Sequence and Spatiotemporal Expression Analysis of CLE-Motif Containing Genes from the Reniform Nematode (Rotylenchulus reniformis Linford & Oliveira).
ABSTRACT: The reniform nematode, Rotylenchulus reniformis, is a sedentary semi-endoparasitic species with a host range that encompasses more than 77 plant families. Nematode effector proteins containing plant-ligand motifs similar to CLAVATA3/ESR (CLE) peptides have been identified in the Heterodera, Globodera, and Meloidogyne genera of sedentary endoparasites. Here, we describe the isolation, sequence analysis, and spatiotemporal expression of three R. reniformis genes encoding putative CLE motifs named Rr-cle-1, Rr-cle-2, and Rr-cle-3. The Rr-cle cDNAs showed >98% identity with each other and the predicted peptides were identical with the exception of a short stretch of residues at the carboxy(C)-terminus of the variable domain (VD). Each RrCLE peptide possessed an amino-terminal signal peptide for secretion and a single C-terminal CLE motif that was most similar to Heterodera CLE motifs. Aligning the Rr-cle cDNAs with their corresponding genomic sequences showed three exons with an intron separating the signal peptide from the VD and a second intron separating the VD from the CLE motif. An alignment of the RrCLE1 peptide with Heterodera glycines and Heterodera schachtii CLE proteins revealed a high level of homology within the VD region associated with regulating in planta trafficking of the processed CLE peptide. Quantitative RT-PCR (qRT-PCR) showed similar expression profiles for each Rr-cle transcript across the R. reniformis life-cycle with the greatest transcript abundance being in sedentary parasitic female nematodes. In situ hybridization showed specific Rr-cle expression within the dorsal esophageal gland cell of sedentary parasitic females.
Project description:?-1,4-endoglucanses, a.k.a. cellulases, are parasitism genes that facilitate root penetration and migration by plant-parasitic nematodes. Rotylenchulus reniformis is a sedentary semi-endoparasite for which little molecular data has been collected. In this report, we describe the isolation and characterization of a predicted glycosyl hydrolase family 5 cellulase from R. reniformis that we have named Rr-eng-1. The Rr-eng-1 cDNA was 1,341 bp long and was comprised of a 19 bp 5'-untranslated region (UTR), a 1,245 bp open reading frame (ORF), and an 80 bp 3'-UTR. The Rr-eng-1 genomic sequence was 2,325 bp. Alignment of the cDNA and genomic sequences revealed seven introns and eight exons for Rr-eng-1. BLASTN analysis showed the Rr-eng-1 cDNA was most homologous to the Hg-eng-6 mRNA from Heterodera glycines. Southern blot analysis indicated that at least three Rr-eng-1-like sequences were present in the R. reniformis genome. Translation of the Rr-eng-1 ORF yielded a 414 amino acid peptide (Rr-ENG-1) having an N-terminal signal sequence for secretion. No cellulose binding module (CBM) was detected in Rr-ENG-1; however, a putative CBM linker sequence N-terminal to the catalytic domain was present. Rr-ENG-1 was most homologous to Hg-ENG-6 but also shared a number of intron splice positions with Mi-ENG-2. Quantitative RT-PCR indicated that Rr-eng-1 was highly expressed in the J2 and adult vermiform life-stages with a sharp decline in expression detected in sedentary females.
Project description:Sedentary plant-parasitic nematodes (PPNs) induce and maintain an intimate relationship with their host, stimulating cells adjacent to root vascular tissue to re-differentiate into unique and metabolically active 'feeding sites'. The interaction between PPNs and their host is mediated by nematode effectors. We describe the discovery of a large and diverse family of effector genes, encoding C-TERMINALLY ENCODED PEPTIDE (CEP) plant hormone mimics (RrCEPs), in the syncytia-forming plant parasite Rotylenchulus reniformis. The particular attributes of RrCEPs distinguish them from all other CEPs, regardless of origin. Together with the distant phylogenetic relationship of R. reniformis to the only other CEP-encoding nematode genus identified to date (Meloidogyne), this suggests that CEPs probably evolved de novo in R. reniformis. We have characterized the first member of this large gene family (RrCEP1), demonstrating its significant up-regulation during the plant-nematode interaction and expression in the effector-producing pharyngeal gland cell. All internal CEP domains of multi-domain RrCEPs are followed by di-basic residues, suggesting a mechanism for cleavage. A synthetic peptide corresponding to RrCEP1 domain 1 is biologically active and capable of up-regulating plant nitrate transporter (AtNRT2.1) expression, whilst simultaneously reducing primary root elongation. When a non-CEP-containing, syncytia-forming PPN species (Heterodera schachtii) infects Arabidopsis in a CEP-rich environment, a smaller feeding site is produced. We hypothesize that CEPs of R. reniformis represent a two-fold adaptation to sustained biotrophy in this species: (i) increasing host nitrate uptake, whilst (ii) limiting the size of the syncytial feeding site produced.
Project description:In this article, we present the cloning of two CLAVATA3/ESR (CLE)-like genes, HsCLE1 and HsCLE2, from the beet cyst nematode Heterodera schachtii, a plant-parasitic cyst nematode with a relatively broad host range that includes the model plant Arabidopsis. CLEs are small secreted peptide ligands that play important roles in plant growth and development. By secreting peptide mimics of plant CLEs, the nematode can developmentally reprogramme root cells for the formation of unique feeding sites within host roots for its own benefit. Both HsCLE1 and HsCLE2 encode small secreted polypeptides with a conserved C-terminal CLE domain sharing highest similarity to Arabidopsis CLEs 1-7. Moreover, HsCLE2 contains a 12-amino-acid CLE motif that is identical to AtCLE5 and AtCLE6. Like all other plant and nematode CLEs identified to date, HsCLEs caused wuschel-like phenotypes when overexpressed in Arabidopsis, and this activity was abolished when the proteins were expressed without the CLE motif. HsCLEs could also function in planta without a signal peptide, highlighting the unique, yet conserved function of nematode CLE variable domains in trafficking CLE peptides for secretion. In a direct comparison of HsCLE2 overexpression phenotypes with those of AtCLE5 and AtCLE6, similar shoot and root phenotypes were observed. Exogenous application of 12-amino-acid synthetic peptides corresponding to the CLE motifs of HsCLEs and AtCLE5/6 suggests that the function of this class of CLEs may be subject to complex endogenous regulation. When seedlings were grown on high concentrations of peptide (10 µm), root growth was suppressed; however, when seedlings were grown on low concentrations of peptide (0.1 µm), root growth was stimulated. Together, these findings indicate that AtCLEs1-7 may be the target peptides mimicked by HsCLEs to promote parasitism.
Project description:The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.
Project description:Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular procambium/cambium are regulated by CLE-receptor kinase-WOX signaling modules. Previous data showed that cyst nematode CLE-like effector proteins delivered into host cells through a stylet, act as ligand mimics of plant A-type CLE peptides and are pivotal for successful parasitism. Here we report the identification of a new class of CLE peptides from cyst nematodes with functional similarity to the B-type CLE peptide TDIF (tracheary element differentiation inhibitory factor) encoded by the CLE41 and CLE44 genes in Arabidopsis. We further demonstrate that the TDIF-TDR (TDIF receptor)-WOX4 pathway, which promotes procambial meristem cell proliferation, is involved in beet cyst nematode Heterodera schachtii parasitism. We observed activation of the TDIF pathway in developing feeding sites, reduced nematode infection in cle41 and tdr-1 wox4-1 mutants, and compromised syncytium size in cle41, tdr-1, wox4-1 and tdr-1 wox4-1 mutants. By qRT-PCR and promoter:GUS analyses, we showed that the expression of WOX4 is decreased in a clv1-101 clv2-101 rpk2-5 mutant, suggesting that WOX4 is a potential downstream target of nematode CLEs. Exogenous treatment with both nematode A-type and B-type CLE peptides induced massive cell proliferation in wild type roots, suggesting that the two types of CLEs may regulate cell proliferation during feeding site formation. These findings highlight an important role of the procambial cell proliferation pathway in cyst nematode feeding site formation.
Project description:The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) is a semi-endoparasitic nematode that is a pathogen of numerous major crops such as cotton and soybean. Here, the authors present transcriptome assemblies of the egg, second-stage juvenile (J2), J3, vermiform adult, and sedentary female life stages of this important plant pathogen.The reniform nematode (Rotylenchulus reniformis Linford and Oliveira) is a semi-endoparasitic nematode that is a pathogen of numerous major crops such as cotton and soybean. Here, the authors present transcriptome assemblies of the egg, second-stage juvenile (J2), J3, vermiform adult, and sedentary female life stages of this important plant pathogen.
Project description:Plants can achieve amazing lifespans because of their continuous and repetitive formation of new organs by stem cells present within meristems. The balance between proliferation and differentiation of meristem cells is largely regulated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptide hormones. One of the well-characterized CLE peptides, CLE41/TDIF (tracheary elements differentiation inhibitory factor), functions to suppress tracheary element differentiation and promote procambial cell proliferation, playing important roles in vascular development and wood formation. The recognition mechanisms of TDIF or other CLE peptides by their respective receptors, however, remain largely elusive. Here we report the crystal structure of TDIF in complex with its receptor PXY, a leucine-rich repeat receptor kinase (LRR-RK). Our structure reveals that TDIF mainly adopts an "?"-like conformation binding to the inner surface of the LRR domain of PXY. Interaction between TDIF and PXY is predominately mediated by the relatively conserved amino acids of TDIF. Structure-based sequence alignment showed that the TDIF-interacting motifs are also conserved among other known CLE receptors. Our data provide a structural template for understanding the recognition mechanism of CLE peptides by their receptors, offering an opportunity for the identification of receptors of other uncharacterized CLE peptides.
Project description:BACKGROUND: Plants encode a large number of leucine-rich repeat receptor-like kinases. Legumes encode several LRR-RLK linked to the process of root nodule formation, the ligands of which are unknown. To identify ligands for these receptors, we used a combination of profile hidden Markov models and position-specific iterative BLAST, allowing us to detect new members of the CLV3/ESR (CLE) protein family from publicly available sequence databases. RESULTS: We identified 114 new members of the CLE protein family from various plant species, as well as five protein sequences containing multiple CLE domains. We were able to cluster the CLE domain proteins into 13 distinct groups based on their pairwise similarities in the primary CLE motif. In addition, we identified secondary motifs that coincide with our sequence clusters. The groupings based on the CLE motifs correlate with known biological functions of CLE signaling peptides and are analogous to groupings based on phylogenetic analysis and ectopic overexpression studies. We tested the biological function of two of the predicted CLE signaling peptides in the legume Medicago truncatula. These peptides inhibit the activity of the root apical and lateral root meristems in a manner consistent with our functional predictions based on other CLE signaling peptides clustering in the same groups. CONCLUSION: Our analysis provides an identification and classification of a large number of novel potential CLE signaling peptides. The additional motifs we found could lead to future discovery of recognition sites for processing peptidases as well as predictions for receptor binding specificity.
Project description:CLE peptides, named for the CLV3/ESR-related peptide family, participate in intercellular-signaling pathways. Here we investigated members of the CLE-like (CLEL) gene family that encode peptide precursors recently designated as root growth factors [Matsuzaki Y et al. (2010) Science 329:1065-1067]. CLEL precursors share a similar domain structure with CLE precursors (i.e., they contain a putative N-terminal signal peptide and a C-terminal conserved 13-amino-acid CLEL motif with a variable middle portion). Our evidence shows that, unlike root growth factor, CLEL peptides are (i) unmodified and (ii) function in the regulation of the direction of root growth and lateral root development. Overexpression of several CLEL genes in Arabidopsis resulted in either long roots or long and wavy roots that also showed altered lateral root patterning. Exogenous application of unmodified synthetic 13-amino-acid peptides derived from two CLEL motifs resulted in similar phenotypic changes in roots of wild-type plants. In CLEL peptide-induced long roots, the root apical meristem (RAM) was enlarged and consisted of an increased number of cells, compared with wild-type root apical meristems. The wavy-root phenotype appeared to be independent of other responses of the roots to the environment (e.g., gravitropism, phototropism, and thigmotropism). Results also showed that the inhibition of lateral initiation by CLEL overexpression was not overcome by the application of auxin. These findings establish CLEL as a peptide family with previously unrecognized regulatory functions controlling the pattern of root growth and lateral root development in plants.
Project description:CLE peptides are key regulators of cell proliferation and differentiation in plant shoots, roots, vasculature, and legume nodules. They are C-terminally encoded peptides that are post-translationally cleaved and modified from their corresponding pre-propeptides to produce a final ligand that is 12-13 amino acids in length. In this study, an array of bionformatic and comparative genomic approaches was used to identify and characterize the complete family of CLE peptide-encoding genes in two of the world's most important crop species, soybean and common bean. In total, there are 84 CLE peptide-encoding genes in soybean (considerably more than the 32 present in Arabidopsis), including three pseudogenes and two multi-CLE domain genes having six putative CLE domains each. In addition, 44 CLE peptide-encoding genes were identified in common bean. In silico characterization was used to establish all soybean homeologous pairs, and to identify corresponding gene orthologues present in common bean and Arabidopsis. The soybean CLE pre-propeptide family was further analysed and separated into seven distinct groups based on structure, with groupings strongly associated with the CLE domain sequence and function. These groups provide evolutionary insight into the CLE peptide families of soybean, common bean, and Arabidopsis, and represent a novel tool that can aid in the functional characterization of the peptides. Transcriptional evidence was also used to provide further insight into the location and function of all CLE peptide-encoding members currently available in gene atlases for the three species. Taken together, this in-depth analysis helped to identify and categorize the complete CLE peptide families of soybean and common bean, established gene orthologues within the two legume species, and Arabidopsis, and provided a platform to help compare, contrast, and identify the function of critical CLE peptide hormones in plant development.