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.     

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.     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

Strigolactones: Internal and external signals in plant symbioses?

As the newest plant hormone, strigolactone research is undergoing an exciting expansion. In less than five years, roles for strigolactones have been defined in shoot branching, secondary growth, root growth and nodulation, to add to the growing understanding of their role in arbuscular mycorrhizae and parasitic weed interactions.¹ Strigolactones are particularly fascinating as signaling molecules as they can act both inside the plant as an endogenous hormone and in the soil as a rhizosphere signal.^2-4 Our recent research has highlighted such a dual role for strigolactones, potentially acting as both an endogenous and exogenous signal for arbuscular mycorrhizal development.⁵ There is also significant interest in examining strigolactones as putative regulators of responses to environmental stimuli, especially the response to nutrient availability, given the strong regulation of strigolactone production by nitrate and phosphate observed in many species.^5,6 In particular, the potential for strigolactones to mediate the ecologically important response of mycorrhizal colonization to phosphate has been widely discussed. However, using a mutant approach we found that strigolactones are not essential for phosphate regulation of mycorrhizal colonization or nodulation.⁵ This is consistent with the relatively mild impairment of phosphate control of seedling root growth observed in Arabidopsis strigolactone mutants.⁷ This contrasts with the major role for strigolactones in phosphate control of shoot branching of rice and Arabidopsis^8,9 and indicates that the integration of strigolactones into our understanding of nutrient response will be complex. New data presented here, along with the recent discovery of phosphate specific CLE peptides,¹⁰ indicates a potential role for PsNARK, a component of the autoregulation of nodulation pathway, in phosphate control of nodulation.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

Neutralization of Clostridium difficile toxin A using antibody combinations

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.^1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.^3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.⁵ Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A⁻ B⁺”) suggest a critical role for toxin B in pathogenicity.^6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,^8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.^9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,^11–14 and is responsible for cell surface recognition and endocytosis.^12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.^2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue^19,20 and various non-antibiotic treatments are under investigation.^20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,^26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.^20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.³¹ Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.^33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴