quasi-instant association with BAK1 (Gómez-Gómez and Boller, 2000; Zipfel et al., 2006; Chinchilla et al., 2007; Heese et al., 2007; Schulze et al., 2010; Roux et al., 2011; Sun et al., 2013). BAK1 is required for optimal downstream immune signaling events, such as mitogen-activated protein kinase (MAPK) activation, reactive oxygen species (ROS) bursts, callose depositions, induction of immune genes, and induced resistance. Similarly, BAK1 is a positive regulator of innate immune responses triggered by the Arabidopsis LRR-RLKs PLANT ELICITOR PEPTIDE1 RECEPTOR1 (PEPR1) and PEPR2 that bind the Arabidopsis-derived damage-associated molecular pattern A. thaliana Peptide1 (AtPep1; Krol et al., 2010; Postel et al., 2010; Roux et al., 2011) and by the tomato (Solanum lycopersicum) LRR receptor-like protein Ve1 that recognizes Ave1 derived from Verticillium spp. (Fradin et al., 2009; de Jonge et al., 2012). Consistent with the role of BAK1 downstream of numerous PRRs, BAK1 is required for full immunity to a number of bacterial, fungal, oomycete, and viral pathogens (Heese et al., 2007; Kemmerling et al., 2007; Fradin et al., 2009; Chaparro-Garcia et al., 2011; Roux et al., 2011; Kørner et al., 2013).Notably, it has been recently shown that the ortholog of BAK1 in Nicotiana attenuata regulates the induction of jasmonic acid (JA) accumulation upon herbivory (Yang et al., 2011a). However, immunity to insects was not affected when BAK1 was silenced, and the observed effect on JA accumulation may be due to an indirect effect on brassinosteroid (BR) responses, for which BAK1 is also an important positive regulator (Li et al., 2002; Nam and Li, 2002). Therefore, it is currently unclear if BAK1 is involved in the early recognition of insect-derived elicitors leading to immunity.We discovered that the key regulatory LRR-RLK BAK1 participates in plant defense to an insect herbivore. We found that extracts of GPA trigger plant defense responses in Arabidopsis that are characteristic of PTI. Arabidopsis bak1 mutant plants are compromised in defense to GPA, which colonizes Arabidopsis, and to pea aphid, for which Arabidopsis is a nonhost. BAK1 is required for ROS bursts, callose deposition, and induced resistance in Arabidopsis upon perception of aphid-derived elicitors. One of the defense genes induced by GPA-derived extracts encodes PHYTOALEXIN DEFICIENT3 (PAD3), a cytochrome P450 that catalyzes the conversion of dihydrocamalexic acid to camalexin, which is a major Arabidopsis phytoalexin that is toxic to GPA (Kettles et al., 2013). PAD3 expression is required for Arabidopsis-induced resistance to GPA, independently of BAK1 and ROS. Our results provide evidence that innate immunity to insect herbivores may rely on the early perception of elicitors by cell surface-localized PRR.     

线粒体抗病毒信号蛋白(MAVS)在宿主天然免疫信号通路中的调节作用

线粒体抗病毒信号蛋白(MAVS)作为一种接头蛋白在调节宿主天然免疫信号通路过程中扮演重要角色．Toll样受体(TLR)和RIG-Ⅰ样受体(RLR)等细胞模式识别受体识别入侵的病原体并将信号传递给MAVS,MAVS通过刺激下游的TBK1复合体和IKK复合体分别活化NF-κB和IRF3等信号通路,进而激活干扰素α／β表达,诱发细胞内抗感染天然免疫反应．MAVS除定位线粒体外,也可定位于过氧化物酶体上．MAVS在细胞内的不同定位决定了其在早期快速和持续性抗病毒天然免疫中的不同调节机制．MAVS只有同时定位在过氧化物酶体和线粒体上才可诱导干扰素刺激基因(ISG)快速且稳定地表达．本文通过对MAVS的发现、结构、细胞定位及其在天然免疫信号通路中的调控机制等最新进展进行综述,以期揭示MAVS蛋白在细胞内天然免疫信号通路中的重要调节作用,为研究病毒逃逸宿主天然免疫的机制和研究新型抗病毒免疫治疗策略提供新思路．     

Arabidopsis RECEPTOR-LIKE PROTEIN30 and Receptor-Like Kinase SUPPRESSOR OF BIR1-1/EVERSHED Mediate Innate Immunity to Necrotrophic Fungi

Effective plant defense strategies rely in part on the perception of non-self determinants, so-called microbe-associated molecular patterns (MAMPs), by transmembrane pattern recognition receptors leading to MAMP-triggered immunity. Plant resistance against necrotrophic pathogens with a broad host range is complex and yet not well understood. Particularly, it is unclear if resistance to necrotrophs involves pattern recognition receptors. Here, we partially purified a novel proteinaceous elicitor called SCLEROTINIA CULTURE FILTRATE ELICITOR1 (SCFE1) from the necrotrophic fungal pathogen Sclerotinia sclerotiorum that induces typical MAMP-triggered immune responses in Arabidopsis thaliana. Analysis of natural genetic variation revealed five Arabidopsis accessions (Mt-0, Lov-1, Lov-5, Br-0, and Sq-1) that are fully insensitive to the SCFE1-containing fraction. We used a forward genetics approach and mapped the locus determining SCFE1 sensitivity to RECEPTOR-LIKE PROTEIN30 (RLP30). We also show that SCFE1-triggered immune responses engage a signaling pathway dependent on the regulatory receptor-like kinases BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1) and SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR). Mutants of RLP30, BAK1, and SOBIR1 are more susceptible to S. sclerotiorum and the related fungus Botrytis cinerea. The presence of an elicitor in S. sclerotiorum evoking MAMP-triggered immune responses and sensed by RLP30/SOBIR1/BAK1 demonstrates the relevance of MAMP-triggered immunity in resistance to necrotrophic fungi.