Receptor-like Cytoplasmic Kinase Research Articles

Arabidopsis class A S-acyl transferases modify the pollen receptors LIP1 and PRK1 to regulate pollen tube guidance.

Protein S-acylation catalyzed by protein S-acyl transferases (PATs) is a reversible lipid modification regulating protein targeting, stability, and interaction profiles. PATs are encoded by large gene families in plants, and many proteins including receptor-like cytoplasmic kinases (RLCKs) and receptor-like kinases (RLKs) are subject to S-acylation. However, few PATs have been assigned substrates, and few S-acylated proteins have known upstream enzymes. We report that Arabidopsis (Arabidopsis thaliana) class A PATs redundantly mediate pollen tube guidance and participate in the S-acylation of POLLEN RECEPTOR KINASE1 (PRK1) and LOST IN POLLEN TUBE GUIDANCE1 (LIP1), a critical RLK or RLCK for pollen tube guidance, respectively. PAT1, PAT2, PAT3, PAT4, and PAT8, collectively named PENTAPAT for simplicity, are enriched in pollen and show similar subcellular distribution. Functional loss of PENTAPAT reduces seed set due to male gametophytic defects. Specifically, pentapat pollen tubes are compromised in directional growth. We determine that PRK1 and LIP1 interact with PENTAPAT, and their S-acylation is reduced in pentapat pollen. The plasma membrane (PM) association of LIP1 is reduced in pentapat pollen, whereas point mutations reducing PRK1 S-acylation affect its affinity with its interacting proteins. Our results suggest a key role of S-acylation in pollen tube guidance through modulating PM receptor complexes.

Improvement of Broad-Spectrum Disease-Resistant Rice by the Overexpression of BSR1 via a Moderate-Strength Constitutive Promoter and a Pathogen-Inducible Promoter.

Conferring crops with resistance to multiple diseases is crucial for stable food production. Genetic engineering is an effective means of achieving this. The rice receptor-like cytoplasmic kinase BSR1 mediates microbe-associated molecular pattern-induced immunity. In our previous study, we demonstrated that rice lines overexpressing BSR1 under the control of the maize ubiquitin promoter exhibited broad-spectrum resistance to rice blast, brown spot, leaf blight, and bacterial seedling rot. However, unfavorable phenotypes were observed, such as a decreased seed germination rate and a partial darkening of husked rice. Herein, we present a strategy to address these unfavorable phenotypes using an OsUbi7 constitutive promoter with moderate expression levels and a pathogen-inducible PR1b promoter. Rice lines expressing BSR1 under the influence of both promoters maintained broad-spectrum disease resistance. The seed germination rate and coloration of husked rice were similar to those of the wild-type rice.

Fortify the defense frontline: MAPKs phosphorylate receptor-like cytoplasmic kinase to maintain plant resistance in soybean

Mitogen-activated protein kinase (MAPK) activation is one of the significant immune events that respond to pathogens in plants. A MAPK cascade often contains a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK/MKK), and a MAPK. The well-characterized MAPK cascade, to date, is the MAPKKK3/4/5-MKK4/5-MPK3/6 module. Soybean cyst nematodes (SCN) is one of the most devastating soybean pathogens. However, the early immune components contributing to soybean resistance to SCN and the role of the MAPK cascade in the soybean–SCN interaction remain unclear. A recent study published in Plant Cell discovered that GmMPK3/6 phosphorylates a receptor-like cytoplasmic kinase (RLCK), CDG1-LIKE1 (GmCDL1), and maintains the stability of GmCDL1 in soybean. Remarkably, GmCDL1 enhances GmMPK3/6 activation and resistance to SCN by phosphorylating GmMAPKKK5 and activating the GmMAPKKK5-GmMKK4-GmMPK3/6 cascade. In addition, two L-type lectin receptor kinases (LecRKs), GmLecRK02g and GmLecRK08g, are involved in the GmCDL1 function after the perception of SCN. taken together, this study not only discovers a complete early immune pathway that responds to SCN infection in soybean, but also reveals a molecular mechanism by which plants maintain the activation of the MAPK cascade and resistance.

Signaling of Plant Defense Mediated by Receptor-like Kinases, Receptor-like Cytoplasmic Protein Kinases and MAPKs Triggered by Fungal Chitin in Horticultural Crops

Fresh horticultural products are economically significant foods that are highly demanded by consumers worldwide; however, they are highly perishable and susceptible to deterioration by fungi, which contribute to their short shelf-life and cause significant post-harvest losses. Among the alternatives suggested for fungal control in plants is the elicitation of the innate plant defense mechanism, which may be activated when specific molecules of the phytopathogen, such as chitin, are recognized. Chitin is a long-chain polymer of N-acetyl-α-D-glucosamine of the fungal cell wall; it possesses biological activity by eliciting the plant immune response. This molecule and its oligosaccharides are recognized through transmembrane receptors known as receptor-like kinases (RLKs) and receptor-like proteins (RLPs). Mediated by receptor-like cytoplasmic kinases (RLCKs), which bind to the intracellular domain of these receptors, they initiate intracellular signal transduction via MAP kinases, triggering the plant defense response. In model plants, such as Oryza sativa (rice) and Arabidopsis thaliana, the set of RLK/RLP-RLCK-MAP kinases is involved in plant immunity triggered by chitin. Furthermore, in horticultural products, research into the molecular events between these three elements has suggested that similar processes occur. However, little is known about these molecular events in fruits. Against this background, the present review provides the most recent and relevant findings on the molecular associations of these three elements in the response to fungal chitin in plants and outlines which elements could participate in this signaling process in horticultural crops.

Pollen-expressed RLCKs control pollen tube burst

In angiosperms, the pollen tube enters the receptive synergid cell, where they rupture to release its cytoplasm along with two sperm cells. Although this interaction is complex, the exact signal transducers that trigger the bursting of pollen tubes are not well understood. In this study, we identify three homologous Receptor-Like Cytoplasmic Kinase (RLCK) expressed in pollen tubes, named Delayed Burst 1/2/3 (DEB1/2/3) in Arabidopsis, which play a crucial role in this process. These genes produce proteins localized on the plasma membrane, and knockout of them causes delayed pollen tube burst and the entrance of additional pollen tubes into the embryo sac due to fertilization recovery. We show that DEBs interact with the Ca2+ pump ACA9, influencing the dynamics of cytoplasmic Ca2+ in pollen tubes through phosphorylation. These results highlight the importance of DEBs as key signal transducers and the critical function of the DEB-ACA9 axis in the timely pollen tube burst in synergids.

S-nitrosylation of a receptor-like cytoplasmic kinase regulates plant immunity.

Perception of pathogen/microbial-associated molecular patterns (P/MAMPs) by plant cell surface receptors leads to a sustained burst of reactive oxygen species (ROS), a key feature of P/MAMP-triggered immunity (PTI). Here we report that P/MAMP recognition leads to a rapid nitrosative burst, initiating the accumulation of nitric oxide (NO), subsequently leading to S-nitrosylation of the receptor-like cytoplasmic kinase (RLCK), botrytis-induced kinase 1 (BIK1), at Cys80. This redox-based, posttranslational modification, promotes the phosphorylation of BIK1, subsequently resulting in BIK1 activation and stabilization. Further, BIK1 S-nitrosylation increases its physical interaction with RBOHD, the source of the apoplastic oxidative burst, promoting ROS formation. Our data identify mechanistic links between rapid NO accumulation and the expression of PTI, providing insights into plant immunity.

Rice NLR protein XinN1, induced by a pattern recognition receptor XA21, confers enhanced resistance to bacterial blight.

Rice CC-type NLR XinN1, specifically induced by a PRR XA21, activates defense pathways against Xoo. Plants have evolved two layers of immune systems regulated by two different types of immune receptors, cell surface located pattern recognition receptors (PRRs) and intracellular nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs). Plant PRRs recognize conserved molecular patterns from diverse pathogens, resulting in pattern-triggered immunity (PTI), whereas NLRs are activated by effectors secreted by pathogens into plant cells, inducing effector-triggered immunity (ETI). Rice PRR, XA21, recognizes a tyrosine-sulfated RaxX peptide (required for activation of XA21-mediated immunity X) as a molecular pattern secreted by Xanthomonas oryzae pv. oryzae (Xoo). Here, we identified a rice NLR gene, XinN1, that is specifically induced during the XA21-mediated immune response against Xoo. Transgenic rice plants overexpressing XinN1 displayed increased resistance to infection by Xoo with reduced lesion length and bacterial growth. Overexpression of autoactive mutant of XinN1 (XinN1D543V) also displayed increased resistance to Xoo, accompanied with severe growth retardation and cell death. In rice protoplast system, overexpression of XinN1 or XinN1D543V significantly elevated reactive oxygen species (ROS) production and cytosolic-free calcium (Ca2+) accumulations. In addition, XinN1 overexpression additionally elevated the ROS burst caused by the interaction between XA21 and RaxX-sY and induced the transcription of PTI signaling components, including somatic embryogenesis receptor kinases (OsSERKs) and receptor-like cytoplasmic kinases (OsRLCKs). Our results suggest that XinN1 induced by the PRR XA21 activates defense pathways and provides enhanced resistance to Xoo in rice.

BRASSINOSTEROID-SIGNALING KINASE1 associates with and is required for cysteine protease RESPONSE TO DEHYDRATION 19-mediated disease resistance in Arabidopsis

The receptor-like cytoplasmic kinase BRASSINOSTEROID-SIGNALING KINASE1 (BSK1) interacts with pattern recognition receptor (PRR) FLAGELLIN SENSING2 (FLS2) and positively regulates plant innate immunity in Arabidopsis thaliana. However, the molecular components involved in BSK1-mediated immune signaling remain largely unknown. To further explore the molecular mechanism underlying BSK1-mediated disease resistance, we screened two cysteine proteases, RESPONSE TO DEHYDRATION 19 (RD19) and RD19-LIKE 2 (RDL2), as BSK1-binding partners. Overexpression of RD19, but not RDL2, displayed an autoimmune phenotype, presenting programmed cell death and enhanced resistance to multiple pathogens. Interestingly, RD19-mediated immune activation depends on BSK1, as knockout of BSK1 in RD19-overexpressing plants rescued their autoimmunity and abolished the increased resistance. Furthermore, we found that BSK1 plays a positive role in maintaining RD19 protein abundance in Arabidopsis. Our results provide new insights into BSK1-mediated immune signaling and reveal a potential mechanism by which BSK1 stabilizes RD19 to promote effective immune output.

OsACA9, an Autoinhibited Ca2+-ATPase, Synergically Regulates Disease Resistance and Leaf Senescence in Rice.

Calcium (Ca2+) is a versatile intracellular second messenger that regulates several signaling pathways involved in growth, development, stress tolerance, and immune response in plants. Autoinhibited Ca2+-ATPases (ACAs) play an important role in the regulation of cellular Ca2+ homeostasis. Here, we systematically analyzed the putative OsACA family members in rice, and according to the phylogenetic tree of OsACAs, OsACA9 was clustered into a separated branch in which its homologous gene in Arabidopsis thaliana was reported to be involved in defense response. When the OsACA9 gene was knocked out by CRISPR/Cas9, significant accumulation of reactive oxygen species (ROS) was detected in the mutant lines. Meanwhile, the OsACA9 knock out lines showed enhanced disease resistance to both rice bacterial blight (BB) and bacterial leaf streak (BLS). In addition, compared to the wild-type (WT), the mutant lines displayed an early leaf senescence phenotype, and the agronomy traits of their plant height, panicle length, and grain yield were significantly decreased. Transcriptome analysis by RNA-Seq showed that the differentially expressed genes (DEGs) between WT and the Osaca9 mutant were mainly enriched in basal immune pathways and antibacterial metabolite synthesis pathways. Among them, multiple genes related to rice disease resistance, receptor-like cytoplasmic kinases (RLCKs) and cell wall-associated kinases (WAKs) genes were upregulated. Our results suggest that the Ca2+-ATPase OsACA9 may trigger oxidative burst in response to various pathogens and synergically regulate disease resistance and leaf senescence in rice.

Mitogen-activated protein kinases MPK3 and MPK6 phosphorylate receptor-like cytoplasmic kinase CDL1 to regulate soybean basal immunity.

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe), one of the most devastating soybean (Glycine max) pathogens, causes significant yield loss in soybean production. Nematode infection triggers plant defense responses; however, the components involved in the upstream signaling cascade remain largely unknown. In this study, we established that a mitogen-activated protein kinase (MAPK) signaling module, activated by nematode infection or wounding, is crucial for soybeans to establish SCN resistance. GmMPK3 and GmMPK6 directly interact with CDG1-LIKE1 (GmCDL1), a member of the receptor-like cytoplasmic kinase (RLCK) subfamily VII. These kinases phosphorylate GmCDL1 at Thr-372 to prevent its proteasome-mediated degradation. Functional analysis demonstrated that GmCDL1 positively regulates immune responses and promotes SCN resistance in soybeans. GmMPK3-mediated and GmMPK6-mediated phosphorylation of GmCDL1 enhances GmMPK3 and GmMPK6 activation and soybean disease resistance, representing a positive feedback mechanism. Additionally, 2 L-type lectin receptor kinases, GmLecRK02g and GmLecRK08g, associate with GmCDL1 to initiate downstream immune signaling. Notably, our study also unveils the potential involvement of GmLecRKs and GmCDL1 in countering other soybean pathogens beyond nematodes. Taken together, our findings reveal the pivotal role of the GmLecRKs-GmCDL1-MAPK regulatory module in triggering soybean basal immune responses.

The ZmWAKL-ZmWIK-ZmBLK1-ZmRBOH4 module provides quantitative resistance to gray leaf spot in maize.

Gray leaf spot (GLS), caused by the fungal pathogens Cercospora zeae-maydis and Cercospora zeina, is a major foliar disease of maize worldwide (Zea mays L.). Here we demonstrate that ZmWAKL encoding cell-wall-associated receptor kinase-like protein is the causative gene at the major quantitative disease resistance locus against GLS. The ZmWAKLY protein, encoded by the resistance allele, can self-associate and interact with a leucine-rich repeat immune-related kinase ZmWIK on the plasma membrane. The ZmWAKLY/ZmWIK receptor complex interacts with and phosphorylates the receptor-like cytoplasmic kinase (RLCK) ZmBLK1, which in turn phosphorylates its downstream NADPH oxidase ZmRBOH4. Upon pathogen infection, ZmWAKLY phosphorylation activity is transiently increased, initiating immune signaling from ZmWAKLY, ZmWIK, ZmBLK1 to ZmRBOH4, ultimately triggering a reactive oxygen species burst. Our study thus uncovers the role of the maize ZmWAKL-ZmWIK-ZmBLK1-ZmRBOH4 receptor/signaling/executor module in perceiving the pathogen invasion, transducing immune signals, activating defense responses and conferring increased resistance to GLS.

The zinc finger protein DHHC09 S-acylates the kinase STRK1 to regulate H2O2 homeostasis and promote salt tolerance in rice.

Soil salinity results in oxidative stress and heavy losses to crop production. The S-acylated protein SALT TOLERANCE RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (STRK1) phosphorylates and activates CATALASE C (CatC) to improve rice (Oryza sativa L.) salt tolerance, but the molecular mechanism underlying its S-acylation involved in salt signal transduction awaits elucidation. Here, we show that the DHHC-type zinc finger protein DHHC09 S-acylates STRK1 at Cys5, Cys10, and Cys14 and promotes salt and oxidative stress tolerance by enhancing rice H2O2-scavenging capacity. This modification determines STRK1 targeting to the plasma membrane or lipid nanodomains and is required for its function. DHHC09 promotes salt signaling from STRK1 to CatC via transphosphorylation, and its deficiency impairs salt signal transduction. Our findings demonstrate that DHHC09 S-acylates and anchors STRK1 to the plasma membrane to promote salt signaling from STRK1 to CatC, thereby regulating H2O2 homeostasis and improving salt stress tolerance in rice. Moreover, overexpression of DHHC09 in rice mitigates grain yield loss under salt stress. Together, these results shed light on the mechanism underlying the role of S-acylation in RLK/RLCK-mediated salt signal transduction and provide a strategy for breeding highly salt-tolerant rice.

Green leaf volatiles co-opt proteins involved in molecular pattern signalling in plant cells.

The green leaf volatiles (GLVs) Z-3-hexen-1-ol (Z3-HOL) and Z-3-hexenyl acetate (Z3-HAC) are airborne infochemicals released from damaged plant tissues that induce defenses and developmental responses in receiver plants, but little is known about their mechanism of action. We found that Z3-HOL and Z3-HAC induce similar but distinctive physiological and signaling responses in tomato seedlings and cell cultures. In seedlings, Z3-HAC showed a stronger root growth inhibition effect than Z3-HOL. In cell cultures, the two GLVs induced distinct changes in MAP kinase (MAPK) activity and proton fluxes as well as rapid and massive changes in the phosphorylation status of proteins within 5 min. Many of these phosphoproteins are involved in reprogramming the proteome from cellular homoeostasis to stress and include pattern recognition receptors, a receptor-like cytoplasmic kinase, MAPK cascade components, calcium signaling proteinsand transcriptional regulators. These are well-known components of damage-associated molecular pattern (DAMP) signaling pathways. These rapid changes in the phosphoproteome may underly the activation of defense and developmental responses to GLVs. Our data provide further evidence that GLVs function like DAMPs and indicate that GLVs coopt DAMP signaling pathways.

Structural and biochemical basis of FLS2-mediated signal activation and transduction in rice

The receptor-like kinase FLAGELLIN-SENSITIVE 2 (FLS2) functions as a bacterial flagellin receptor localized on the cell membrane of plants. In Arabidopsis, the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) cooperates with FLS2 to detect the flagellin epitope flg22, resulting in formation of a signaling complex that triggers plant defense responses. However, the co-receptor responsible for recognizing and signaling the flg22 epitope in rice remains to be determined, and the precise structural mechanism underlying FLS2-mediated signal activation and transduction has not been clarified. This study presents the structural characterization of a kinase-dead mutant of the intracellular kinase domain of OsFLS2 (OsFLS2-KDD1013A) in complex with ATP or ADP, resolved at resolutions of 1.98 Å and 2.09 Å, respectively. Structural analysis revealed that OsFLS2 can adopt an active conformation in the absence of phosphorylation, although it exhibits only weak basal catalytic activity for autophosphorylation. Subsequent investigations demonstrated that OsSERK2 effectively phosphorylates OsFLS2, which reciprocally phosphorylates OsSERK2, leading to complete activation of OsSERK2 and rapid phosphorylation of the downstream substrate receptor-like cytoplasmic kinases OsRLCK176 and OsRLCK185. Through mass spectrometry experiments, we successfully identified critical autophosphorylation sites on OsSERK2, as well as sites transphosphorylated by OsFLS2. Furthermore, we demonstrated the interaction between OsSERK2 and OsFLS2, which is enhanced in the presence of flg22. Genetic evidence suggests that OsRLCK176 and OsRLCK185 may function downstream of the OsFLS2-mediated signaling pathway. Our study reveals the molecular mechanism by which OsFLS2 mediates signal transduction pathways in rice and provides a valuable example for understanding RLK-mediated signaling pathways in plants.

The OsBSK1-2-MAPK module regulates blast resistance in rice

Receptor-like cytoplasmic kinase OsBSK1-2 was reported to play an important role in regulation of response to rice blast, but the signaling pathway remained unknown. In this study, we identified OsMAPKKK18 and previously uncharacterized MAPKKKs OsMAPKKK16 and OsMAPKKK19 that interact with OsBSK1-2. Expression of all three MAPKKKs was induced by Magnaporthe oryzae infection, and all three induced cell death when transiently expressed in Nicotiana benthamiana leaves. Knockout of OsMAPKKK16 and OsMAPKKK18 compromised blast resistance and overexpression of OsMAPKKK19 increased blast resistance, indicating that all three MAPKKKs are involved in regulation of rice blast response. Furthermore, both OsMAPKKK16 and OsMAPKKK19 interacted with and phosphorylated OsMKK4 and OsMKK5, and chitin-induced MAPK activation was suppressed in osmapkkk16 and osbsk1-2 mutants. OsMAPKKK18 was earlier reported to interact with and phosphorylate OsMKK4 and affect chitin-induced MAPK activation, suggesting that OsBSK1-2 is involved in regulation of immunity through multiple MAPK signaling pathways. Unlike BSK1 in Arabidopsis, OsBSK1-2 was not involved in response to avirulent M. oryzae strains. Taken together, our results revealed important roles of OsMAPKKK16/18/19 and a OsBSK1-2-OsMAPKKK16/18/19-OsMKK4/5 module in regulating response to rice blast.

The chromosome-scale genome and the genetic resistance machinery against insect herbivores of the Mexican toloache, Datura stramonium.

Plant resistance refers to the heritable ability of plants to reduce damage caused by natural enemies, such as herbivores and pathogens, either through constitutive or induced traits like chemical compounds or trichomes. However, the genetic architecture-the number and genome location of genes that affect plant defense and the magnitude of their effects-of plant resistance to arthropod herbivores in natural populations remains poorly understood. In this study, we aimed to unveil the genetic architecture of plant resistance to insect herbivores in the annual herb Datura stramonium (Solanaceae) through quantitative trait loci mapping. We achieved this by assembling the species' genome and constructing a linkage map using an F2 progeny transplanted into natural habitats. Furthermore, we conducted differential gene expression analysis between undamaged and damaged plants caused by the primary folivore, Lema daturaphila larvae. Our genome assembly resulted in 6,109 scaffolds distributed across 12 haploid chromosomes. A single quantitative trait loci region on chromosome 3 was associated with plant resistance, spanning 0 to 5.17 cM. The explained variance by the quantitative trait loci was 8.44%. Our findings imply that the resistance mechanisms of D. stramonium are shaped by the complex interplay of multiple genes with minor effects. Protein-protein interaction networks involving genes within the quantitative trait loci region and overexpressed genes uncovered the key role of receptor-like cytoplasmic kinases in signaling and regulating tropane alkaloids and terpenoids, which serve as powerful chemical defenses against D. stramonium herbivores. The data generated in our study constitute important resources for delving into the evolution and ecology of secondary compounds mediating plant-insect interactions.

The OsCPK17-OsPUB12-OsRLCK176 module regulates immune homeostasis in rice.

Plant immunity is fine-tuned to balance growth and defense. However, little is yet known about molecular mechanisms underlying immune homeostasis in rice (Oryza sativa). In this study, we reveal that a rice calcium-dependent protein kinase (CDPK), OsCPK17, interacts with and stabilizes the receptor-like cytoplasmic kinase (RLCK) OsRLCK176, a close homolog of Arabidopsis thaliana BOTRYTIS-INDUCED KINASE 1 (AtBIK1). Oxidative burst and pathogenesis-related gene expression triggered by pathogen-associated molecular patterns are significantly attenuated in the oscpk17 mutant. The oscpk17 mutant and OsCPK17-silenced lines are more susceptible to bacterial diseases than the wild-type plants, indicating that OsCPK17 positively regulates rice immunity. Furthermore, the plant U-box (PUB) protein OsPUB12 ubiquitinates and degrades OsRLCK176. OsCPK17 phosphorylates OsRLCK176 at Ser83, which prevents the ubiquitination of OsRLCK176 by OsPUB12 and thereby enhances the stability and immune function of OsRLCK176. The phenotypes of the ospub12 mutant in defense responses and disease resistance show that OsPUB12 negatively regulates rice immunity. Therefore, OsCPK17 and OsPUB12 reciprocally maintain OsRLCK176 homeostasis and function as positive and negative immune regulators, respectively. This study uncovers positive cross talk between CDPK- and RLCK-mediated immune signaling in plants and reveals that OsCPK17, OsPUB12, and OsRLCK176 maintain rice immune homeostasis.

Genome-wide identification of GhRLCK-VII subfamily genes in Gossypium hirsutum and investigation of their functions in resistance to Verticillium wilt

BackgroundThe receptor-like cytoplasmic kinases subfamily VII (RLCK-VII) is critical in regulating plant growth, development, and pattern-triggered immunity. However, a comprehensive exploration of these genes in the allotetraploid Gossypium hirsutum is still lacking. This study aimed to identify RLCK-VII genes in G. hirsutum and investigate their evolutionary history, structural features, expression patterns, and role in plant defense.ResultsSeventy-two RLCK-VII genes in the G. hirsutum genome were unveiled and classified into nine groups following their phylogenetic analysis with Arabidopsis thaliana. Group VII-1 was the largest, accounting for 28%, while Groups VII-2 and VII-3 had only one member each. The analysis using MCScanX revealed that these 72 genes formed 166 collinear gene pairs and were resided on 26 chromosomes of G. hirsutum, suggesting that they were derived from whole genome segmental duplication events. Their calculated Ka/Ks values were below one, implying the occurrence of purification selection during the evolution and inhibition of gene function differentiation/loss. All members of the RLCK-VII subfamily possessed two conserved domains, PKinase-Tyr and PKinase, and several conserved PBS1 kinase subdomains, individually included in one of the ten motifs identified using MEME. The RNA-Seq results showed that RLCK-VII genes exhibited different spatiotemporal expression, indicating their involvement in cotton growth, development, and defense responses to Verticillium dahliae. The transcription patterns of RLCK-VII genes found by RNA-Seq were further validated using qRT-PCR assays after inoculating “20B12” (cotton cultivar) with “V991” (V. dahliae). The virus-induced gene silencing (VIGS) assays uncovered that two RLCK-VII genes (Gohir.A13G227248 and Gohir.A10G219900) were essential to G. hirsutum resistance to Verticillium wilt.ConclusionsThese observations offer valuable insight into the attributes and roles of RLCK-VII genes in G. hirsutum, potentially enable the breeding of new cotton cultivars with enhanced resistance to Verticillium wilt.

Genome-Wide Identification and Expression Analysis of RLCK-VII Subfamily Genes Reveal Their Roles in Stress Responses of Upland Cotton.

Receptor-like cytoplasmic kinase VII (RLCK-VII) subfamily members are vital players in plant innate immunity and are also involved in plant development and abiotic stress tolerance. As a widely cultivated textile crop, upland cotton (Gossypium hirsutum) attaches great importance to the cotton industry worldwide. To obtain details of the composition, phylogeny, and putative function of RLCK-VII genes in upland cotton, genome-wide identification, evolutionary event analysis, and expression pattern examination of RLCK-VII subfamily genes in G. hirsutum were performed. There are 129 RLCK-VII members in upland cotton (GhRLCKs) and they were divided into nine groups based on their phylogenetic relationships. The gene structure and sequence features are relatively conserved within each group, which were divided based on their phylogenetic relationships, and consistent with those in Arabidopsis. The phylogenetic analysis results showed that RLCK-VII subfamily genes evolved in plants before the speciation of Arabidopsis and cotton, and segmental duplication was the major factor that caused the expansion of GhRLCKs. The diverse expression patterns of GhRLCKs in response to abiotic stresses (temperature, salt, and drought) and V. dahliae infection were observed. The candidates that may be involved in cotton's response to these stresses are highlighted. GhRLCK7 (GhRLCK7A and D), which is notably induced by V. dahliae infection, was demonstrated to positively regulate cotton defense against V. dahliae by the loss-of-function assay in cotton. These findings shed light on the details of the RLCK-VII subfamily in cotton and provide a scaffold for the further function elucidation and application of GhRLCKs for the germplasm innovation of cotton.

BIK1 protein homeostasis is maintained by the interplay of different ubiquitin ligases in immune signaling

Pathogen-associated molecular patterns (PAMPs) trigger plant innate immunity that acts as the first line of inducible defense against pathogen infection. A receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) functions as a signaling hub immediately downstream of multiple pattern recognition receptors (PRRs). It is known that PLANT U-BOX PROTEIN 25 (PUB25) and PUB26 ubiquitinate BIK1 and mediate BIK1 degradation. However, how BIK1 homeostasis is maintained is not fully understood. Here, we show that two closely related ubiquitin ligases, RING DOMAIN LIGASE 1 (RGLG1) and RGLG2, preferentially associate with the hypo-phosphorylated BIK1 and promote the association of BIK1 with the co-receptor for several PRRs, BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). PUB25 interacts with RGLG2 and mediates its degradation. In turn, RGLG2 represses the ubiquitin ligase activity of PUB25. RGLG1/2 suppress PUB25-mediated BIK1 degradation, promote BIK1 protein accumulation, and positively regulate immune signaling in a ubiquitin ligase activity-dependent manner. Our work reveals how BIK1 homeostasis is maintained by the interplay of different ubiquitin ligases.