Perception of root‐active CLE peptides requires CORYNE function in the phloem vasculature

Arabidopsis root development is orchestrated by signaling pathways that consist of different CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide ligands and their cognate CLAVATA (CLV) and BARELY ANY MERISTEM (BAM) receptors. How and where different CLE peptides trigger specific morphological or physiological changes in the root is poorly understood. Here, we report that the receptor‐like protein CLAVATA 2 (CLV2) and the pseudokinase CORYNE (CRN) are necessary to fully sense root‐active CLE peptides. We uncover BAM3 as the CLE45 receptor in the root and biochemically map its peptide binding surface. In contrast to other plant peptide receptors, we found no evidence that SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) proteins act as co‐receptor kinases in CLE45 perception. CRN stabilizes BAM3 expression and thus is required for BAM3‐mediated CLE45 signaling. Moreover, protophloem‐specific CRN expression complements resistance of the crn mutant to root‐active CLE peptides, suggesting that protophloem is their principal site of action. Our work defines a genetic framework for dissecting CLE peptide signaling and CLV/BAM receptor activation in the root.     

CLE genes may act in a variety of tissues/cells and involve other signaling cascades in addition to CLV3-WUS-like pathways

CLE, which is the term for the CLV3/ESR-related gene family, is thought to participate in CLAVATA3-WUSCHEL (CLV3-WUS) and CLV3-WUS-like signaling pathways to regulate meristem activity in plant. Although some CLE genes are expressed in meristems, many CLE genes appear to express in a variety of tissues/cells. Here we report that CLE14 and CLE20 express in various specific tissues/cells outside the shoot/root apical meristem (SAM/RAM), including in highly differentiated cells, and at different developmental stages. Overexpressing CLE14 or CLE20 also causes multiple phenotypes, which is consistent with its expression pattern in Arabidopsis. These results suggest that CLE genes may play multiple roles and involve other signaling cascades in addition to the CLV3-WUS and CLV3-WUS-like pathways.Key words: CLE, CLAVATA3-WUSCHEL, cell signaling and development, root apical meristem, arabidopsisIntercellular communication and coordination between adjacent cell populations are critical for cell-fate specification, as well as for meristem organization and maintenance. In the shoot apical meristem (SAM), local signaling, which involves the CLAVATA3-WUSCHEL (CLV3-WUS) negative feedback loop, controls stem cell homeostasis and SAM activity.¹ As well, it has been suggested that a CLV3-WUS-like negative feedback pathway operates to control root apical meristem (RAM) activity. This view is supported by the facts that a WUS-related homeobox gene, WOX5, is expressed in cells of the quiescent center (QC) in the RAM, and that loss-of-function of WOX5 in the QC leads to the differentiation of the adjacent root cap initials (RCI), whereas gain-of-function blocks the differentiation of derivatives of the RCI in the root.² Additional support for the function in the RAM of a CLV3-WUS-like pathway, comes from observations that CLE genes (collectively referred to as the CLV3/ESR-relate gene family) are not only expressed in the RAM,^3,4 but also, that overexpression of some CLE genes triggers premature termination of the RAM.⁵ In this regard it has been recently reported that CLE40, which expresses in the differentiating daughter cells of the distal root stem cells, restricts WOX5 expression and promotes differentiation of stem cells in the RAM.⁶ Taken together these data suggest a CLV3-WUS-like feedback loop acts to negatively regulate RAM activity in plants.Our previous results have shown that CLE14 and CLE20 express in specific cells of roots, and that overexpression of CLE14 or CLE20 in Arabidopsis triggers early termination of the RAM in a CLAVATA1 (CLV1)-independent, but CLAVATA2 (CLV2)-dependent manner.^7,8 We also showed that both CLE14 and CLE20 peptides inhibit, irreversibly, root growth by reducing cell division rates in the RAM.⁷ CLV2 and CRN (a receptor-like protein kinase, also known as SOL2, isolated as a suppressor of root-specific overexpression of CLE19) are required for CLE14 and CLE20 peptide functions in vitro.^9,10 Using computational modeling approaches we further demonstrated that 12-amino-acid CLE14 and CLE20 peptides may function through a potential heterodimer/heterotetramer CLV2-CRN complex.⁷CLV3 expresses exclusively in the stem cells of the SAM, and it has been consistently shown that the CLV3 peptide is required for homeostasis of the stem cells and for the maintenance of the SAM.¹ Although some CLE genes are found to express in meristems, many CLE genes appear to express in an array of tissues and cells, including highly differentiated tissues/cells.^3,4 In this report we show that CLE14 and CLE20 express in specific tissues outside the RAM and SAM of Arabidopsis, including highly differentiated cells, and at different developmental stages. Overexpressing CLE14 or CLE20 also causes multiple phenotypes, which is consistent with its expression pattern in Arabidopsis. These results suggest that CLE genes may play multiple roles in regulating the developmental fate of cells, which includes, but is not limited to, stem cells, and also may be involved in other signaling cascades in addition to the CLV3-WUS pathway.     

Nematode CLE signaling in Arabidopsis requires CLAVATA2 and CORYNE

Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ESR (CLE)-like effector proteins. These proteins have been shown to act as ligand mimics of plant CLE peptides and are required for successful nematode infection; however, the receptors for nematode CLE-like peptides have not been identified. Here we demonstrate that CLV2 and CORYNE (CRN), members of the receptor kinase family, are required for nematode CLE signaling. Exogenous peptide assays and overexpression of nematode CLEs in Arabidopsis demonstrated that CLV2 and CRN are required for perception of nematode CLEs. In addition, promoter-reporter assays showed that both receptors are expressed in nematode-induced syncytia. Lastly, infection assays with receptor mutants revealed a decrease in both nematode infection and syncytium size. Taken together, our results indicate that perception of nematode CLEs by CLV2 and CRN is not only required for successful nematode infection but is also involved in the formation and/or maintenance of nematode-induced syncytia.     

Evidence for functional conservation, sufficiency, and proteolytic processing of the CLAVATA3 CLE domain

Arabidopsis (Arabidopsis thaliana) CLAVATA3 (CLV3) is hypothesized to act as a ligand for the CLV1 receptor kinase in the regulation of stem cell specification at shoot and flower meristems. CLV3 is a secreted protein, with an amino-terminal signal sequence and a conserved C-terminal domain of 15 amino acids, termed the CLE (CLV3/ESR-related) domain, based on its similarity to a largely unstudied protein family broadly present in land plants. We have tested the function of 13 Arabidopsis CLEs in vivo and found a significant variability in the ability of CLEs to replace CLV3, ranging from complete to no complementation. The best rescuing CLE depends on CLV1 for function, while other CLEs act independently of CLV1. Domain-swap experiments indicate that differences in function can be traced to the CLE domain within these proteins. Indeed, when the CLE domain of CLV3 is placed downstream of an unrelated signal sequence, it is capable of fully replacing CLV3 function. Finally, we have detected proteolytic activity in extracts from cauliflower (Brassica oleracea) that process both CLV3 and CLE1 at their C termini. For CLV3, processing appears to occur at the absolutely conserved arginine-70 found at the beginning of the CLE domain. We propose that CLV3 and other CLEs are C-terminally processed to generate an active CLE peptide.     

CLE14/CLE20 peptides may interact with CLAVATA2/CORYNE receptor-like kinases to irreversibly inhibit cell division in the root meristem of Arabidopsis

Towards an understanding of the interacting nature of the CLAVATA (CLV) complex, we predicted the 3D structures of CLV3/ESR-related (CLE) peptides and the ectodomain of their potential receptor proteins/kinases, and docking models of these molecules. The results show that the ectodomain of CLV1 can form homodimers and that the 12-/13-amino-acid CLV3 peptide fits into the binding clefts of the CLV1 dimers. Our results also demonstrate that the receptor domain of CORYNE (CRN), a recently identified receptor-like kinase, binds tightly to the ectodomain of CLV2, and this likely leads to an increased possibility for docking with CLV1. Furthermore, our docking models reveal that two CRN-CLV2 ectodomain heterodimers are able to form a tetramer receptor complex. Peptides of CLV3, CLE14, CLE19, and CLE20 are also able to bind a potential CLV2-CRN heterodimer or heterotetramer complex. Using a cell-division reporter line, we found that synthetic 12-amino-acid CLE14 and CLE20 peptides inhibit, irreversibly, root growth by reducing cell division rates in the root apical meristem, resulting in a short-root phenotype. Intriguingly, we observed that exogenous application of cytokinin can partially rescue the short-root phenotype induced by over-expression of either CLE14 or CLE20 in planta. However, cytokinin treatment does not rescue the short-root phenotype caused by exogenous application of the synthetic CLE14/CLE20 peptides, suggesting a requirement for a condition provided only in living plants. These results therefore imply that the CLE14/CLE20 peptides may act through the CLV2-CRN receptor kinase, and that their availabilities and/or abundances may be affected by cytokinin activity in planta.     

CLE25 peptide regulates phloem initiation in Arabidopsis through a CLERK‐CLV2 receptor complex

The phloem, located within the vascular system, is critical for delivery of nutrients and signaling molecules throughout the plant body. Although the morphological process and several factors regulating phloem differentiation have been reported, the molecular mechanism underlying its initiation remains largely unknown. Here, we report that the small peptide‐coding gene, CLAVATA 3 (CLV3)/EMBEYO SURROUNDING REGION 25 (CLE25), the expression of which begins in provascular initial cells of 64‐cell‐staged embryos, and continues in sieve element‐procambium stem cells and phloem lineage cells, during post‐embryonic root development, facilitates phloem initiation in Arabidopsis. Knockout of CLE25 led to delayed protophloem formation, and in situ expression of an antagonistic CLE25_G6T peptide compromised the fate‐determining periclinal division of the sieve element precursor cell and the continuity of the phloem in roots. In stems of CLE25_G6T plants the phloem formation was also compromised, and procambial cells were over‐accumulated. Genetic and biochemical analyses indicated that a complex, consisting of the CLE‐RESISTANT RECEPTOR KINASE (CLERK) leucine‐rich repeat (LRR) receptor kinase and the CLV2 LRR receptor‐like protein, is involved in perceiving the CLE25 peptide. Similar to CLE25, CLERK was also expressed during early embryogenesis. Taken together, our findings suggest that CLE25 regulates phloem initiation in Arabidopsis through a CLERK‐CLV2 receptor complex.     

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

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

The Involvement of Mammalian and Plant FK506-Binding Proteins (FKBPs) in Development

The FK506-binding proteins (FKBPs) are peptidyl prolyl cis/trans isomerases and the information gathered in the last 10 years reveals their involvement in diverse biological systems affecting the function and structure of target proteins. Members of the FKBP family were shown to be growth-regulated and participate in signal transduction. In this review we have chosen to focus on a few examples of the mammalian and plant systems in which members of the FKBP family have been demonstrated to affect the function of proteins or development. The technologies that enable production of knockout mice, Arabidopsis mutants and overexpression in transgenic organisms have revealed the contribution of FKBP to development in higher eukaryotes. It appears that members of the FKBP family have conserved some of their basic functions in the animal and plant kingdom, whereas other functions became unique. Studies that will take advantage of the full genome sequence available for Arabidopsis and the human genome, DNA chip technologies and the use of transgenic complementation system will contribute to the elucidation of the molecular mechanism and biological function of FKBPs.     

CLE polypeptide signaling gene expression in Arabidopsis embryos

The CLAVATA3 (CLV3)/ESR-related (CLE) family of small polypeptides mediate intercellular signaling events in plants. The biological roles of several CLE family members have been characterized, but the function of the majority still remains elusive. We recently performed a systematic expression analysis of 23 Arabidopsis CLE genes to gain insight into the developmental processes they may potentially regulate during vegetative and reproductive growth. Our study revealed that each Arabidopsis tissue expresses one or more CLE genes, suggesting that they might play roles in many developmental and/or physiological processes. Here we determined the expression patterns of nine Arabidopsis CLE gene promoters in mature embryos and compared them to the known expression patterns in seedlings. We found that more than half of these CLE genes have similar expression profiles at the embryo and seedling stages, whereas the rest differ dramatically. The implications of these findings in understanding the biological processes controlled by these CLE genes are discussed.Key words: arabidopsis, CLE, embryo, polypeptide, signalingThe CLE genes encode small, secreted polypeptides characterized by a highly conserved 14 amino-acid region at their carboxyl termini called the CLE domain.¹ To date 32 family members have been identified in Arabidopsis, yet only three have been assigned functions: CLV3, CLE40 and CLE41 have been implicated in stem cell homeostasis in shoot, root and vascular meristems, respectively.^2–5 Overexpression studies indicated that CLE genes may regulate additional biological processes as diverse as root and shoot growth, phyllotaxis, apical dominance and leaf shape and size control.^6,7 This hypothesis is consistent with our recent expression analysis of Arabidopsis A-type CLE genes,⁸ in which we found that all examined tissues expressed one or more CLE genes, in overlapping patterns. Each CLE promoter exhibited a highly distinct and specific activity profile, and many showed complex expression dynamics during vegetative and reproductive growth.Consistent with their roles in meristem maintenance, CLV3 and CLE40 are expressed early in embryogenesis when meristem initiation and organization take place.^3,5 Yet there are no other reports of CLE gene expression in Arabidopsis embryos, and therefore it is not known to what extent this family of small peptides regulates intercellular signaling events during embryogenesis. We addressed this question by analyzing the expression patterns of selected CLE promoters in mature embryos and compared them with those in 11-day-old seedlings. We chose nine CLE genes whose promoters are active in different tissues of the seedling.⁸ Transgenic dried seeds carrying a single CLE promoter sequence driving the expression of the uidA reporter gene were imbibed in water for four days, the embryos dissected out of their seed coats, and beta-glucuronidase (GUS) reporter assays performed.⁹ Stained embryos were cleared with chloral hydrate¹⁰ and visualized using a Zeiss Axiophot microscope.Five of the CLE genes analyzed showed similar promoter expression patterns in mature embryos and in seedlings. In embryos, the CLE11, 13, 16 and 17 promoters drove GUS activity in specific patterns in the root. CLE11 and CLE13 promoter activity was detected in the root cap and root apical meristem (Fig. 1A and B), CLE16 promoter activity was observed in the root cap and above the root apical meristem (Fig. 1C), and CLE17 promoter activity was seen weakly in the root apical meristem (Fig. 1D). Each of these CLE genes exhibited a similar expression pattern in seedling roots.⁸ CLE17 was additionally expressed in the embryo shoot apex and at the cotyledon margins (Fig. 1D). Similarly, in seedlings CLE17 was expressed in the vegetative shoot apex, and at the margins of the cotyledons and fully expanded leaves.⁸ In embryos, CLE27 promoter activity was strong in the hypocotyl, as well as in the medial region of the cotyledons along the main vein (Fig. 1E). In seedlings, CLE27 was strongly expressed in the hypocotyl and exhibited patchy expression in both cotyledons and leaves.⁸ Our analysis reveals that the expression of these CLE genes is established early during development and remains constant at later stages, suggesting that they may perform the same function throughout the Arabidopsis life cycle.Open in a separate windowFigure 1GUS reporter activity driven by the promoters of (A) CLE11, (B) CLE13, (C) CLE16, (D) CLE17, (E) CLE27, (F) CLE1, (G) CLE12, (H) CLE18 and (I) CLE25 in mature Arabidopsis embryos. Arrowhead indicates GUS activity in the root cap and the arrow indicates GUS activity in the root apical meristem. Scale bar, 100 µm.Remarkably, the other four CLE promoters drove embryo expression patterns that were strongly divergent from what was observed in seedlings. We found that the CLE1 promoter was active in the embryo throughout the hypocotyl and in the central region of the cotyledons (Fig. 1F), but was observed in seedlings solely in the vasculature of fully differentiated roots and at the root tips.⁸ CLE12 promoter activity in embryos was observed throughout the hypocotyl and the cotyledons (Fig. 1G), whereas in seedlings it was detected weakly in the leaf vasculature and more strongly in the root vasculature.⁸ In contrast, the CLE18 and CLE25 promoters did not drive reporter activity in mature embryos (Fig. 1H and I), despite being broadly and strongly expressed in seedlings.⁸These four CLE gene promoters show dynamic shifts in their activity between different developmental stages. From our data we infer that CLE1 activity in hypocotyls and cotyledons is required solely during embryogenesis, and that the gene then acquires a distinct function in post-embryonic root development. Similarly CLE12 appears to acquire a post-embryonic function in the root vasculature, and its broad activity in the embryonic leaves becomes restricted to the leaf vasculature following germination. Finally, the absence of CLE18 and CLE25 promoter activity in mature embryos suggests that they may be dispensable for embryo formation, and might either specifically regulate post-embryonic signaling events in certain tissues or be involved in mediating responses to environmental stimuli to which embryos are not subjected. Alternatively, they may be expressed earlier during embryogenesis and become repressed during seed dormancy.Our spatio-temporal expression analysis of a small group of CLE genes in mature embryos and seedlings indicates that the majority of these signaling molecules exert their roles beginning early in development, potentially contributing to tissue patterning and organization. Yet whereas some appear to contribute to the same biological processes throughout the plant life cycle, others seem to function in different tissues at different developmental stages. In addition, each CLE promoter studied here is active in vegetative and/or reproductive tissues that are not present in embryos, such as trichomes (CLE16 and CLE17) and style (CLE1).⁸ This observation suggests that CLE genes are widely recruited to new tissue-specific signaling functions during the course of plant development.     

Identification and comprehensive analysis of the CLV3/ESR‐related (CLE) gene family in Brassica napus L.

• The CLE (CLAVATA3/ESR) gene family, encoding a group of small secretory peptides, plays important roles in cell‐to‐cell communication, thereby controlling a broad spectrum of development processes. The CLE family has been systematically characterized in some plants, but not in Brassica napus.
• In the present study, 116 BnCLE genes were identified in the B. napus genome, including seven unannotated, six incorrectly predicted and five multi‐CLE domain‐encoding genes. These BnCLE members were separated into seven distinct groups based on phylogenetic analysis, which might facilitate the functional characterization of the peptides.
• Further characterization of CLE pre‐propeptides revealed 31 unique CLE peptides from 45 BnCLE genes, which may give rise to distinct roles of BnCLE and expansion of the gene family. The biological activity of these unique CLE dodecamer peptides was tested further through in vitro peptide assays. Variations in several important residues were identified as key contributors to the functional differentiation of BnCLE and expansion of the gene family in B. napus. Expression profile analysis helped to characterize possible functional redundancy and sub‐functionalization among the BnCLE members.
• This study presents a comprehensive overview of the CLE gene family in B. napus and provides a foundation for future evolutionary and functional studies.

     

The 14-amino acid CLV3, CLE19, and CLE40 peptides trigger consumption of the root meristem in Arabidopsis through a CLAVATA2-dependent pathway

CLAVATA3 (CLV3), CLV3/ESR19 (CLE19), and CLE40 belong to a family of 26 genes in Arabidopsis thaliana that encode putative peptide ligands with unknown identity. It has been shown previously that ectopic expression of any of these three genes leads to a consumption of the root meristem. Here, we show that in vitro application of synthetic 14-amino acid peptides, CLV3p, CLE19p, and CLE40p, corresponding to the conserved CLE motif, mimics the overexpression phenotype. The same result was observed when CLE19 protein was applied externally. Interestingly, clv2 failed to respond to the peptide treatment, suggesting that CLV2 is involved in the CLE peptide signaling. Crossing of the CLE19 overexpression line with clv mutants confirms the involvement of CLV2. Analyses using tissue-specific marker lines revealed that the peptide treatments led to a premature differentiation of the ground tissue daughter cells and misspecification of cell identity in the pericycle and endodermis layers. We propose that these 14-amino acid peptides represent the major active domain of the corresponding CLE proteins, which interact with or saturate an unknown cell identity-maintaining CLV2 receptor complex in roots, leading to consumption of the root meristem.     

CLAVATA3-like genes are differentially expressed in grape vine (Vitis vinifera) tissues

The CLAVATA3 (CLV3)/endosperm surrounding region [(ESR) CLE] peptides function as intercellular signaling molecules that regulate various physiological and developmental processes in diverse plant species. We identified five CLV3-like genes from grape vine (Vitis vinifera var. Pinot Noir): VvCLE 6, VvCLE 25-1, VvCLE 25-2, VvCLE 43 and VvCLE TDIF. These CLV3-like genes encode short proteins containing 43–128 amino acids. Except VvCLE TDIF, grape vine CLV3-like proteins possess a consensus amino acid sequence known as the CLE domain. Phylogenic analysis suggests that the VvCLE 6, VvCLE25-1, VvCLE25-2 and VvCLE43 genes have evolved from a single common ancestor to the Arabidopsis CLV3 gene. Expression analyses showed that the five grape CLV3-like genes are expressed in leaves, stems, roots and axillary buds with significant differences in their levels of expression. For example, while all of them were strongly expressed in axillary buds, VvCLE6 and VvCLE43 expression prevailed in roots, and VvCLE25-1, VvCLE25-2 and VvCLE TDIF expression in stems. The differential expression of the five grape CLV3-like peptides suggests that they play different roles in different organs and developmental stages.     

A large family of genes that share homology with CLE domain in Arabidopsis and rice

Thirty-one CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) proteins are encoded in the Arabidopsis genome, and they are supposed to function as dodecapeptides with two hydroxyproline residues. Twenty-six synthetic CLE peptides, corresponding to the predicted products of the 31 CLE genes, were examined in Arabidopsis and rice. Nineteen CLE peptides induced root meristem consumption, resulting in the short root phenotype in Arabidopsis and rice, whereas no CLE peptides affected the shoot apical meristem in rice. Database searches revealed 47 putative CLE genes in the rice genome. Three of the rice CLE genes, OsCLE502, OsCLE504 and OsCLE506, encode CLE proteins with multiple CLE domains, which are not found in the Arabidopsis genome, and polyproline region was found between these CLE domains. These results indicate conserved and/or diverse CLE functions in each plant species.Key words: CLE, CLAVATA, meristem, SAM, RAM, peptideIntercellular communication is a fundamental mechanism for coordinating the development of complex bodies of multicellular organisms such as plants and animals. Peptide signaling in plants has been largely overlooked for many years, despite the importance of peptide signaling in animals, yeast and other organisms. The recent identification of several peptide hormones indicated the importance of cell-cell communication^1,2 in defense responses,³ cell proliferation,⁴ cell differentiation,⁵ shoot apical meristem (SAM) size regulation,⁶ self-incompatibility in crucifer species,⁷ and stomatal patterning.⁸CLAVATA3 (CLV3), and tracheary element differentiation inhibitory factor (TDIF) were shown to be involved as CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) members, and they function as dodecapeptides.^5,6 Chemically biosynthesized peptides could be a powerful tool for examining the CLE peptide functions. Twenty-six putative CLE peptides encoded in the Arabidopsis genome were investigated. Nineteen CLE peptides functioned not only in Arabidopsis but also in rice to reduce root apical meristem (RAM) size, whereas no Arabidopsis peptides affected rice SAM size in our assay system. However, 10 CLE peptides exhibited a strong effect on the Arabidopsis SAM.⁹ This may indicate that the CLE peptides function less redundantly in the SAM than in the RAM, and that some Arabidopsis CLE peptides can bind less effectively to rice receptors due to the sequence differences between Arabidopsis and rice.Sequencing of the rice genome has finished, and rice genes encoding putative CLE domains were database searched from RAP-DB (http://rapdb.lab.nig.ac.jp) using the Arabidopsis CLE sequences of 12 amino acid residues as queries. The resulting rice sequences were used as queries to repeat the step in an iterative manner. The search was terminated when no novel sequences were retrieved. A total of 47 putative CLE genes were found in the database search (Fig. 1).Open in a separate windowFigure 1Alignment of the deduced polypeptides of the rice CLE gene family. The conserved dodecapeptide CLE region is boxed, and a CLE-related 13 amino acid sequence in OsCLE505 is underlined. Proline rich region is shown in gray. N-terminal 31 amino acid residues of CLE506, MSSISYFLVAMLLCN GFGFIVSAQVVGGGSS, are not shown because of limited space.The coding regions of CLV3 and CLE40 genes are interrupted by two introns in Arabidopsis,¹⁰ and the application of these synthetic peptides to wild type plants induced SAM consumption. In rice, nine CLE genes, OsCLE201, OsCLE305, OsCLE402, OsCLE502, OsCLE506, OsCLE507, OsCLE47, OsCLE603 and FON2/FON4, have multiple exons. FON2/FON4 has been reported to regulate floral meristem size in a similar manner to the CLV3 in Arabidopsis,^11,12 and another eight CLE genes might be involved in meristem size regulation.Three rice CLE genes, OsCLE502, OsCLE504 and OsCLE506, encode CLE proteins carrying multiple CLE domains (Fig. 1), although no Arabidopsis gene encodes such a CLE protein. In addition to the results described in Kinoshita et al.,⁹ four more putative CLE domains, OsCLE502C (REVPSGPDPITS), OsCLE502D (REVPSGPDPITS), OsCLE502E (RKVHHKALGIAS) and OsCLE506F (RLTPIGPDPIHN), were found in these rice CLEs. The two sequence-related rice CLE genes, CLE504 and CLE505, are located at interval of no more than about 2 kb on chromosome 5, suggesting that they have arisen by local gene duplication events. However, OsCLE504 has two putative CLE domains, whereas OsCLE505 has one apparent CLE domain and another CLE-like motif composed of an unusual 13 amino acid residues, HDVPSSGPSPVHN, which should correspond to OsCLE504A (Fig. 1). The CLE-like peptide might not function any longer as a CLE peptide due to an additional amino acid. It is possible that these sister CLE genes might acquire different functions during their evolutionary steps. OsCLE506 encodes six CLE domains and it might function in rice-specific physiological events, but not in morphological events, because the transgenic plants harboring the RNAi construct of the OsCLE506 gene did not show any obvious morphological abnormalities (Sawa et al., unpublished results).Most of the conserved CLE domains were located at or near the C-terminal end of the sequences we identified, but every CLE domain located at the middle region of the OsCLE502, OsCLE504 and OsCLE506 proteins was followed by an acidic amino acid residue and a characteristic polyproline region (Fig. 1). The polyproline sequence often adopts a rigid, rod-like secondary structure called as a polyproline type II helix, which is capable of serving as a protein-protein interaction domain or organizing unfolded polypeptides.^13,14 In the case of the yeast peptide pheromone, alpha factor, four copies of 13 amino acid peptides are produced from one precursor.¹⁵ The KEX2-encoded endoprotease cleaves the precursor after pairs of basic residues such as lysine and arginine and these basic residues are chopped out by a carboxypeptidase, KEX1.¹³ Thus, CLE proteins with multiple CLE domains might be precursors and require a similar maturation steps. In this context, the polyproline region might serve as or provide a recognition site for the first endoprotease. Many CLE precursors have lysine and/or arginine residues just after the CLE domain, and this also supports the idea that carboxypeptidases are responsible for the C-terminal maturation step.CLE proteins are currently one of the best described families of small polypeptides in plants; however, precise molecular details, such as CLE peptide maturation, movement, reception and signaling in a target cell, remain to be solved. Further genetic and biochemical analyses of the CLE family would give insights to help unveil not only the molecular mechanisms, but also the diversity and evolution of intercellular communication in plants.     

Antagonistic Peptide Technology for Functional Dissection of CLV3/ESR Genes in Arabidopsis

In recent years, peptide hormones have been recognized as important signal molecules in plants. Genetic characterization of such peptides is challenging since they are usually encoded by small genes. As a proof of concept, we used the well-characterized stem cell-restricting CLAVATA3 (CLV3) to develop an antagonistic peptide technology by transformations of wild-type Arabidopsis (Arabidopsis thaliana) with constructs carrying the full-length CLV3 with every residue in the peptide-coding region replaced, one at a time, by alanine. Analyses of transgenic plants allowed us to identify one line exhibiting a dominant-negative clv3-like phenotype, with enlarged shoot apical meristems and increased numbers of floral organs. We then performed second dimensional amino acid substitutions to replace the glycine residue individually with the other 18 possible proteinaceous amino acids. Examination of transgenic plants showed that a glycine-to-threonine substitution gave the strongest antagonistic effect in the wild type, in which over 70% of transgenic lines showed the clv3-like phenotype. Among these substitutions, a negative correlation was observed between the antagonistic effects in the wild type and the complementation efficiencies in clv3. We also demonstrated that such an antagonistic peptide technology is applicable to other CLV3/EMBRYO SURROUNDING REGION (CLE) genes, CLE8 and CLE22, as well as in vitro treatments. We believe this technology provides a powerful tool for functional dissection of widely occurring CLE genes in plants.In animals, small peptides are important signal molecules in neural and endocrinal systems (Feld and Hirschberg, 1996; Edlund and Jessell, 1999). In recent years, over a dozen different types of peptide hormones have been identified in plants, regulating both developmental and adaptive responses, usually through interacting with Leu-rich repeat receptor kinases localized in plasma membranes of neighboring cells (Boller and Felix, 2009; De Smet et al., 2009; Katsir et al., 2011). These peptides are often produced from genes with small open reading frames, after posttranslational processing (Matsubayashi, 2011). In addition, peptide hormones, such as CLAVATA3/EMBRYO SURROUNDING REGION (CLV3/ESR [CLE]), systemin, PHYTOSULFOKINE, AtPEP1, and EPIDERMAL PATTERNING FACTOR1 (EPF1), often have paralogs in genomes (Cock and McCormick, 2001; Yang et al., 2001; Pearce and Ryan, 2003; Huffaker et al., 2007; Hara et al., 2007). Bioinformatics analyses revealed that the Arabidopsis (Arabidopsis thaliana) genome contains 33,809 small open reading frames (Lease and Walker, 2006).CLV3 acts as a secreted 12- or 13-amino acid glycosylated peptide (Kondo et al., 2006; Ohyama et al., 2009) to restrict the number of stem cells in shoot apical meristems (SAMs), through a CLV1-CLV2-SOL2 (for SUPPRESSOR OF LLP1 2, also called CORYNE)-RECEPTOR-LIKE PROTEIN KINASE2 (RPK2) receptor kinase-mediated pathway (Clark et al., 1993; Jeong et al., 1999; Miwa et al., 2008; Müller et al., 2008; Kinoshita et al., 2010; Zhu et al., 2010). All CLE family members, of which there are 83 in Arabidopsis and 89 in rice (Oryza sativa), carry a putative signal peptide and share a conserved 12-amino acid core CLE motif (Oelkers et al., 2008). Overexpression of CLE genes often shows a common dwarf and short-root phenotype (Strabala et al., 2006; Jun et al., 2010), which may not reflect their endogenous functions. Due to redundancies and difficulties in identifying mutants of these small genes, studies of CLE members are challenging. Only a few CLE genes have been genetically characterized, in particular, CLV3, CLE8, CLE40, and CLE41 in Arabidopsis and FLORAL ORGAN NUMBER4 (FON4), FON2-LIKE CLE PROTEIN1 (FCP1), and FON2 SPARE1 in rice (Fletcher et al., 1999; Hobe et al., 2003; Chu et al., 2006; Suzaki et al., 2008, 2009; Etchells and Turner, 2010; Fiume and Fletcher, 2012), while functions of other CLE members remain unknown.As a proof of concept, we used the well-characterized CLV3 gene to develop an antagonistic peptide technology for functionally dissecting CLE family members in Arabidopsis. A series of constructs carrying Ala substitutions in every amino acid residue in the core CLE motif of CLV3, expressed under the endogenous CLV3 regulatory elements, were made and introduced to wild-type Arabidopsis by transformation. This allowed us to identify the conserved Gly residue in the middle of the CLE motif was vulnerable for generating the dominant-negative clv3-like phenotype. We then performed second dimensional amino acid substitutions to replace the Gly with all other 18 possible proteinaceous amino acids, one at a time, and observed that the substitution of the Gly residue by Thr generated the strongest dominant-negative clv3-like phenotype. Further experiments showed that this technology can potentially be applied to in vitro-synthesized peptides and for functional characterization of other CLE members.     

In Planta Processing and Glycosylation of a Nematode CLAVATA3/ENDOSPERM SURROUNDING REGION-Like Effector and Its Interaction with a Host CLAVATA2-Like Receptor to Promote Parasitism

Like other biotrophic plant pathogens, plant-parasitic nematodes secrete effector proteins into host cells to facilitate infection. Effector proteins that mimic plant CLAVATA3/ENDOSPERM SURROUNDING REGION-related (CLE) proteins have been identified in several cyst nematodes, including the potato cyst nematode (PCN); however, the mechanistic details of this cross-kingdom mimicry are poorly understood. Plant CLEs are posttranslationally modified and proteolytically processed to function as bioactive ligands critical to various aspects of plant development. Using ectopic expression coupled with nanoliquid chromatography-tandem mass spectrometry analysis, we show that the in planta mature form of proGrCLE1, a multidomain CLE effector secreted by PCN during infection, is a 12-amino acid arabinosylated glycopeptide (named GrCLE1-1Hyp4,7g) with striking structural similarity to mature plant CLE peptides. This glycopeptide is more resistant to hydrolytic degradation and binds with higher affinity to a CLAVATA2-like receptor (StCLV2) from potato (Solanum tuberosum) than its nonglycosylated forms. We further show that StCLV2 is highly up-regulated at nematode infection sites and that transgenic potatoes with reduced StCLV2 expression are less susceptible to PCN infection, indicating that interference of the CLV2-mediated signaling pathway confers nematode resistance in crop plants. These results strongly suggest that phytonematodes have evolved to utilize host cellular posttranslational modification and processing machinery for the activation of CLE effectors following secretion into plant cells and highlight the significance of arabinosylation in regulating nematode CLE effector activity. Our finding also provides evidence that multidomain CLEs are modified and processed similarly to single-domain CLEs, adding new insight into CLE maturation in plants.Plants are vulnerable to attack by plant-parasitic nematodes. The cyst-forming endoparasitic nematodes (Globodera and Heterodera spp.) are among the most damaging plant pathogens, causing tremendous crop losses globally (Chitwood, 2003). Cyst nematodes have evolved an intimate parasitic relationship with their hosts by transforming normal root cells into a unique feeding structure called a syncytium that serves as the sole nutritive source required for subsequent growth and development (Hussey and Grundler, 1998; Davis et al., 2004). Cyst nematodes are soil-borne pathogens. Once infective juveniles hatch in the soil, they penetrate into the roots of host plants and select a single cell near the root vasculature to initiate a syncytium. The syncytium forms by the fusion of cells adjacent to the initial syncytial cell through extensive cell wall dissolution and develops into a large fused cell that is highly metabolically active and contains numerous enlarged nuclei and nucleoli (Endo, 1964). Like other plant pathogens, cyst nematodes use secreted effector proteins to facilitate plant parasitism. Effector proteins, originating from the nematode esophageal gland cells (two subventral and one dorsal) and secreted into root tissues through the nematode stylet (a mouth spear), represent important molecular signals that manipulate various host cellular processes to redifferentiate normal root cells into a syncytium (Davis et al., 2004; Mitchum et al., 2008, 2013).Genes encoding effector proteins with sequence similarity to plant CLAVATA3/ENDOSPERM SURROUNDING REGION-related (CLE) proteins have recently been cloned from several cyst nematode species, including the potato cyst nematode (PCN [Globodera rostochiensis; Gr]; Wang et al., 2001, 2011; Gao et al., 2003; Lu et al., 2009), a regulated and devastating pest of potato (Solanum tuberosum [St]) and tomato (Solanum lycopersicum) crops. Plant CLE proteins, identified from diverse monocot and dicot species (Cock and McCormick, 2001; Oelkers et al., 2008), are a class of peptide hormones that regulate many aspect of plant growth and development (Yamada and Sawa, 2013). Plant CLE genes encode small proteins that contain an N-terminal signal peptide, an internal variable domain, and either a single or multiple conserved C-terminal CLE domain(s) (Cock and McCormick, 2001; Kinoshita et al., 2007; Oelkers et al., 2008). The Arabidopsis (Arabidopsis thaliana [At]) genome encodes at least 32 single-domain CLEs, of which CLAVATA3 (CLV3) is the best characterized member. CLV3 is found to interact with three major membrane-associated receptor complexes, CLV1, CLV2-CORYNE (CRN), and RECEPTOR LIKE PROTEIN KINASE2 (RPK2; Clark et al., 1993; Jeong et al., 1999; Müller et al., 2008; Kinoshita et al., 2010; Zhu et al., 2010), to control the fate of stem cells in the shoot apical meristem (Fletcher et al., 1999). Among the three CLV3 receptors, CLV1 and RPK2 are leucine-rich repeat (LRR) receptor-like kinases, whereas CLV2 is an LRR receptor-like protein that acts together with a membrane-associated protein kinase, CRN, to transmit the CLV3 signal. The 96-amino acid CLV3 precursor is proteolytically processed into a mature 13-amino acid arabinosylated glycopeptide derived from its CLE domain, in which one (at position 7) of the two Hyp residues (at positions 4 and 7) is further modified by the addition of three units of l-Ara (Ohyama et al., 2009). The mature CLV3 glycopeptide exhibits full biological activity and binds to the LRR domain of CLV1 more strongly than nonarabinosylated forms (Ohyama et al., 2009). Hyp arabinosylation, a posttranslational modification unique to plants, also has been observed in mature CLE2 and CLE9 peptides from Arabidopsis as well as in CLE-ROOT SIGNAL2, an Arabidopsis CLE2 ortholog that controls nodulation in Lotus japonicus (Lj; Ohyama et al., 2009; Shinohara et al., 2012; Okamoto et al., 2013), where the arabinoside chains are revealed to have important roles in biological activity, receptor binding, and peptide conformation (Shinohara and Matsubayashi, 2013). Many Arabidopsis CLE genes are expressed in roots (Sharma et al., 2003; Jun et al., 2010), and evidence is emerging that CLE-receptor signaling pathways regulate root meristem function (Stahl et al., 2009, 2013; Meng and Feldman, 2010).Nematode CLE genes are expressed exclusively within the dorsal gland cell and encode secreted proteins with the characteristic CLE motif(s) at their C termini (Mitchum et al., 2008; Lu et al., 2009; Wang et al., 2011). Outside the conserved CLE motif, there is no sequence similarity between nematode and plant CLE proteins. The dramatic up-regulation of CLE genes in parasitic stages of the nematode life cycle (Wang et al., 2001, 2010b, 2011; Gao et al., 2003; Lu et al., 2009), along with the observation that transgenic plants expressing double-stranded RNA complementary to nematode CLE genes are less susceptible to nematode infection (Patel et al., 2008), have made it clear that CLE effectors play a critical role in nematode parasitism. Nematode-encoded CLE genes are the only CLE genes that have been identified outside the plant kingdom. Several lines of evidence suggest that nematode CLEs function as peptide mimics of endogenous plant CLEs. First, overexpression of nematode CLE genes in Arabidopsis generated phenotypes similar to those of plant CLE gene overexpression (Wang et al., 2005, 2011; Lu et al., 2009). Second, expression of nematode-encoded CLE genes in the shoot apical meristem of an Arabidopsis clv3-2 null mutant partially or completely rescued the mutant phenotypes (Lu et al., 2009; Wang et al., 2010b). Lastly, recent studies showed that Arabidopsis receptors, including CLV1, CLV2-CRN, and RPK2, are expressed in syncytia induced by the beet cyst nematode (BCN; Heterodera schachtii) and that receptor mutants fail to respond to BCN CLE peptides and show increased resistance to BCN infection (Replogle et al., 2011, 2013), further bolstering the notion of nematode-secreted CLE effectors as peptide mimics of endogenous plant CLEs and the importance of nematode CLE signaling in plant parasitism.Plant CLE precursors undergo posttranslational modifications and proteolytic processing to become bioactive CLE peptides (Shinohara and Matsubayashi, 2010; Shinohara et al., 2012; Okamoto et al., 2013). To fulfill a role as peptide mimics of plant CLEs, nematode CLEs are presumably recognized by the existing host modification and processing machinery for maturation. However, until now, the bioactive form of nematode-secreted CLEs that acts in planta has not been described. In addition, cyst nematodes are specialist feeders. Many agriculturally important nematode species, such as PCN, the soybean cyst nematode (Heterodera glycines), and the cereal cyst nematode (Heterodera avenae), fail to infect Arabidopsis. The mechanism of perception of nematode-secreted CLEs in crop plants still awaits investigation. In this study, we explored the molecular basis of CLE mimicry in the PCN-potato pathosystem. Using ectopic expression coupled with nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis, we determined that the in planta mature form of proGrCLE1, a representative and multidomain CLE effector secreted from PCN during infection (Lu et al., 2009), is a 12-amino acid arabinosylated glycopeptide (hereafter referred to as GrCLE1-1Hyp4,7g) similar in structure to bioactive plant CLE peptides. We further cloned a CLV2-like gene from potato (hereafter referred to as StCLV2). We found that the GrCLE1-1Hyp4,7g glycopeptide binds directly to the StCLV2 ectodomain with high affinity and that transgenic potato lines with reduced StCLV2 expression are less susceptible to PCN infection. Our data provide direct evidence that nematode-secreted CLE effectors can be recognized by existing host cellular machinery to become bioactive mimics of endogenous plant CLE signals and suggest that cyst nematodes tap into the conserved CLV2 signaling pathway to promote successful infection of crop plants.