Ubiquitination regulates pattern-triggered immune responses in plants, but the underlying mechanisms remain incompletely understood. In Arabidopsis, the E3 ubiquitin ligase PUB4 has been implicated in growth, development, and stress responses, and is known to interact with and be phosphorylated by CERK1, resulting in the positive regulation of chitin-triggered immunity. In this study, we identified ubiquitinated proteins enriched from chitin-treated microsomal fractions and discovered the receptor kinase FERONIA as a potential target of PUB4. FERONIA interacted with PUB4 both in vivo and in vitro, as evidenced by yeast two-hybrid, BiFC, and co-immunoprecipitation assays. An in vitro ubiquitination assay demonstrated that PUB4 ubiquitinates the cytoplasmic domain of FERONIA. Moreover, transient expression assays in wild-type and pub4-2 mutant protoplasts revealed that PUB4 mediates chitin-induced FERONIA degradation. Unlike PUB4, which positively regulates chitin-induced defense responses, FERONIA negatively regulates them. For example, fer4 mutants exhibited elevated basal and chitin-induced ROS and callose levels. This suggests that FERONIA suppresses immune signaling under steady-state conditions, and that PUB4-mediated degradation of FERONIA relieves this inhibition. These findings clearly reveal a novel mechanism by which PUB4 regulates chitin-induced immunity driven by CERK1 through targeted degradation of FERONIA. This highlights the balance between positive and negative regulators of plant immune signaling.

Spider mites (Tetranychus urticae) are a major threat to economically important crops. Here, we investigated the potential of tetranins, in particular Tet3 and Tet4, as T. urticae protein-type elicitors that stimulate plant defense. Truncated Tet3 and Tet4 proteins showed efficacy in activating the defense gene pathogenesis-related 1 (PR1) and inducing phytohormone production in leaves of Phaseolus vulgaris. In particular, Tet3 caused a drastically higher Ca2+ influx in leaves, but a lower reactive oxygen species (ROS) generation compared to other tetranins, whereas Tet4 caused a low Ca2+ influx and a high ROS generation in the host plants. Such specific and non-specific elicitor activities were examined by knockdown of Tet3 and Tet4 expressions in mites, confirming their respective activities and in particular showing that they function additively or synergistically to induce defense responses. Of great interest is the fact that Tet3 and Tet4 expression levels were higher in mites on their preferred host, P. vulgaris, compared to the levels in mites on the less-preferred host, Cucumis sativus, whereas Tet1 and Tet2 were constitutively expressed regardless of their host. Furthermore, mites that had been hosted on C. sativus induced lower levels of PR1 expression, Ca2+ influx and ROS generation, i.e., Tet3- and Tet4-responsive defense responses, in both P. vulgaris and C. sativus leaves compared to the levels induced by mites that had been hosted on P. vulgaris. Taken together, these findings show that selected tetranins respond to variable host cues that may optimize herbivore fitness by altering the anti-mite response of the host plant.

Plants defend themselves against herbivores by recognizing herbivore-derived elicitors and activating intracellular signaling. In Arabidopsis, the receptor-like kinase HAK1 recognizes the poly-saccharide elicitor (FrA) from Spodoptera litura larvae, leading to the expression of defense-related genes such as PDF1.2. During this process, the cytoplasmic kinase CRK2 phosphorylates PBL27, triggers the ERF13 expression via ethylene signaling and subsequently leads to PDF1.2 expression. Herein, we investigated four cytoplasmic kinases from the same receptor-like cytoplasmic kinase (RLCK) VII family as PBL27 that interacts with CRK2. Among them, PBL11, like PBL27, is phosphorylated by CRK2 and induces PDF1.2 expression but does not affect ERF13 expression. The weight gain of S. litura larvae on PBL11-deficient mutant plants was only slightly higher than that of wild-type plants, suggesting that PBL11 may function as a minor RLCK that supports the defense response.

JAV1-associated ubiquitin ligase 1 (JUL1) is a RING-type E3 ubiquitin ligase that catalyzes ubiquitination of JAV1, a jasmonate signaling repressor, in Arabidopsis thaliana in response to herbivore attack. Here we present a new insight into the nature of JUL1 as a multi-targeting enzyme for not only JAV1 but also transcription factors (TFs) screened using in vitro and in vivo protein interaction assays. Reporter assays using protoplasts showed that the JUL1-interacting TFs (JiTFs), including ERF15, bZIP53 and ORA59, were involved in transcriptional activation of jasmonate-responsive PDF1.2 and abscisic acid-responsive GEA6. Likewise, assays using mutant plants suggested that the 3 JiTFs were indeed responsible for transcriptional regulation of PDF1.2 and/or GEA6, and ERF15 and ORA59 were substantially responsible for the anti-herbivore trait. In vitro protein ubiqutination assays showed that JUL1 catalyzed ubiqutination of JAV1 but not any of the TFs. This was in accord with the finding that JUL1 abolished JAV1′s interference with ERF15 function, according to the reporter assay. Moreover, of great interest is our finding that ERF15 but not bZIP53 or ORA59 serves as a scaffold for the JAV1/JUL1 system, indicating that there is narrow selectivity of the transcriptional reprogramming by the JAV1/JUL1 system.

Plants defend against folivores by responding to folivore-derived elicitors following activation of signaling cascade networks. In Arabidopsis, HAK1, a receptor-like kinase, responds to polysaccharide elicitors (Frα) that are present in oral secretions of Spodoptera litura larvae to upregulate defense genes (e.g., PDF1.2) mediated through downstream cytoplasmic kinase PBL27. Here, we explored whether other protein kinases, including CPKs and CRKs, function with PBL27 in the intracellular signaling network for anti-herbivore responses. We showed that CRK2 and CRK3 were found to interact with PBL27, but CPKs did not. Although transcripts of PDF1.2 were upregulated in leaves of wild-type Arabidopsis plants in response to mechanical damage with Frα, this failed in CRK2- and PBL27-deficient mutant plants, indicating that the CRK2/PBL27 system is predominantly responsible for the Frα-responsive transcription of PDF1.2 in S. litura-damaged plants. In addition to CRK2-phosphorylated ERF13, as shown previously, ethylene signaling in connection to CRK2-phosphorylated PBL27 was predicted to be responsible for transcriptional regulation of a gene for ethylene response factor 13 (ERF13). Taken together, these findings show that CRK2 regulates not only ERF13 phosphorylation but also PBL27-dependent de novo synthesis of ERF13, thus determining active defense traits against S. litura larvae via transcriptional regulation of PDF1.2.

The nonexpressor of pathogenesis-related (NPR) gene family is well known to play a crucial role in transactivation of TGA transcription factors for salicylic acid (SA)-responsive genes, including pathogenesis-related protein 1 (PR1), during plants' immune response after pathogen attack in the model dicot Arabidopsis thaliana. However, little is known about NPR gene functions in monocots. We therefore explored the functions of NPRs in SA signaling in the model monocot Brachypodium distachyon. BdNPR1 and BdNPR2/3 share structural similarities with A. thaliana AtNPR1/2 and AtNPR3/4 subfamilies, respectively. The transcript level of BdNPR2 but not BdNPR1/3 appeared to be positively regulated in leaves in response to methyl salicylate. Reporter assays in protoplasts showed that BdNPR2 positively regulated BdTGA1-mediated activation of PR1. This transactivation occurred in an SA-dependent manner through SA binding at Arg468 of BdNPR2. In contrast, BdNPR1 functioned as a suppressor of BdNPR2/BdTGA1-mediated transcription of PR1. Collectively, our findings reveal that the TGA-promoted transcription of SA-inducible PR1 is orchestrated by the activator BdNPR2 and the repressor BdNPR1, which function competitively in B. distachyon.

Cell cultures established from various plant species have been used for a range of physiological and biochemical studies. Homogeneity of cell types and size of clusters in the cell culture often gave a clearer and simpler results compared to those obtained with the whole plant. On the other hand, possible variability of physiological conditions and responsiveness to external stimuli between the cell lines could be problematic for comparative studies. Aiming at combining the usefulness of plant cell culture with the rich information and genetic resources of Arabidopsis, we systemically examined the methods/conditions to establish cell lines for comparative studies, which could be applicable to a variety of genetic resources. Arabidopsis cell lines thus established from the meristem of mature seeds showed reproducible and comparable MAMP responses such as ROS generation and defense-related gene expression. MAMP responses of the cultured cells showed the specificity depending on the presence/absence of the corresponding MAMP receptor. Pharmacological study with a protein kinase inhibitor, K252a, also showed the usefulness of the cell culture for such studies. These results indicated the usefulness of the method to establish Arabidopsis cell lines, which are useful for comparative studies between genetic resources.

Plants respond to herbivory by perceiving herbivore danger signal(s) (HDS(s)), including “elicitors”, that are present in herbivores’ oral secretions (OS) and act to induce defense responses. However, little is known about HDS-specific molecules and intracellular signaling. Here we explored soybean receptor-like kinases (RLKs) as candidates that might mediate HDS-associated RLKs’ (HAKs’) actions in leaves in response to OS extracted from larvae of a generalist herbivore, Spodoptera litura . Fractionation of OS yielded Frα, which consisted of polysaccharides. The GmHAKs composed of their respective homomultimers scarcely interacted with Frα. Moreover, Arabidopsis HAK1 homomultimers interacted with cytoplasmic signaling molecule PBL27, resulting in herbivory resistance, in an ethylene-dependent manner. Altogether, our findings suggest that HAKs are herbivore-specific RLKs mediating HDS-transmitting, intracellular signaling through interaction with PBL27 and the subsequent ethylene signaling for plant defense responses in host plants.

Reactive oxygen species generation is one of the most popular index of plant immune responses. Leaf disk assay has been commonly used for MAMP/elicitor-induced ROS analysis by many groups. However, the reproducibility of the leaf disk assay relies on the skills of the people engaged in the experiments and the experiment itself seems not suitable for some plant species, which had a tough leaf structure and lower penetration efficiency of MAMPs/elicitors. In this study, we prepared a handmade leaf cutter to cut out the leaf fragments with uniform size and slits. The use of such fragments obtained by the new leaf cutter as well as the increase of the number of leaf fragments for each experiment improved the reliability and reproducibility of the leaf disk assay. This cutter was also successfully applied to rice leaf disk assay, indicating the applicability to other plant spices.

The two-spotted spider mite (Tetranychus urticae) is a plant-sucking arthropod herbivore that feeds on a wide array of cultivated plants. In contrast to the well-characterized classical chewing herbivore salivary elicitors that promote plant defense responses, little is known about sucking herbivores' elicitors. To characterize the sucking herbivore elicitors, we explored putative salivary gland proteins of spider mites by using an Agrobacterium-mediated transient expression system or protein infiltration in damaged bean leaves. Two candidate elicitors (designated as tetranin1 (Tet1) and tetranin2 (Tet2)) triggered early leaf responses (cytosolic calcium influx and membrane depolarization) and increased the transcript abundances of defense genes in the leaves, eventually resulting in reduced survivability of T. urticae on the host leaves as well as induction of indirect plant defenses by attracting predatory mites. Tet1 and/or Tet2 also induced jasmonate, salicylate and abscisic acid biosynthesis. Notably, Tet2-induced signaling cascades were also activated via the generation of reactive oxygen species. The signaling cascades of these two structurally dissimilar elicitors are mostly overlapping but partially distinct and thus they would coordinate the direct and indirect defense responses in host plants under spider mite attack in both shared and distinct manners.

Lysin motif (LysM) receptor-like kinase CERK1 is a co-receptor essential for plant immune responses against carbohydrate microbe-associated molecular patterns (MAMPs). Concerning the immediate downstream signaling components of CERK1, receptor-like cytoplasmic kinases such as PBL27 and other RLCK VII members have been reported to regulate immune responses positively. In this study, we report that a novel CERK1-interacting E3 ubiquitin ligase, PUB4, is also involved in the regulation of MAMP-triggered immune responses. Knockout of PUB4 resulted in the alteration of chitin-induced defense responses, indicating that PUB4 positively regulates reactive oxygen species generation and callose deposition but negatively regulates MAPK activation and defense gene expression. On the other hand, detailed analyses of a double knockout mutant of pub4 and sid2, a mutant of salicylic acid (SA) synthesis pathway, showed that the contradictory phenotype of the pub4 mutant was actually caused by abnormal accumulation of SA in this mutant and that PUB4 is a positive regulator of immune responses. The present and recent findings on the role of PUB4 indicate that PUB4 is a unique E3 ubiquitin ligase involved in the regulation of both plant immunity and growth/development.

While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1(cyt), showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GSTAtCERK1( S493D)(cyt), transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.

Lysin motif (LysM) receptor-like proteins/kinases (LysM-RLPs/RLKs) are well known to play an important role in the induction of defense or symbiosis signaling through the recognition of carbohydrate ligands in plants. Chitin elicitor receptor kinase 1 (CERK1) is the receptor-like kinase (RLK) essential for chitin-induced defense signaling in Arabidopsis and rice. At/OsCERK1 is also known to be important for peptidoglycan (PGN)-induced defense signaling. To induce chitin and PGN responses, CERK1 forms a receptor complex with corresponding LysM-type receptors such as CEBiP, LYM1/3 or LYP4/6. Furthermore, OsCERK1 in rice also plays a key role in lipopolysaccharide (LPS) signaling, and AtCERK1 in Arabidopsis is involved in unbranched β-glucan signaling. In any case, CERK1 plays a crucial role in the activation of defense signaling by these ligands and is an essential hub-RLK/co-receptor in these systems. After chitin perception, autophosphorylation of the CERK1 kinase domain is essential for inducing immune signaling, and some functionally important phosphorylation sites have been identified. In addition, several CERK1-interacting proteins and their contribution to the downstream signaling have been reported. In particular, phosphorylation of receptor-like cytoplasmic kinases (RLCKs) by CERK1 is important for the activation of chitin signaling. These findings have contributed to our understanding of the early steps of chitin signaling. This review focuses on current knowledge about CERK1-mediated receptor complex formation and subsequent intracellular signaling.

Plant cell surface receptor-like kinases (RLKs) mediate the signals from microbe-associated molecular patterns (MAMPs) that induce immune responses. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, is a common MAMP perceived by animals and plants; however, the plant receptors/co-receptors are unknown except for LORE, a bulb-type lectin S-domain RLK (B-lectin SD1-RLK) in Arabidopsis. OsCERK1 is a multifunctional RLK in rice that contains lysin motifs (LysMs) and is essential for the perception of chitin, a fungal MAMP, and peptidoglycan, a bacterial MAMP. Here, we analyzed the relevance of OsCERK1 to LPS perception in rice. Using OsCERK1-knockout mutants (oscerk1), we evaluated hydrogen peroxide (H2O2) production and gene expression after LPS treatment. We also examined the LPS response in knockout mutants for the B-lectin SD1-RLK genes in rice and for all LysM-protein genes in Arabidopsis. Compared with wild-type rice cells, LPS responses in oscerk1 cells were mostly diminished. By contrast, rice lines mutated in either of three B-lectin SD1-RLK genes and Arabidopsis lines mutated in the LysM-protein genes responded normally to LPS. From these results, we conclude that OsCERK1 is an LPS receptor/co-receptor and that the LPS perception systems of rice and Arabidopsis are significantly different.

Plants possess the ability to recognize microbe-associated molecular patterns (MAMPs) and PAMPs through the PRRs, and initiate pattern-triggered immunity. MAMPs are derived from cell-envelope components, secreted materials and cytosolic proteins from bacteria, oomycetes or fungi, and some MAMPs play a similar function in the innate immunity in mammals. Chitin is a representative fungal MAMP and triggers defense signaling in a wide range of plant species. The chitin receptors CEBiP and CERK1 on the plasma membrane have LysM (lysin motif) in their ectodomains. These molecules play an important role for the defense responses in rice and Arabidopsis, strictly recognizing the size and acetylated form of chitin oligosaccharides. However, related LysM receptors also play major roles for the signaling in root nodule and arbuscular mycorrhizal symbiosis. This review summarizes current knowledge on the molecular mechanisms of the defense and symbiosis signaling mediated by LysM receptors, including the activation steps of chitin-induced defense signaling downstream of LysM receptors.

Pattern recognition receptors on the plant cell surface mediate the recognition of microbe/damage-associated molecular patterns (MAMPs/DAMPs) and activate downstream immune signaling. Autophosphorylation of signaling receptor-like kinases is a critical event for the activation of downstream responses but the function of each phosphorylation site in the regulation of immune signaling is not well understood. In this study, 41 Ser/Thr/Tyr and 15 Ser/Thr residues were identified as in vitro and in vivo autophosphorylation sites of Arabidopsis CERK1, which is essential for chitin signaling. Comprehensive analysis of transgenic plants expressing mutated CERK1 genes for each phosphorylation site in the cerk1-2 background indicated that the phosphorylation of T479 in the activation segment and Y428 located upstream of the catalytic loop is important for the activation of chitin-triggered defense responses. Contribution of the phosphorylation of T573 to the chitin responses was also suggested. In vitro evaluation of kinase activities of mutated kinase domains indicated that the phosphorylation of T479 and T573 is directly involved in the regulation of kinase activity of CERK1 but the phosphorylation of Y428 regulates chitin signaling independently of the regulation of kinase activity. These results indicated that the phosphorylation of specific residues in the kinase domain contributes to the regulation of downstream signaling either through the regulation of kinase activity or the different mechanisms, e.g. regulation of protein-protein interactions.