No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Molecular characterisation and developmental expression of a cellulose-binding protein gene in the soybean cyst nematode Heterodera glycines
Abstract
Secretory proteins encoded by 'parasitism genes' expressed in the oesophageal gland cells of plant-parasitic nematodes play key roles in nematode infection and parasitism of host plants. A cellulose-binding protein-encoding cDNA, designated Hg-cbp-1, was cloned from a Heterodera glycines oesophageal gland-cell long-distance PCR cDNA library. The cDNA hybridised to genomic DNA of H. glycines in Southern blots, and the genomic sequence of Hg-cbp-1 contained only one intron. The Hg-cbp-1 cDNA contained an open reading frame encoding 132 amino acids, with a predicted signal peptide sequence for secretion and a cellulose-binding domain. Bacterial expressed recombinant HG-CBP-1, minus the signal peptide sequence, had no hydrolytic activity on carboxymethyl-cellulose but was able to bind to cellulose. The developmental expression of Hg-cbp-1, determined by real-time reverse transcriptase PCR, showed that Hg-cbp-1 is expressed throughout the parasitic cycle of H. glycines, with a relatively higher expression level in developing parasitic stages.
... Oligo (dT) 25 magnetic beads (DynaI, Lake Success, NY) were used to isolate mRNA from the aspirated gland-cell cytoplasm (Fig. 1A). The isolated mRNA was then eluted with 5:1 diethyl pyrocarbonate (DEPC)-treated H 2 O at 70°C for 2 min (26). ...
... Sequences were collected on an ABI 3700 Sequencer (Applied Biosystems, Foster City, CA). Base calls and quality scores were generated from the raw chromotograph files using Phred (24,25). Our own script was used (seqclean) for primary vector-linker cleanup and for removing low quality sequences. ...
... The top hit (E-value of 4.00E-09) for GLAND10 was the functionally characterized cellulose-binding protein effector of H. schachtii (GenBank accession ABY49997.1) (25,34), however, 67% of the amino acids are different. GLAND15 hit to another previously identified putative H. glycines gland protein, G23G11 (GenBank accession AAP30771.1; ...
Heterodera glycines, the soybean cyst nematode, is the number one pathogen of soybean (Glycine max). This nematode infects soybean roots and forms an elaborate feeding site in the vascular cylinder. H. glycines produces an arsenal of effector proteins in the secretory esophageal gland cells. More than sixty H. glycines candidate effectors were identified in previous gland cell-mining projects. However, it is likely that additional candidate effectors remained unidentified. With the goal of identifying remaining H. glycines candidate effectors, we constructed and sequenced a large gland cell cDNA library resulting in 11,814 ESTs. After bioinformatic filtering for candidate effectors using a number of criteria, in situ hybridizations were performed in H. glycines whole mount specimens to identify candidate effectors whose mRNA exclusively accumulated in the esophageal gland cells, which is a hallmark of many nematode effectors. This approach resulted in the identification of eighteen new H. glycines esophageal gland cell-specific candidate effectors. Of these candidate effectors, eleven sequences were pioneers without similarities to known proteins while seven sequences had similarities to functionally annotated proteins in databases. These putative homologies provided the bases for the development of hypotheses about potential functions in the parasitism process.
... Nematode GHF5 endoglucanases can show activity against hemicelluloses, but the activity seems limited to (1,4)-β linked polysaccharides (Gao et al., 2004b). The GHF45 endoglucanases of B. xylophilus are also active against glucomannan (Shibuya and Kikuchi, 2008). ...
... The GHF45 endoglucanases of B. xylophilus are also active against glucomannan (Shibuya and Kikuchi, 2008). A limited activity of some endoglucanases against lichenan has also been observed (Bera-Maillet et al., 2000;Gao et al., 2004b;Shibuya and Kikuchi, 2008). Some endoglucanases are able to hydrolyze xylan, as demonstrated for endoglucanases of Heterodera glycines (Gao et al., 2004b). ...
... A limited activity of some endoglucanases against lichenan has also been observed (Bera-Maillet et al., 2000;Gao et al., 2004b;Shibuya and Kikuchi, 2008). Some endoglucanases are able to hydrolyze xylan, as demonstrated for endoglucanases of Heterodera glycines (Gao et al., 2004b). In addition, specific endoxylanase enzymes have been identified in a limited number of nematodes: in Meloidogyne species and in Radopholus similis (Abad et al., 2008;Haegeman et al., 2009a;Mitreva-Dautova et al., 2006;Opperman et al., 2008). ...
Evidence from as early as the 1950’s that phytoparasitic nematodes could secrete enzymes to facilitate penetration of the
stylet through the host cell wall or to promote nematode migration within plant tissues was confirmed in 1998 with the report
of the first genes encoding endogenous endo-1,4-β-glucanases isolated from animals, the phytoparasitic cyst nematodes. Expressed
gene analyses and recent genome sequencing efforts have since identified genes encoding cellulases, hemicellulases, pectinases,
and chitinases in many species of phytoparasitic nematodes. A cellulose-binding module present in some nematode endoglucanases
also exists independently of the hydrolytic domain in some species and has promoted the discovery of the first expansins outside
of the plant kingdom. Database homologies and phylogenetic analyses suggest the potential for independent ancient horizontal
gene transfer events of genes encoding a number of nematode cell wall-modifying proteins from microbes present in the nematode’s
ecological niche, including soil bacterial sources for root-feeding nematodes and fungal sources for shoot and fungal-feeding
nematodes. Subsequent gene duplication and domain shuffling activity has apparently led to the evolutionary expansion of families
of genes encoding cell wall-modifying proteins within nematode genomes. In almost all cases, expression of these cell wall-modifying
proteins is restricted to the nematode oesophageal gland cells for secretion from the stylet during the migratory stages of
the nematode parasitic cycle.
KeywordsCellulase-Chitinase-Oesophageal gland cells-Expansin-Fungal cell wall-Horizontal gene transfer-Pectinase-Plant cell wall-Secretions-Xylanase
... Among the subventral gland-produced parasitism proteins of H. glycines was a short protein with high similarity to the cellulose binding domain (CBD) of previously identified H. glycines endoglucanases (Smant et al., 1998). Additionally, actual binding to cellulose was documented experimentally; therefore, this protein was termed cellulose binding protein, or Hg CBP (Gao et al., 2004). Cyst nematode endoglucanases produced in the subventral glands and secreted during the migratory phase are thought to be key parasitic tools to breach root cell walls (Smant et al., 1998;de Boer et al., 1999;Wang et al., 1999;Goellner et al., 2001). ...
... Strong evidence for secretion and involvement of Mi CBP-1 in pathogenesis was provided by the detection of this protein in stylet secretions. Hg CBP from H. glycines was the second such cDNA from a nematode (Gao et al., 2003(Gao et al., , 2004 and the first CBP identified in nematodes consisting only of a signal peptide and a CBD. Like Mi CBP-1, recombinant Hg CBP protein had no hydrolytic activity on carboxymethyl-cellulose but was able to bind to cellulose in an in vitro assay. ...
... Hg CBP does not have similarity to expansins, and its functional roles in plant-nematode interactions still remain elusive. However, the developmental expression patterns of Hg CBP during different nematode life stages suggest a role in pathogenesis most likely during the establishment and maintenance of the nematode feeding sites (Gao et al., 2004). ...
Plant-parasitic cyst nematodes secrete a complex of cell wall-digesting enzymes, which aid in root penetration and migration. The soybean cyst nematode Heterodera glycines also produces a cellulose binding protein (Hg CBP) secretory protein. To determine the function of CBP, an orthologous cDNA clone (Hs CBP) was isolated from the sugar beet cyst nematode Heterodera schachtii, which is able to infect Arabidopsis thaliana. CBP is expressed only in the early phases of feeding cell formation and not during the migratory phase. Transgenic Arabidopsis expressing Hs CBP developed longer roots and exhibited enhanced susceptibility to H. schachtii. A yeast two-hybrid screen identified Arabidopsis pectin methylesterase protein 3 (PME3) as strongly and specifically interacting with Hs CBP. Transgenic plants overexpressing PME3 also produced longer roots and exhibited increased susceptibility to H. schachtii, while a pme3 knockout mutant showed opposite phenotypes. Moreover, CBP overexpression increases PME3 activity in planta. Localization studies support the mode of action of PME3 as a cell wall-modifying enzyme. Expression of CBP in the pme3 knockout mutant revealed that PME3 is required but not the sole mechanism for CBP overexpression phenotype. These data indicate that CBP directly interacts with PME3 thereby activating and potentially targeting this enzyme to aid cyst nematode parasitism.
... Genes consisting solely of the expansin domain are also present in these nematodes. CBM2 domains are also present in endogenous cellulases of G. rostochiensis and Heterodera glycines Gao et al., 2004). By contrast, in spite of the availability of large EST datasets for B. ...
... It is therefore possible that the CBM2 domain was acquired by an ancestor of root-knot and cyst nematodes as part of a cellulase encoding gene and that during subsequent evolution of the group it has been duplicated and transferred to the expansin-like genes. Support for this model is provided by the existence of genes consisting solely of the CBM2 domain in both cyst and root knot nematodes (Gao et al., 2004). The fact that the expansin-like protein from B. xylophilus is so similar to the expansins from G. rostochiensis and not like any other proteins in any other nematode species, suggests that it was acquired via horizontal gene transfer by a common ancestor of cyst nematodes and Bursaphelenchus. ...
The full text of my PhD thesis is available in the following link:
https://biblio.ugent.be/publication/470947/file/1881101
... GH31 with α-glucosides, GH38 with α-mannosidase (Class II) and GH47 with exoacting α-1, 2-mannosidases activities were present in all of the five species analyzed (Fig. 3). Some of the GH3, GH5 and GH45 family enzymes were also reported to have the hemicellulase activity apart from the cellulose activity [40,41]. There are reports of limited activity of GH5 family against the 1,4-β linked polysaccharides [41]. ...
... Some of the GH3, GH5 and GH45 family enzymes were also reported to have the hemicellulase activity apart from the cellulose activity [40,41]. There are reports of limited activity of GH5 family against the 1,4-β linked polysaccharides [41]. The GH45 family has been reported to have activity against the glucomannon in the pine wood nematode, B. xylophilus [40]. ...
Background:
The plant cell wall serves as a primary barrier against pathogen invasion. The success of a plant pathogen largely depends on its ability to overcome this barrier. During the infection process, plant parasitic nematodes secrete cell wall degrading enzymes (CWDEs) apart from piercing with their stylet, a sharp and hard mouthpart used for successful infection. CWDEs typically consist of cellulases, hemicellulases, and pectinases, which help the nematode to infect and establish the feeding structure or form a cyst. The study of nematode cell wall degrading enzymes not only enhance our understanding of the interaction between nematodes and their host, but also provides information on a novel source of enzymes for their potential use in biomass based biofuel/bioproduct industries. Although there is comprehensive information available on genome wide analysis of CWDEs for bacteria, fungi, termites and plants, but no comprehensive information available for plant pathogenic nematodes. Herein we have performed a genome wide analysis of CWDEs from the genome sequenced phyto pathogenic nematode species and developed a comprehensive publicly available database.
Results:
In the present study, we have performed a genome wide analysis for the presence of CWDEs from five plant parasitic nematode species with fully sequenced genomes covering three genera viz. Bursaphelenchus, Glorodera and Meloidogyne. Using the Hidden Markov Models (HMM) conserved domain profiles of the respective gene families, we have identified 530 genes encoding CWDEs that are distributed among 24 gene families of glycoside hydrolases (412) and polysaccharide lyases (118). Furthermore, expression profiles of these genes were analyzed across the life cycle of a potato cyst nematode. Most genes were found to have moderate to high expression from early to late infectious stages, while some clusters were invasion stage specific, indicating the role of these enzymes in the nematode's infection and establishment process. Additionally, we have also developed a Nematode's Plant Cell Wall Degrading Enzyme (NCWDE) database as a platform to provide a comprehensive outcome of the present study.
Conclusions:
Our study provides collective information about different families of CWDEs from five different sequenced plant pathogenic nematode species. The outcomes of this study will help in developing better strategies to curtail the nematode infection, as well as help in identification of novel cell wall degrading enzymes for biofuel/bioproduct industries.
... The resistance or susceptible interaction(s) between the nematode and soybean affected by Rhg1 was not induced until females stopped moving through the roots and established a feeding site comprising several giant cells [28][29][30]. Full resistance to SCN, based on the combined action of the genes at Rhg1 and one or more additional Rhg loci, was manifest as; cell wall appositions to surround the feeding site; failure to supply the feeding site a tracheary element; and a necrosis as the feeding site develops. If the resistance allele at Rhg1 was present normal rates of root growth were slightly depressed but the above ground stunting, yellowing and early senescence did not occur. ...
... For soybean transformation, the cassette included pSBHB94 that was a 9.772 kbp insert sub-cloned from BAC B21d09 by nebulization, size fractionated to 9-11 kbp and ligated into pHOS2. The plasmid pSBHB94 encompassed the sequences found from 30,194 bp in BAC B73p06. Transformation, selection and plant regeneration were conducted after [67]. ...
... The resistance or susceptible interaction(s) between the nematode and soybean affected by Rhg1 was not induced until females stopped moving through the roots and established a feeding site comprising several giant cells [28][29][30]. Full resistance to SCN, based on the combined action of the genes at Rhg1 and one or more additional Rhg loci, was manifest as; cell wall appositions to surround the feeding site; failure to supply the feeding site a tracheary element; and a necrosis as the feeding site develops. If the resistance allele at Rhg1 was present normal rates of root growth were slightly depressed but the above ground stunting, yellowing and early senescence did not occur. ...
... For soybean transformation, the cassette included pSBHB94 that was a 9.772 kbp insert sub-cloned from BAC B21d09 by nebulization, size fractionated to 9-11 kbp and ligated into pHOS2. The plasmid pSBHB94 encompassed the sequences found from 30,194 bp in BAC B73p06. Transformation, selection and plant regeneration were conducted after [67]. ...
Soybean (Glycine max (L. Merr.)) resistance to any population of Heterodera glycines (I.), or Fusarium virguliforme (Akoi, O'Donnell, Homma & Lattanzi) required a functional allele at Rhg1/Rfs2. H. glycines, the soybean cyst nematode (SCN) was an ancient, endemic, pest of soybean whereas F. virguliforme causal agent of sudden death syndrome (SDS), was a recent, regional, pest. This study examined the role of a receptor like kinase (RLK) GmRLK18-1 (gene model Glyma_18_02680 at 1,071 kbp on chromosome 18 of the genome sequence) within the Rhg1/Rfs2 locus in causing resistance to SCN and SDS.
A BAC (B73p06) encompassing the Rhg1/Rfs2 locus was sequenced from a resistant cultivar and compared to the sequences of two susceptible cultivars from which 800 SNPs were found. Sequence alignments inferred that the resistance allele was an introgressed region of about 59 kbp at the center of which the GmRLK18-1 was the most polymorphic gene and encoded protein. Analyses were made of plants that were either heterozygous at, or transgenic (and so hemizygous at a new location) with, the resistance allele of GmRLK18-1. Those plants infested with either H. glycines or F. virguliforme showed that the allele for resistance was dominant. In the absence of Rhg4 the GmRLK18-1 was sufficient to confer nearly complete resistance to both root and leaf symptoms of SDS caused by F. virguliforme and provided partial resistance to three different populations of nematodes (mature female cysts were reduced by 30-50%). In the presence of Rhg4 the plants with the transgene were nearly classed as fully resistant to SCN (females reduced to 11% of the susceptible control) as well as SDS. A reduction in the rate of early seedling root development was also shown to be caused by the resistance allele of the GmRLK18-1. Field trials of transgenic plants showed an increase in foliar susceptibility to insect herbivory.
The inference that soybean has adapted part of an existing pathogen recognition and defense cascade (H.glycines; SCN and insect herbivory) to a new pathogen (F. virguliforme; SDS) has broad implications for crop improvement. Stable resistance to many pathogens might be achieved by manipulation the genes encoding a small number of pathogen recognition proteins.
... Many of the cyst and root-knot nematode cellulases are composed of a carbohydrate binding domain (CBD) from carbohydrate binding module family II (CBM2) coupled to a catalytic domain most similar to (bacterial) glycosyl hydrolase family 5 (GHF5) proteins. In addition, cyst and root-knot nematodes express proteins consisting solely of the CBD, and these are also CBM2-like proteins (e.g., Gao et al., 2004). ...
... Plant expansins also usually consist of an expansin domain coupled to a CBD, although in these proteins the CBD is not a CBM2-like protein and is found at the C-terminus. CBM2 domains are present in other cyst and root-knot nematode proteins; in cellulases and as stand-alone proteins Gao et al., 2004) but no proteins containing such domains have been described from any Bursaphelenchus species, in spite of the availability of large EST datasets for B. xylophilus and B. mucronatus (Kikuchi et al., 2007) and characterised cell wall degrading enzymes (Kikuchi et al., 2004(Kikuchi et al., , 2006. It is possible that future genome sequencing projects will reveal carbohydrate binding domains within Bursaphelenchus but the sequence features described to date allow a speculative model of evolution of the expansin gene family to be suggested. ...
We report the cloning and characterisation of genes encoding expansin-like proteins from the pine wood nematodes, Bursaphelenchus xylophilus and B. mucronatus . A small family of genes is present in both species and the Bursaphelenchus genes are most similar to expansins and expansin-like proteins from the potato cyst nematode Globodera rostochiensis and root-knot nematodes. Molecular modelling suggests that the genes could encode a protein with a structure similar to that of functionally characterised expansins. Expression analysis showed that the Bursaphelenchus expansin-like genes are expressed solely in the pharyngeal gland cells, implying a role in the host-parasite interaction, most likely in assisting migration through the plant. Some G. rostochiensis and root-knot nematode expansins are composed of a carbohydrate-binding domain coupled to an expansin domain but no carbohydrate binding domain is present on any of the Bursaphelenchus sequences. We suggest a model for evolution of the expansin gene family within the plant-parasitic nematodes of the Tylenchida and Aphelenchida
... In addition, similar proteins were detected in other plant-parasitic nematodes [e.g., H. glycines (GenPept AAN32887) and M. incognita (GenPept AAC05133)], and the sequence is more similar to these sequences than to the CBD of nematode endoglucanases. Previous analyses (Ding et al. 1998;Gao et al. 2004) indicate that the CBPs are secreted by plant-parasitic nematodes and may have a role in parasitism. The specific function of this protein is not yet known, but recombinant CBD was found to modulate the elongation of different plant cells in vitro (Shpigel et al. 1998). ...
... Hg-ENG5 differs significantly in its catalytic domain compared to the other cloned endoglucanases of H. schachtii [Hs-ENG1 (GenPept CAC12958) and Hs-ENG2 (GenPept CAC12959)] (De Meutter et al. 1999). In contrast to these endoglucanases, Hg-ENG5 possesses activity toward xylan (Gao et al. 2004), which is a major component of hemicellulose of the plant cell wall. CL279contig1 showed only weak similarity to previously cloned endoglucanases (Hs-ENG1; E=3e−6), and the homology was restricted to the CBD of Hs-ENG1. ...
The beet cyst nematode Heterodera schachtii is an important pathogen worldwide, but its molecular characterization has been limited to studying individual genes of interest. We undertook a high-throughput genomic approach and drastically increased the number of available sequences for this parasite. A total of 2,662 expressed sequence tags were grouped into 1,212 clusters representing a nonredundant catalog of H. schachtii genes. Implementing a bioinformatic workflow, we identified 50 sequences coding for candidate secreted proteins. All of these contain a putative signal peptide required for entry into the secretory pathway and lack any transmembrane domain. Included are previously postulated cell-wall-degrading enzymes and other parasitism-related genes. Moreover, we provide the first report of an arabinogalactan endo-1,4-beta-galactosidase enzyme (EC 3.2.1.89) in animals. As sequence data increase at a rapid rate, developing high-throughput genomic screening is a necessity. The in silico approach described here is an effective way to identify putative secreted proteins and prioritize candidates for further studies.
... Genes encode for enzymes that degrade or modify plant cell walls enzymes, such as chitinases [79], pectinases [80,81], cellulases or -1,4-endoglucanases [79,82], and xylanases [45]. These cell-wall-degrading enzymes facilitate the migration of infective J2 nematodes through plant roots by smoothing the cell wall. ...
Citation: Khan, A.; Chen, S.; Fatima, S.; Ahamad, L.; Siddiqui, M.A. Biotechnological Tools to Elucidate the Mechanism of Plant and Nematode Interactions. Plants 2023, 12, 2387. https://doi. Abstract: Plant-parasitic nematodes (PPNs) pose a threat to global food security in both the developed and developing worlds. PPNs cause crop losses worth a total of more than USD 150 billion worldwide. The sedentary root-knot nematodes (RKNs) also cause severe damage to various agricultural crops and establish compatible relationships with a broad range of host plants. This review aims to provide a broad overview of the strategies used to identify the morpho-physiological and molecular events that occur during RKN parasitism. It describes the most current developments in the transcriptomic, proteomic, and metabolomic strategies of nematodes, which are important for understanding compatible interactions of plants and nematodes, and several strategies for enhancing plant resistance against RKNs. We will highlight recent rapid advances in molecular strategies, such as gene-silencing technologies, RNA interference (RNAi), and small interfering RNA (siRNA) effector proteins, that are leading to considerable progress in understanding the mechanism of plant-nematode interactions. We also take into account genetic engineering strategies, such as targeted genome editing techniques, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas-9) system, and quantitative trait loci (QTL), to enhance the resistance of plants against nematodes.
... An example of this are the cellulose-binding proteins (CBPs), that play a role in the cell wall modifications during the sedentary phase. Both in RKNs and CNs CBPs have been identified (Adam et al., 2008;Gao et al., 2004). One CBP secreted by the CN H. schachtii was functionally analyzed and has been demonstrated to interact with pectin methylesterase protein3 (Pme3). ...
The root-knot nematode Meloidogyne graminicola is able to infect over a 100 plant species with a clear preference for monocots and is a well-known pathogen of the staple crop rice. The nematode is able to survive in the root under flooded conditions but proliferates in aerobic cultivation. Water scarcity and the demand for higher rice production causes a shift from irrigated towards aerobic rice cultivation and therefore it becomes more problematic to control this pathogen. New insights in the molecular basis of the interaction may lead to a better control of M. graminicola. While invading the root, the nematode secretes a cocktail of many different effector proteins in the host tissue which changes the cellular regulation of the plant cells. In this thesis project we are focusing on five putative effectors that show a high expression in the pre-parasitic juveniles. This indicates a role in the early stage of infection and therefore these effectors were investigated on different aspects of parasitism with a focus on immune suppressing capacities. Each research chapter shows the outcome of several molecular techniques, like RNAi applied to nematodes to test virulence, subcellular localization, PTI and ETI assays, yeast two-hybrid assays and the use of effector overexpressing lines. The thesis is divided in seven chapters, starting with an introduction describing the morphology, life cycle and molecular studies of M. graminicola. In addition, the host plant rice, nematode effectors and the immune response of the plant are described. In the first research chapter an infection assay with M. graminicola was performed on the model plant Nicotiana benthamiana. This plant species is widely used in effector studies, including those of M. graminicola. We demonstrate in this chapter that N. benthamiana can be considered as a host for M. graminicola in contrast to the closely related plant species N. tabacum. In chapter 3, the effector Mg52 is presented as an immune suppressing effector which is secreted by the nematode in the apoplast and the cytoplasm. In the apoplast the effector suppresses the ROS response induced by the elicitor flg22, while in the cytoplasm it suppresses the R2/Avr2 and Mi-1.2T557S induced hypersensitive response. A target protein in rice was identified which plays a role in the ABA induced cell response to abiotic stress. Hormone measurements in the Mg52 overexpressing line showed an interesting accumulation of ABA compared to the wild type, when the plants were not exposed to salt stress. Chapter 4 describes two effectors, Mg41 and Mg42, that both have a calcium dependent carbohydrate binding domain (C-type lectins). RNAi showed a decrease in expression for both genes, but only for Mg41 this affected the infectivity of the nematodes. The effectors did not seem to play a role in the suppression of the hypersensitive response induced by the tested vii R/Avr pairs, but may play a role in the suppression of PTI, although it was quite variable between experiments and needs further research. In Chapter 5 the effector Mg50 was described, a protein which was not identified in other nematodes species yet. RNAi resulted in a decreased virulence of the nematodes and the overexpressing line was more susceptible for infection of M. graminicola. None of the immune response assays showed a role for Mg50 in the suppression of the immune system of the plant. But potentially a target protein was identified in rice. In Chapter 6 the effector Mg8 was functionally analyzed. However, the results obtained with this effector were limited. Finally, in the last chapter of this thesis, Chapter 7, we discuss the outcome of the research described in this thesis whereby the different effectors are compared to each other and M. graminicola effectors that have recently been published. In addition, some molecular techniques used in this study are further discussed. Knowledge on effectors and on how effectors manipulate plant cells is mostly still in the pioneer phase. This thesis provides some insight in the function of a selection of effectors of M. graminicola. However, to fully understand the function of these proteins and the complex interaction between M. graminicola and rice, much more research is needed.
... ve Heterodera spp. nematod cinslerinde yer alan, farklı türlerde cellulase'lar tespit edilmiştir (Ding et al., 1998;Gao et al., 2004;Ithal et al., 2007;Jones et al., 2009;Haegeman et al., 2012). Cellulase'lara ilave olarak, nematod salgılarında hücre duvarı yapısındaki diğer polimerler (Pectin, xylan gibi) üzerinde etkili enzimler tespit edilmiştir. ...
Bitki paraziti nematodlar, gelişim ve üremeleri için gerekli olan besin maddelerini elde edebilmek amacıyla konukçularıyla özel parazitik ilişkiler geliştirmişlerdir. Günümüze kadar yaklaşık 4100 adet bitki parazit nematod türü tanımlanmıştır. Sabit endoparazitik nematodlar içerisinden kök-ur nematodları (Meloidogyne spp.) ve kist nematodları (Globodera spp., ve Heterodera spp.) ekonomik olarak en zararlı bitki paraziti nematod grupları olarak kabul edilmektedir. Sabit endoparazit nematodlar, konukçularında özelleşmiş beslenme hücreleri oluşturmaktadırlar. Nematodlar tarafından üretilen salgılar enfeksiyonda önemli rol oynamaktadırlar. Efektör olarak adlandırılan bu salgılar birçok fonksiyona sahiptir. Bu derleme, sabit endoparazitik nematodların efektör molekülleri hakkında bilgi vermek için hazırlanmıştır.
... Once delivered into plant cells, these effectors localize to specific cellular compartments and target host proteins to execute their virulence functions. Compelling experimental evidence indicates that cyst nematode effectors alter cell wall structure to facilitate nematode migration and syncytium formation (40,59,146,153), mimic host proteins in both structure and mode of action to disrupt cellular processes while evading plant immune systems (106,122,123,148,154), modulate the activity of key components of the auxin signaling pathway to induce celltype-specific patterns of auxin-responsive gene expression in the syncytium (61, 87), and suppress defense signaling pathways (55, 60, 113). The ability of cyst nematode effectors to directly or indirectly suppress host immune responses and defense signaling seems to be a common function of many effectors (57). ...
Soybean hosts a wide variety of pathogens that cause significant yield losses. The importance of soybean as a major oilseed crop has led to research focused on its interactions with pathogens, such as Soybean mosaic virus, Pseudomonas syringae, Phytophthora sojae, Phakopsora pachyrhizi, and Heterodera glycines. Pioneering work on soybean's interactions with these organisms, which represent the five major pathogen groups (viruses, bacteria, oomycetes, fungi, and nematodes), has contributed to our understanding of the molecular mechanisms underlying virulence and immunity. These mechanisms involve conserved and unique features that validate the need for research in both soybean and homologous model systems. In this review, we discuss identification of effectors and their functions as well as resistance gene-mediated recognition and signaling. We also point out areas in which model systems and recent advances in resources and tools have provided opportunities to gain deeper insights into soybean-pathogen interactions. Expected final online publication date for the Annual Review of Phytopathology Volume 54 is August 04, 2016. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
... Meloidogyne spp. secrete cellulose-binding proteins (CBP), that consist of a signal peptide for secretion, in some cases an unknown N-terminal sequence and a CBM (Ding et al., 1998;Gao et al., 2004a;Hewezi et al., 2008;Adam et al., 2008). Recently, a functional study suggested that CBP targets and/or activates a plant pectin methylesterase, to reduce the level of methylesterification of pectin in the cell wall. ...
Plant-parasitic nematodes cause a considerable amount of crop damage worldwide. These nematodes all have a stylet to damage plant cells mechanically, but through which they also secrete proteins important for their infection process. Most studies on genes encoding these so-called parasitism proteins were conducted on sedentary plant-parasitic nematodes which manipulate the plant to induce a specialized nematode feeding site. In this doctoral thesis parasitism genes of the less specialized migratory endoparasitic nematodes were investigated. The focus was on plant cell wall modifying enzymes that are being secreted by the nematode to enable migration through the rigid plant cell wall.
The main component of the plant cell wall is cellulose, the most abundant sugar polymer on earth. In nematodes, cellulases or endo-1,4-β-glucanases which can degrade cellulose were mainly identified in sedentary species. A large part of this thesis is about the characterization of endoglucanases in migratory endoparasitic nematodes, namely in the species Radopholus similis, Ditylenchus africanus and Pratylenchus coffeae. Additionally, a detailed model for the evolution of the gene structure of endoglucanases within nematodes was proposed.
The second most abundant sugar polymer in a plant cell wall is xylan. An endoxylanase from R. similis putatively able to degrade xylan was characterized. When this gene was downregulated in nematodes by RNAi, the infection on the plant was reduced by 60%, showing that the xylanase is important for the nematodes to infect their host plant.
Another technique to identify parasitism genes is expressed sequence tag analysis, by randomly sequencing pieces of genes that are being expressed by the nematode. This approach was applied to D. africanus and 4847 ESTs were analyzed.
Scientific research sometimes leads to unexpected discoveries. During an EST analysis of R. similis several ESTs were found with significant similarity to sequences from a Wolbachia endosymbiotic bacterium. This finding was further investigated and the presence of an intracellular Wolbachia bacterium in the nematode was demonstrated. Although this bacterium is often found in filarial nematodes, it is the first time it is discovered in a plant-parasitic nematode. Future research will be needed to determine what role this endosymbiont plays within the nematode.
... Genes that enable nematodes to parasitize plants have been identified and explored by several research groups using different molecular tools. Genes encoding different cell wall degrading enzymes from different species of nematodes including sedentary nematodes such as M. incognita, (Bére-Maileet et al., 2000), Globodera rostochiensis (Smant et al., 1998), (Goellner et al., 2000), H. glycines (Smant et al., 1998;Gao et al., 2004), H. avenae (Long et al., 2012), as well as migratory nematodes, such as P. penetrans and P. coffeae (Uehara et al., 2001, R. similis , B. xylophilus (Kikuchi et al., 2004;Kikuchi et al., 2005;Kikuchi et al., 2011) and ...
... Sedentary parasitic nematodes also secrete other effector proteins, such as CELLULOSE-BINDING PROTEINs (CBPs), that function in plant cell wall modifications during the sedentary phase of parasitism. While CBPs from both cyst and root-knot nematodes do not contain any catalytic domain required for their activity, they were able to bind to cellulose in in vitro assays (Ding et al., 1998;Gao et al., 2004). Functional characterization of a CBP from the sugar beet (Beta vulgaris) cyst nematode Heterodera schachtii revealed that CBP functions through a direct association with Arabidopsis (Arabidopsis thaliana) PECTIN METHYLESTERASE PROTEIN3 (PME3; Hewezi et al., 2008). ...
Plant-parasitic cyst and root-knot nematodes synthesize and secrete a suite of effector proteins into infected host cells and tissues. These effectors are the major virulence determinants mediating the transformation of normal root cells into specialized feeding structures. Compelling evidence indicates that these effectors directly hijack or manipulate refined host physiological processes to promote the successful parasitism of host plants. Here, we provide an update on recent progress in elucidating the molecular functions of nematode effectors. In particular, we emphasize how nematode effectors modify plant cell wall structure, mimic the activity of host proteins, alter auxin signaling, and subvert defense signaling and immune responses. In addition, we discuss the emerging evidence suggesting that nematode effectors target and recruit various components of host posttranslational machinery in order to perturb the host signaling networks required for immunity and to regulate their own activity and subcellular localization. © 2015 American Society of Plant Biologists. All rights reserved.
... Secretome contains products of diverse parasitism genes. The genes encode for plant cell wall degrading/modifying enzymes including cellulases or β-1,4endoglucanases (Smant et al., 1998;Goellner et al., 2000;Gao et al., 2002;Gao et al., 2004), chitinases (Gao et al., 2002), xylanases (Opperman et al., 2008), pectate lyases and pectinases Opperman et al., 2008), and expansins (Qin et al., 2004;Kudla et al., 2005). These cell wall degrading enzymes help the infective J2 nematodes to migrate through the plant roots by softening the cell wall. ...
Plant parasitic nematodes are obligate parasites causing serious reduction in crop yields. Several economically important species parasitize various plant species, but the root knot and cyst nematodes belonging to the Heteroderidae family are especially dangerous. Plant parasitic nematodes result in crop losses of over $150 billion worldwide. This review gives an account of morpho-physiological and molecular events during parasitism of root-knot and cyst nematodes. It describes the transcriptomes and parasitomes of various nematodes indicating that the effector proteins are crucial for the compatible plant nematode interactions. Various sequencing techniques used in plant-nematode genomics and transcriptomics are discussed. Moreover, the dynamics of host transcriptomes in response to infection with different nematode species have been reported. The host transcriptomes have unrevealed many candidate genes, which are involved in both compatible and incompatible plant nematode interactions. The strategy of manipulation of expression of the genes induced and suppressed by the nematodes in the feeding sites has also been suggested for enhancement of resistance against nematodes. This review will provide the researchers with the information regarding transcriptional changes in the nematodes as well as host plants, which would be important for the induction of resistance against nematodes in different crop plants. © 2015 Friends Science Publishers
... These changes are mediated by the activity of both cell wall-biosynthetic and cell wall-degrading enzymes. It is well known that during migration in root tissues infective larvae of cyst nematodes secrete numerous hydrolytic enzymes or modifying proteins for digesting cell wall polymers such as endo->-1,4-glucanases (Smant et al. 1998;Gao et al. 2004), pectate lyases (Popeijus et al. 2000), and expansins (Qin et al. 2004). But the expression of these proteins was no more detectable, since the larvae selected the initial syncytial cell and became sedentary. ...
Globodera rostochiensis, as well as other cyst nematodes, induces formation of a multinucleate feeding site, called syncytium, in host roots. In tomato roots infected with a potato cyst nematode, the syncytium is initiated in the cortex or pericycle. Progressive cell wall dissolution and subsequent fusion of protoplasts of newly incorporated cells lead to syncytium formation. Expansion and development of a syncytium strongly depends on modifications of a cell wall, including its degradation, elongation, thickening, and formation of ingrowths within it in close contact with tracheary elements. Recent reports have demonstrated that during formation of syncytium, numerous genes of plant origin, coding for cell wall-modifying enzymes are up-regulated. In this research, we studied a detailed distribution and function of two tomato β-1,4- endoglucanases in developing feeding sites induced by G. rostochiensis. In situ localization of tomato LeCel7 and LeCel8 transcripts and proteins demonstrated that these enzymes were specifically up-regulated within syncytium and in the cells adjacent to the syncytium. In non-infected roots an expression of LeCel7 and LeCel8 was observed in the root cap and lateral root primordia. Our data confirm that cell wall-modifying enzymes of plant origin have a role in a modification of cell wall within syncytia, and demonstrate that plant endoglucanases are involved in syncytia formation.
... ENG-1 and ENG-2 were found to be released during penetration and intracellular migration by SCN (De Boer et al., 1999;Wang et al., 1999;Yan et al., 2001) and by the tobacco cyst nematode (Goellner et al., 2001). Gao et al. (2004) showed that three EGases are secreted by H. glycines during infection of soybean. Two other EGases were also secreted by the migratory root lesion nematode Pratylenchus penetrans (Uehara et al., 2001). ...
Plant endo-β-1,4-glucanases (EGases) include cell-wall-modifying enzymes that are involved in nematode-induced growth of syncytia (feeding structures) in nematode-infected roots. EGases in the alpha and beta subfamilies contain signal peptides and are secreted, while those in the gamma subfamily have a membrane-anchoring domain and are not secreted. The Arabidopsis α-EGase At1g48930, designated AtCel6, is known to be down-regulated by beet cyst nematode (Heterodera schachtii) in Arabidopsis roots, while another α-EGase, AtCel2, is up-regulated. Here we report that ectopic expression of AtCel6 in soybean roots reduced susceptibility to both soybean cyst nematode (SCN; Heterodera glycines) and root knot nematode (Meloidogyne incognita). Suppression of GmCel7, the soybean homolog of AtCel2, in soybean roots also reduced susceptibility to SCN. In contrast, in studies on two γ-EGases, both ectopic expression of AtKOR2 in soybean roots and suppression of the soybean homolog of AtKOR3 had no significant effect on SCN parasitism. Our results suggest that secreted α-EGases are likely to be more useful than membrane-bound γ-EGases in the development of an SCN-resistant soybean through gene manipulation. Furthermore, this study provides evidence that Arabidopsis shares molecular events of cyst nematode parasitism with soybean, and confirms the suitability of the Arabidopsis-H. schachtii interaction as a model for the soybean-H. glycines pathosystem.
... However, another plant-parasitic nematode Bursaphelenchus xylophilus, which is located in Clade 10 as described by van Megen et al. [8] and is not directly related to the Clade 12 Tylenchid plant parasites, has GH45 cellulases rather than GH5 [9]. These two GH families show little amino acid similarity to each other and have distinct kinetic mechanisms, catalytic residues and three dimensional structures although both catalyze the breakdown of similar substrate; cellulose and hemicelluloses [10,11]. ...
Horizontal gene transfer (HGT) has been suggested as the mechanism by which various plant parasitic nematode species have obtained genes important in parasitism. In particular, cellulase genes have been acquired by plant parasitic nematodes that allow them to digest plant cell walls. Unlike the typical glycoside hydrolase (GH) family 5 cellulase genes which are found in several nematode species from the order Tylenchida, members of the GH45 cellulase have only been identified in a cluster including the families Parasitaphelenchidae (with the pinewood nematode Bursaphelenchus xylophilus) and Aphelenchoididae, and their origins remain unknown.
In order to investigate the distribution and evolution of GH45 cellulase genes in nematodes and fungi we performed a wide ranging screen for novel putative GH45 sequences. This revealed that the sequences are widespread mainly in Ascomycetous fungi and have so far been found in a single major nematode lineage. Close relationships between the sequences from nematodes and fungi were found through our phylogenetic analyses. An intron position is shared by sequences from Bursaphelenchus nematodes and several Ascomycetous fungal species.
The close phylogenetic relationships and conserved gene structure between the sequences from nematodes and fungi strongly supports the hypothesis that nematode GH45 cellulase genes were acquired via HGT from fungi. The rapid duplication and turnover of these genes within Bursaphelenchus genomes demonstrate that useful sequences acquired via HGT can become established in the genomes of recipient organisms and may open novel niches for these organisms to exploit.
... To date, 70% of all effectors identified from Heterodera are novel proteins, which presents a considerable challenge in ascribing function. Among those identified from H. glycines and H. schachtii with sequence similarity to known proteins is an elaborate suite of cell wall-modifying proteins (CWMPs), including ENGs De Meutter et al., 2001;Gao et al., 2002aGao et al., , 2004aVanholme et al., 2006), CBPs (Gao et al., 2004b;Vanholme et al., 2006), pectate lyases (PELs; Vanholme et al., 2007), and an arabinogalactan endo-1,4-b-galactosidase (Vanholme et al., 2009b). These nematodes also produce secreted effector proteins with homology to chitinase (CHI; Gao et al., 2002b), chorismate mutase (CM; Bekal et al., 2003;Gao et al., 2003), ubiquitin Tytgat et al., 2004), and venom allergen proteins (VAPs; Gao et al., 2001bGao et al., , 2003, although their functions in nematode parasitism remain unknown. ...
Phytonematodes use a stylet and secreted effectors to modify host cells and ingest nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode effectors, with a particular focus on proteinaceous stylet-secreted effectors of sedentary endoparasitic phytonematodes, for which a wealth of information has surfaced in the past 10 yr. We provide an update on the effector repertoires of several of the most economically important genera of phytonematodes and discuss current approaches to dissecting their function. Lastly, we highlight the latest breakthroughs in effector discovery that promise to shed new light on effector diversity and function across the phylum Nematoda.
... To date, a plethora of these enzymes has been characterised (e.g., endoglucanase , pectate lyases (Popeijus et al., 2000), xylanases (Mitreva-Dautova et al., 2006) and polygalacturonase (Jaubert et al., 2002)). Other proteins secreted by the nematode, such as cellulose binding proteins (Gao et al., 2004) and expansins (Qin et al., 2004), do not have enzymatic activity but can interact with polysaccharides of the cell wall and as such weaken the structure. Despite the battery of pectin-degrading proteins identified in nematodes (de Boer et al., 2002;Doyle & Lambert, 2002;Jaubert et al., 2002;Huang et al., 2005;Kikuchi et al., 2006), it is striking that none of them can tackle the side chains of hairy regions. ...
Plant-parasitic nematodes secrete a plethora of enzymes to degrade polysaccharides of the recalcitrant plant cell wall. Here we report on the presence of a putative endo-1,4-β-galactosidase (EC 3.2.1.89) in cyst nematodes of the genus Heterodera. This enzyme hydrolyses β-1,4-galactan in the hairy regions of pectin and to our knowledge it is the first report of this class of enzymes in animals. The gene was cloned from H. schachtii and subjected to a detailed molecular characterisation. The deduced protein contains a putative signal peptide for secretion, being in agreement with the presumed extracellular function of the mature protein. It has a molecular mass of 33.78 kDa and folds into an (α/β)8 barrel structure typical for glycosyl hydrolases. The two glutamic acids that function as electron donor and acceptor in the active site are conserved. Whole mount in situ hybridisation revealed that the gene is expressed in the subventral pharyngeal glands and the expression was correlated with the onset of parasitism.
... This review focusses on recent progress in effector biology of plant Cell-wall degradation: hydrolyses 1,4b-D-glucosidic linkages in 1,4-b-D-glucans such as cellulose, lichenin and cereal b-Dglucans. Knocking down of the expression of cellulase by soaking in dsRNA led to a reduction in penetration of the plant by the nematodes Smant et al. (1998, Lilley et al. (1999), Rosso et al. (1999), Wang et al. (1999), Goellner et al. (2000), Uehara et al. (2001), Gao et al. (2004) ...
Plant parasitic nematodes infect the roots of a wide range of host plants. Migratory endo- or ectoparasites feed off the roots temporarily, but sedentary endoparasites are biotrophic parasites that invade roots and establish a permanent feeding site by re-directing root cell development. Feeding sites develop after injection of nematode effectors into plant cells through a stylet. In this review, we concentrate on several recently-identified effectors and discuss their possible functions in re-directing root cell development. We give examples of effectors that regulate host gene expression, interact with specific host proteins or mimic plant signalling molecules.
... The occurrence of several nematode genes encoding an expressed CBD joined with a peptide of nonendoglucnanase origin (Ding et al. 1998 ;Gao et al. 2004b) is curious since over-expression of a bacterial CBD gene has been reported to increase elongation of plant cells (Shpigel et al. 1998) . One gene ( GR-exp1 ) expressed in the subventral gland cells of the potato cyst nematode (PCN) that encoded a CBD domain was an expansin-like protein that represented the first confirmed report of such a protein outside the plant kingdom (Qin et al. 2004) . ...
Nematodes are parasites of plants and animals that have evolved diverse and often specific mechanisms to promote a given parasitic
lifestyle (Baldwin et al. 2004; Jasmer et al. 2003), including modifications of developmental and reproductive potential,
dissemination amongst and location of primary or alternate hosts, and survival strategies in the absence of a suitable host
or favorable environment. The genetic pathways underlying these lifecycle adaptations may have parallels with or origins in
nonparasitic nematode species that must also adapt to a dynamic or unstable niche. Distinct to the parasites, however, are
adaptations to obtain organic nutriment while living in or on another organism. The products of such parasitism genes “may be manifested as morphological structures that provide access to parasitism of a particular host (e.g. a nematode stylet)
or they may play critical physiological roles in the interaction of the nematode with its host” (Davis et al. 2000).
... Consequently, both genetic identities and environmental conditions must be measured and rigorously controlled during the host pathogen assays (Arelli 1994). In both resistant and susceptible cultivars, the interaction between the nematode and soybean root passed through several discernable phases (Mahalingam and Skorupska 1996; Davis et al. 2004; Gao et al. 2004). However, resistance or susceptibility of the soybean was not induced until females establish a feeding site. ...
Host resistance to "yellow dwarf" or "moonlight" disease cause by any population (Hg type) of Heterodera glycines I., the soybean cyst nematode (SCN), requires a functional allele at rhg1. The host resistance encoded appears to mimic an apoptotic response in the giant cells formed at the nematode feeding site about 24-48 h after nematode feeding commences. Little is known about how the host response to infection is mediated but a linked set of 3 genes has been identified within the rhg1 locus. This study aimed to identify the role of the genes within the locus that includes a receptor-like kinase (RLK), a laccase and an ion antiporter. Used were near isogeneic lines (NILs) that contrasted at their rhg1 alleles, gene-based markers, and a new Hg type 0 and new recombination events. A syntenic gene cluster on Lg B1 was found. The effectiveness of SNP probes from the RLK for distinguishing homolog sequence variants on LgB1 from alleles at the rhg1 locus on LgG was shown. The resistant allele of the rhg1 locus was shown to be dominant in NILs. None of the recombination events were within the cluster of the three candidate genes. Finally, rhg1 was shown to reduce the plant root development. A model for rhg1 as a dominant multi-gene resistance locus based on the developmental control was inferred.
... These have been identified from a range of nematodes including Meloidogyne, Globodera, Bursaphelenchus, Aphelenchus, Ditylenchus, Xiphinema and Pratylenchus Bellafiore et al., 2008;Danchin et al., 2010;Haegeman et al., 2010Haegeman et al., , 2011aJones et al., 2009;Karim et al., 2009;Kikuchi et al., 2009;Kudla et al., 2005;Qin et al., 2004). Cellulose binding proteins consist of a signal peptide and a carbohydrate binding module and have been identified from RKNs and cyst nematodes (Adam et al., 2008;Ding et al., 1998;Gao et al., 2004). Although their potential role in migration is not clear, it was shown that a cellulose binding protein from Heterodera schachtii is able to bind to a plant pectin methylesterase. ...
Plant pathogens have evolved a variety of different strategies that allow them to successfully infect their hosts. Plant-parasitic nematodes secrete numerous proteins into their hosts. These proteins, called effectors, have various functions in the plant cell. The most studied effectors to date are the plant cell wall degrading enzymes, which have an interesting evolutionary history since they are believed to have been acquired from bacteria or fungi by horizontal gene transfer. Extensive genome, transcriptome and proteome studies have shown that plant-parasitic nematodes secrete many additional effectors. The function of many of these is less clear although during the last decade, several research groups have determined the function of some of these effectors. Even though many effectors remain to be investigated, it has already become clear that they can have very diverse functions. Some are involved in suppression of plant defences, while others can specifically interact with plant signalling or hormone pathways to promote the formation of nematode feeding sites. In this review, the most recent progress in the understanding of the function of plant-parasitic nematode effectors is discussed.
... and Meloidogyne spp. These CBPs consist of a signal peptide, in some cases an unknown N-terminal sequence and a CBM (Adam et al. 2008;Ding et al. 1998;Gao et al. 2004a;. Recently, a functional study suggested that CBP targets and/or activates a plant pectin methylesterase, to reduce the level of methylesterification of pectin in the cell wall. ...
In this article, the characterization and evolution of pseudo-endoglucanases and a putative expansin-like gene in the migratory nematode Ditylenchus africanus are described. Four genes were cloned with a very high similarity to the endoglucanase Da-eng1, which, however, lack a part of the catalytic domain most probably due to homologous recombination. Owing to this deletion, at least one of the catalytic residues of the corresponding protein is missing, and hence these genes are possibly pseudogenes. In two of the pseudo-endoglucanase genes, the deletions cause a frameshift (Da-engdel2, Da-engdel4), while two others (Da-engdel1, Da-engdel3) code for protein sequences with an intact carbohydrate-binding module (CBM). Recombinant proteins for Da-ENG1, Da-ENGDEL1, and Da-ENGDEL3 were demonstrated to bind to cellulose, while only Da-ENG1 showed cellulose-degrading activity. This indicates that Da-ENGDEL1 and Da-ENGDEL3 which lack cellulase activity, could still exert a function similar to cellulose-binding proteins (CBPs). Next to the pseudo-endoglucanases, a putative expansin-like gene (Da-exp1) was identified, consisting of a signal peptide, an expansin-like domain, and a CBM. This domain structure was never found before in nematode expansin-like proteins. Interestingly, the CBM of the expansin-like gene is very similar to the endoglucanase CBMs, and a conserved intron position in the CBM of nematode endoglucanases, expansin-like genes, and CBPs indicates a common origin for these domains. This suggests that domain shuffling is an important mechanism in the evolution of cell wall-modifying enzymes in nematodes.
... Variation among the host plant roots response to SCN has been associated with light, temperature and genetic purity such that genetic identities and environmental conditions must be rigorously controlled during host pathogen assays [2,3]. In both resistant and susceptible cultivars, the interaction between the nematode and soybean root passes through several discernable phases [9,10, 11]. However, resistance or susceptibility of the soybean is not induced until females establish a feeding site. ...
Soybean (Glycine max L. Merr.) resistance to populations (HgType) of _Heterodera glycines I._, the soybean cyst nematode (SCN), requires a functional allele at rhg1. An apoptosis-like response in the giant cells formed around the nematode results 24-48 h after feeding commences. This study aimed to identify the role of the three genes within the rhg1 locus, a receptor like kinase (RLK), a laccase and an ion anti-porter. Used were near isogeneic lines (NILs) that contrasted at their rhg1 alleles. Features of the rhg1 locus, the candidate genes and their nascent transcripts and proteins in roots were elucidated. First, evidence for a syntenic gene cluster was found and the effectiveness of SNP probes for distinguishing the homeolog sequence variant on linkage group (Lg) B1 from alleles at the rhg1 locus on Lg G was shown. Analysis of plant s heterozygous at rhg1 showed that the allele for resistance was dominant. The absence of recombination events among the NILs between the RLK and other 2 genes eliminated the possibility of a monogeneic rhg1 locus. Finally, an effect on root development was discovered. A model for multigeneic resistance based on developmental control of root growth including a mechanism for segregation distortion is presented.
... Endo-1,4-b-glucanases were also found to be secreted by cyst nematodes. ENG-1 and ENG-2 were found to be released during penetration and intracellular migration by the soybean cyst nematode (De Boer et al., 1999; Wang et al., 1999; Yan et al., 2001) and the tobacco cyst nematode (). Gao et al. (2004) showed that three endo-1,4-b-glucanases are secreted by Heterodera glycines during infection of soybean. Secretion of two other endo-1,4-b-glucanases was also shown for the migratory root-lesion nematode Pratylenchus penetrans (Uehara et al., 2001). ...
* In plants, UDP-glucuronic acid is synthesized by the oxidation of UDP-glucose by UDP-glucose dehydrogenase or the oxygenation of free myo-inositol by myo-inositol oxygenase (MIOX). In Arabidopsis, myo-inositol oxygenase is encoded by four genes. Transcriptome analysis of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots revealed that MIOX genes are among the most strongly upregulated genes. * We have used beta-glucuronidase (GUS) analysis, in situ reverse transcription polymerase chain reaction (RT-PCR), and real-time RT-PCR to study the expression of all four MIOX genes in syncytia induced by H. schachtii in Arabidopsis roots. All these methods showed that MIOX genes are strongly induced in syncytia. GeneChip data were analysed for the expression of genes related to the MIOX pathway (mapman). * Two complementary double mutants were used to study the importance of MIOX genes. Results of the infection assay with double mutants in two combinations (Deltamiox1+2, Deltamiox4+5) showed a significant reduction (P < 0.05) in the number of females per plant when compared with the wild-type. Furthermore, syncytia in double mutants were significantly smaller than in wild-type plants. * Our data demonstrate an important role of the MIOX genes for syncytium development and for the development of female nematodes.
... rostochiensis and H. glycines (order Tylenchida)) produce and secrete β-1,4-endoglucanase (cellulases). This finding constituted the starting point of a series of papers reporting a range of plant cell wall-degrading enzymes (CWDE) from plant parasitic nematodes, including pectate lyases (27,28), exo-polygalacturonase (29), endo-ß-1,3-glucanase (30), endoxylanases (31), expansins (32) and cellulose-binding proteins (33,34). Nematodes are devoid of plant cell wall-like structures, and hence it seems safe to state that plant cell wall penetration by parasitic nematodes is the result of mechanical weakening and local depolymerization. ...
Horizontal gene transfer (HGT) implies the non-sexual exchange of genetic material between species – in some cases even across kingdoms. Although common among Bacteria and Archaea, HGTs from pro- to eukaryotes and between eukaryotes were thought to be extremely rare. Recent studies on intracellular bacteria and their hosts seriously question this view. Recipient organisms could benefit from HGT as new gene packages could allow them to broaden or change their diet, colonize new habitats, or survive conditions that previously would have been lethal.
... Hg-Con22 and Hg-Con26 are most similar to pancreatic (Kunitz) trypsin inhibitors (Table 1) and may serve a function in the digestive track (Marchbank et al., 1998) or secreted outside the nematode to inhibit external serine proteases in the host (Brandon and Friedman, 2002). A BLAST search of the uniprot database with Hg-Con28 identified similarity (E = 9 × 10 −14 ) with an H. glycines cellulose binding protein (Gao et al., 2004) that is synthesized in a subventral esophageal gland and, thereby, potentially secreted into the host (Gao et al., 2003). It remains to be determined if the two putative Kunitz trypsin inhibitor-like proteins, the cellulose binding-like protein, or any of the other remaining 6 selected unigenes are synthesized in an esophageal gland or amphid where they might be secreted into the host. ...
Two subtraction libraries were prepared from RNA extracted at early and late stages in the development of soybean cyst nematodes (SCN), Heterodera glycines, in soybean roots. The cDNA from inoculated roots were subtracted with cDNA prepared from non-inoculated roots and SCN eggs, and 384 clones from each library were sequenced. BLAST searches revealed that 191 of the cDNA in the late library were most probably of nematode origin. Alignment of the 191 sequences produced 28 unigenes and 1 singlet. The size of the transcripts for the nematode genes was confirmed by RNA blot hybridization. Thirteen SCN transcripts were selected for further study because they included short open reading frames encoding predicted proteins of <20 kDa with signal peptides at their amino-terminus. Ten of the 13 encode predicted peptides <10 kDa. Although most of the 13 transcripts were fairly abundant in the SCN dbEST, most were of unknown function based on BLAST similarities. Nevertheless, several had characteristics common to anti-microbial peptides, and in situ hybridization indicated that three of the selected transcripts were expressed in the female reproductive system.
... In the phylum Nematodea, CBMs have been found in secretions of the root knot nematode Meloidogyne incognita (51) and in the soybean cyst nematode Heterodera glycines (65), and recently it was demonstrated that nematodes express expansin during plant feeding (110,159). ...
Polysaccharide-degrading microorganisms express a repertoire of hydrolytic enzymes that act in synergy on plant cell wall and other natural polysaccharides to elicit the degradation of often-recalcitrant substrates. These enzymes, particularly those that hydrolyze cellulose and hemicellulose, have a complex molecular architecture comprising discrete modules which are normally joined by relatively unstructured linker sequences. This structure is typically comprised of a catalytic module and one or more carbohydrate binding modules (CBMs) that bind to the polysaccharide. CBMs, by bringing the biocatalyst into intimate and prolonged association with its substrate, allow and promote catalysis. Based on their properties, CBMs are grouped into 43 families that display substantial variation in substrate specificity, along with other properties that make them a gold mine for biotechnologists who seek natural molecular "Velcro" for diverse and unusual applications. In this article, we review recent progress in the field of CBMs and provide an up-to-date summary of the latest developments in CBM applications.
... The intronless HG-eng-5 could possibly result from total erasure of introns as described for eIF2γ genes in some protists, fungi and deuterostomates (Krauss et al., 2005). Besides glycosyl hydrolases, genes encoding cellulose-binding proteins (CBP) have been identified in M. incognita and H. glycines that could participate to the loosening of the plant cell wall during parasitism (Ding et al., 1999;Gao et al., 2004b). Similarly to nematode endoglucanases, these proteins contain a CBD homologous to bacteria family II CBDs but are devoid of catalytic domain. ...
Cellulases from plant parasitic nematodes are encoded by multiple gene families and are thought to originate from horizontal gene transfer. Unraveling the evolution of these genes in the phylum will help understanding the evolution of plant parasitism in nematodes. Here we describe a new gene, named MI-eng-2, that encodes a family 5 glycosyl hydrolase (GHF5) with a predicted signal peptide and devoid of linker domain and cellulose-binding domain. The beta-1,4-endoglucanase activity of the protein MI-ENG-2 was confirmed in vitro and the transcription of the gene was localized in the secretory oesophageal glands of infective juveniles, suggesting that MI-ENG-2 is involved in plant cell wall degradation during parasitism. Phylogenetic and exon/intron structure analyses of beta-1,4-endoglucanase genes in the order Tylenchida strengthen the hypothesis that nematode GHF5 genes result from horizontal gene transfer of a bacterial gene with a cellulose-binding domain. GHF5 gene families in Tylenchida result from gene duplications associated with occasional loss of the cellulose-binding domain and the linker domain during their evolution.
... Therefore, only some of the cyst that reproduce on the roots of a resistant genotype have the potential to form a new Hg type. In both resistant and susceptible cultivars, the interaction between the nematode and soybean root passes through several discernable phases (Mahalingham and Skorupska 1996; Davis et al. 2004; Gao et al. 2004). However, resistance or susceptibility of the soybean is not induced until females establish a feeding site. ...
The rhg1 gene or genes lie at a recessive or co-dominant locus, necessary for resistance to all Hg types of the soybean (Glycine max (L.) Merr.) cyst nematode (Heterodera glycines I.). The aim here was to identify nucleotide changes within a candidate gene found at the rhg1 locus that were capable of altering resistance to Hg types 0 (race 3). A 1.5 +/- 0.25 cM region of chromosome 18 (linkage group G) was shown to encompass rhg1 using recombination events from four near isogenic line populations and nine DNA markers. The DNA markers anchored two bacterial artificial chromosome (BAC) clones 21d9 and 73p6. A single receptor like kinase (RLK; leucine rich repeat-transmembrane-protein kinase) candidate resistance gene was amplified from both BACs using redundant primers. The DNA sequence showed nine alleles of the RLK at Rhg1 in the soybean germplasm. Markers designed to detect alleles showed perfect association between allele 1 and resistance to soybean cyst nematode Hg types 0 in three segregating populations, fifteen additional selected recombination events and twenty-two Plant Introductions. A quantitative trait nucleotide (QTN) [corrected] in the RLK at rhg1 was inferred that alters A87 to V87 in the context of H274 rather than N274. [corrected] Contiguous DNA sequence of 315 kbp of chromosome 18 (about 2 cM) contained additional gene candidates that may modulate resistance to other Hg-types including a variant laccase, a hydrogen-sodium ion antiport and two proteins of unknown function. A molecular basis for recessive and co-dominant resistance that involves interactions among paralagous disease-resistance genes was inferred that would improve methods for developing new nematode-resistant soybean cultivars.
... Endo-1,4-b-glucanases were also found to be secreted by cyst nematodes. ENG-1 and ENG-2 were found to be released during penetration and intracellular migration by the soybean cyst nematode (De Boer et al., 1999; Wang et al., 1999; Yan et al., 2001) and the tobacco cyst nematode (). Gao et al. (2004) showed that three endo-1,4-b-glucanases are secreted by Heterodera glycines during infection of soybean. Secretion of two other endo-1,4-b-glucanases was also shown for the migratory root-lesion nematode Pratylenchus penetrans (Uehara et al., 2001). ...
Cyst nematodes induce root syncytia with specific features such as hypertrophy, increased metabolic activity and fusion with adjacent cells. Cell walls of the syncytia undergo massive changes such as thickening, local dissolution and formation of ingrowths. Cell wall degrading and modifying proteins are apparently involved in syncytium formation but detailed knowledge of this is still limited. Therefore, we studied the regulation and function of the entire Arabidopsis endo-1,4-beta-glucanase gene family in syncytia induced by Heterodera schachtii. Endo-1,4-beta-glucanases hydrolyze the 1,4-beta-glucosidic linkages between glucose residues. Using semi-quantitative and quantitative approaches we identified seven genes that are upregulated in syncytia. Two of these genes, coding for secreted AtCel2 and membrane-bound KOR3, are shoot-specific but show high expression in syncytia at different developmental stages. In silico analysis of the promoter regions of both genes compared with other genes with modified regulation in nematode feeding sites did not reveal specific cis-acting elements that could be related to specific transcription in syncytia. However, motifs responsive to sugar and different plant hormones were identified. Accordingly, treatments with sucrose, gibberellic acid and NAA induced upregulation of AtCel2, whereas ABA triggered downregulation of both AtCel2 and KOR3 in roots. As AtCel2 is related to degradation of the cell wall matrix, we analysed the hemicellulose content in syncytia. The measured values resembled the expression pattern of AtCel2. A distinctly reduced number of females developed in cel2 and kor3 T-DNA mutants, and we therefore conclude that endo-1,4-beta-glucanases play an important role in the formation and function of syncytia.
Root-knot and cyst nematodes are two groups of plant parasitic nematodes that cause the majority of crop losses in agriculture. As a result, these nematodes are the focus of most nematode effector research. Root-knot and cyst nematode effectors are defined as secreted molecules, typically proteins, with crucial roles in nematode parasitism. There are likely hundreds of secreted effector molecules exuded through the nematode stylet into the plant. The current research has shown that nematode effectors can target a variety of host proteins and have impacts that include the suppression of plant immune responses and the manipulation of host hormone signaling. The discovery of effectors that localize to the nucleus indicates that the nematodes can directly modulate host gene expression for cellular reprogramming during feeding site formation. In addition, plant peptide mimicry by some nematode effectors highlights the sophisticated strategies the nematodes employ to manipulate host processes. Here we describe research on the interactions between nematode effectors and host proteins that will provide insights into the molecular mechanisms underpinning plant–nematode interactions. By identifying the host proteins and pathways that are targeted by root-knot and cyst nematode effectors, scientists can gain a better understanding of how nematodes establish feeding sites and subvert plant immune responses. Such information will be invaluable for future engineering of nematode-resistant crops, ultimately fostering advancements in agricultural practices and crop protection.
[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.
Root-knot nematodes pose a significant threat to agricultural production all over the world. They are ubiquitous, polyphagous, sedentary endoparasites and belong to genus, Meloidogyne. These biotrophic pathogens establish and maintain an intimate interaction with their host plants. Effectors are crucial for the entry of RKNs to the root of plants, subsequently leading to the formation of the specialized hypertrophied and multinucleate feeding sites for nematode growth and development. During the establishment and maintenance of nematode feeding sites, effectors are considered to play a critical role in the modulation of developmental and defence signaling pathways in host cells. Chemical nematicides are widely used in RKNs management strategies, despite their threat to environmental and human health. Recently, restrictions on using some chemicals have been enforced. So, there is a need of potential environmentally benign strategies to the management of RKNs. We will highlight most the effective strategies viz., biological control by bacteria and fungi and organic amendment by botanicals and oil cakes. Also taken into account are traditional strategies like cultural method, soil solarization and resistant cultivars. One of the best alternatives to chemical control of RKNs is biological control, which involves the use of predators and parasites to reduce the RKNs at various life stages (eggs, juveniles and adults), as well as the secretion of poisonous diffusible inhibitory metabolites which hamper the reproduction ability of RKNs. Here, we provide a brief review of the biology of the genus, Meloidogyne, plant-nematode interactions and various environmentally benign strategies.
Au Canada, le nematode a kyste du soya, Heterodera glycines (NKS), a d’abord ete detecte en Ontario en 1988 et plus recemment au Quebec en 2013. Il est la principale cause des pertes economiques associees a la production de soya au Canada et aux Etats-Unis. Les moyens de lutte contre ce ravageur sont limites et reposent essentiellement sur l’exploitation des genes de resistance naturels du soya. II est primordial d’etudier la reponse du NKS ainsi que celle de son hote sous le climat du Quebec afin de developper des strategies de lutte efficaces. De plus, les fluctuations de temperature associees aux changements climatiques modifieront les traits biologiques du NKS, la phenologie de son hote et leur interaction. Cela pourrait ainsi affecter l’efficacite et la durabilite des outils de gestion. Dans ce contexte, l’impact des changements climatiques anticipes sur les interactions entre le nematode a kyste du soya et son hote principal, le soya, sera presente.
Substantial progress has been made over the past decade in our understanding of nematode parasitism of plants that is now being applied to devise novel management strategies for nematodes through the use of biotechnology. The main research focus has been on the discovery and functional analysis of genes that enable nematodes to parasitize plants. Plant-parasitic nematodes are equipped with an arsenal of parasitism-associated genes which encode for secreted proteins that are expressed in their oesophageal glands. The expressed proteins are released into the apoplast or cytoplasm of host cells through the nematode stylet for the establishment of successful parasitic associations. Understanding the nature of the secreted products of nematode parasitism genes is expanding our knowledge of what makes a nematode a plant parasite. Moreover, this knowledge has unveiled key targets for the genetic manipulation of plants for developing novel resistance strategies against nematodes.
Plant cell wall signaling, or more generally cell wall performance and integrity control, is thought to play crucial roles in the regulation of plant growth and development in the presence of abiotic and biotic stresses. While, analogous to the well-characterized cell wall integrity response in yeast, the hallmarks of plant cell wall signaling are stress-induced global alterations in the expression of genes related to cell wall biosynthesis and remodeling, its molecular players are only beginning to become defined at the genetic level. Biochemical, molecular, and genetic studies have implicated cell wall signaling with the response to various plant pathogens including fungi and bacteria. Here we speculate how cell wall performance and integrity control might be involved in the infection of roots by sedentary plant-parasitic nematodes. We recapitulate that analogous to various typical cell wall stress scenarios, changes in the expression of cell wall–related genes are a major characteristic of nematode infection.
Sedentary plant-parasitic nematodes induce sophisticated feeding sites in roots of many crop plants causing tremendous yield losses in agriculture worldwide. The most damaging genera are cyst and root-knot nematodes forming syncytia and giant cells embedded in root galls, respectively. In addition to many morphological changes, elaborated cell wall alterations play an important role during the formation of both feeding sites and thus are essential for survival and reproduction of these parasites. Plant-parasitic nematodes were the first animals shown to produce cell wall modifying and degrading proteins and enzymes. Already during the migration through the root tissue, infective juveniles secrete e.g. cellulases and expansins, which, in addition to mechanical cell wall disruption performed with the stylet, considerably facilitate the infection process. At the begining of sedentary stage secretions from the dorsal gland trigger the formation of a feeding site in the central cylinder. These widely unknown substances change the host gene expression affecting, in addition to many others, also cell wall-related processes leading to various architectural modifications in host cells. Several cellulolytic and pectolytic enzymes as well as expansins are specifically expressed and play an important function during the formation of the feeding sites and development of the nematodes. This chapter summarizes data on cell wall modifications in plant tissues infected with cyst and root-knot nematodes as well as on involvement of different proteins, of both nematode and plant origin, during migration and sedentary phase of nematode parasitism.
Nematodes are considered one of the most difficult organisms to identify due to their microscopic size, morphological similarity, limited number of distinguishable taxonomic characters and overlapping morphometric measurements. However, there are few trained nematode taxonomists remaining, primarily due to retirement without replacement and/or young scientists' lack of interest in classical taxonomy. Due to the continuing decline in classical taxonomic expertise of many taxa including Nematoda, there is an increasing reliance on developing molecular-based diagnostic protocols to identify pests and pathogens. To improve the resolution and reliability of nematode phylogenetic and diagnostic studies, they should ideally combine morphological with molecular data. However, few of the published studies have compared phylogenetic trees derived from morphological/morphometric characters with those derived from molecular data. Furthermore, nematode sequences deposited in public databases should have an identification based on morphological and morphometrical characters ascertained by a trained taxonomist. Nevertheless, molecular diagnostics do offer new opportunities in Nematology, such as improving the capacity to deal with large numbers of samples or with species mixtures which have previously been impractical. In conclusion, a balanced molecular and morphological taxonomic approach is required as proposed by the integrative taxonomy concept. Classical taxonomy and molecular diagnostics should be considered as complementary.
The cell wall is a complex structure mainly composed by a cellulose-hemicellulose network embedded in a cohesive pectin matrix. Pectin is synthesized in a highly methyl esterified form and is de-esterified in muro by pectin methyl esterases (PMEs). The degree and pattern of methyl esterification affect the cell wall structure and properties with consequences on both the physiological processes of the plants and their resistance to pathogens. PME activity displays a crucial role in the outcome of the plant-pathogen interactions by making pectin more susceptible to the action of the enzymes produced by the pathogens. This review focuses on the impact of pectin methyl esterification in plant-pathogen interactions and on the dynamic role of its alteration during pathogenesis.
We report a new and improved pPZP vector (pPZP3425) for efficient plant transformation. This vector is derived from the widely
used pPZP100 series of binary Agrobacterium vectors. One disadvantage of these vectors is the use of chloramphenicol resistance for selection in Escherichia coli and Agrobacteria. We have therefore included a kanamycin resistance gene for selection in Agrobacterium. Furthermore, the strong 35S CaMV promoter driving the plant resistance gene has been replaced by the weaker nos promoter because it has been shown that the 35S promoter driving the plant resistance marker can lead to ectopic expression
of the transgene. During replacement of the 35S promoter, the NcoI site within the plant resistance gene has been removed,
and NcoI can now be used for cloning purposes within the expression cassette which consists of an intron-containing gus gene driven by a strong constitutive promoter (35S promoter with doubled enhancer plus omega-element as translational enhancer).
Thus, a single vector can conveniently be used for two purposes: (1) for overexpression of proteins by replacing the gus gene by the coding sequence of choice and (2) for creation of promoter:gus fusions by substituting the constitutive promoter by any other promoter. We demonstrate the usefulness of this vector for
cloning a promoter:gus fusion and in planta transformation of Arabidopsis.
The cellulose-binding protein gene, cbp, of Meloidogyne javanica encodes a protein that contains a signal secretion peptide and is expressed in the eggs and pre-invasive stage J2s but not from adult females. Several highly similar forms were found in the three root knot nematode species, M. javanica, Meloidogyne incognita and Meloidogyne arenaria indicating very limited divergence of the orthologues and paralogues. The genomic sequences included three introns which differed in size between species and between isolates of M. incognita and M. arenaria, however, the coding regions of the various forms were highly similar. In M. javanica only one form of cbp was found. Treatment of pre-invasive stage J2 nematodes with cbp-dsRNA resulted in a reduction in the ability of J2s to penetrate tomato roots and consequently in a reduction in the number of egg masses produced by the cpb-dsRNA treated nematodes compared to the control nematodes, consistent with an important role of CBP in the early stages of penetration and invasion.
The soybean cyst nematode, Heterodera glycines, adversely affects the production of soybean, Glycine max, in many areas of the world, particularly in the United States, where it is the most economically important soybean pathogen. Despite the availability of hundreds of H. glycines-resistant soybean cultivars, the nematode continues to be a major limiting factor in soybean production. The use of nonhost rotation and resistance are the primary means of reducing losses caused by the nematode, but each of these options has disadvantages. As a subject for study of nematode parasitism and virulence, H. glycines provides a useful model despite its obligately parasitic nature. Its obligately sexual reproduction and ready adaptation to resistant cultivars, formerly referred to as "race shift," presents an excellent opportunity for the study of virulence in nematodes. Recent advances in H. glycines genomics have helped identify putative nematode parasitism genes, which, in turn, will aid in the understanding of nematode pathogenicity and virulence and may provide new targets for engineering nematode resistance.
Parasitism of the cyst nematode Heterodera schachtii is characterized by the formation of syncytial feeding structures in the host root. Syncytia are formed by the fusion of root cells, accompanied by local cell wall degradation, fusion of protoplasts and hypertrophy. Expansins are cell wall-loosening proteins involved in growth and cell wall disassembly. In this study, we analysed whether members of the expansin gene family are specifically and developmentally regulated during syncytium formation in the roots of Arabidopsis thaliana. We used PCR to screen a cDNA library of 5-7-day-old syncytia for expansin transcripts with primers differentiating between 26 alpha- and three beta-expansin cDNAs. AtEXPA1, AtEXPA3, AtEXPA4, AtEXPA6, AtEXPA8, AtEXPA10, AtEXPA15, AtEXPA16, AtEXPA20 and AtEXPB3 could be amplified from the library. In a semi-quantitative RT-PCR and a Genechip analysis AtEXPA3, AtEXPA6, AtEXPA8, AtEXPA10 and AtEXPA16 were found to be upregulated specifically in syncytia, but not to be transcribed in surrounding root tissue. Histological analyses were performed with the aid of promoter::GUS lines and in situ RT-PCR. Results from both approaches supported the specific expression pattern. Among the specifically expressed genes, AtEXPA3 and AtEXPA16 turned out to be of special interest as they are shoot-specific in uninfected plants. We conclude that syncytium formation involves the specific regulation of expansin genes, indicating that the encoded expansins take part in cell growth and cell wall disassembly during syncytium formation.
Summary The beet cyst nematodeHeterodera schachtii is able to establish a feeding structure (syncytium) in the vascular tissue of roots and shoots ofArabidopsis thaliana. Histological and ultrastructural studies were performed to assess plant responses during the development of juvenile females under monoxenic conditions. After destructively invading a root the nematode selects and pierces a single procambial cell with its stylet and transforms it into an initial syncytial cell (ISC) by secretory activity. The first most obvious changes in the ISC occur in the vacuolar system and at the wall. Differentiation of a central vacuole is impeded resulting in the formation of numerous small vacuoles. Multivesicular and paramural bodies are formed. An electron translucent material is deposited on the cell wall. Partial dissolution of the cell wall leads to the formation of a syncytium. At the juveniles' last pre-adult developmental stage the syncytium attains its maximum longitudinal and radial extension, occupying a major part of the central cylinder. Its features are indicative of a very high level of metabolic activity. The hypertrophied syncytium is ensheathed by a peridermal cover in which secondary xylem and phloem elements are interspersed. When females die the syncytia degenerate. The ultrastructural and histological features of syncytia described from roots are also found in syncytia induced in aerial parts of the plant.
β-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing β-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37–44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3′ end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.
• endoglucanase
• Globodera
• Heterodera
• in situ hybridization
• secretory protein
Several types of domain occur in beta-1, 4-glycanases. The best characterized of these are the catalytic domains and the cellulose-binding domains. The domains may be joined by linker sequences rich in proline or hydroxyamino acids or both. Some of the enzymes contain repeated sequences up to 150 amino acids in length. The enzymes can be grouped into families on the basis of sequence similarities between the catalytic domains. There are sequence similarities between the cellulose-binding domains, of which two types have been identified, and also between some domains of unknown function. The beta-1, 4-glycanases appear to have arisen by the shuffling of a relatively small number of progenitor sequences.
Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.
The crystal structure of a family-III cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface-exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.
We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks
trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction
schemes and can easily be applied on genome-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal
signal-anchor sequences is also possible, though with lower precision. Predictions can be made on a publicly available WWW
server.
beta-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing beta-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37-44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3' end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.
Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 &mgr;g mL-1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 &mgr;g mL-1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.
The genomic organization of genes encoding beta-1,4-endoglucanases (cellulases) from the plant-parasitic cyst nematodes Heterodera glycines and Globodera rostochiensis (HG-eng1, Hg-eng2, GR-eng1, and GR-eng2) was investigated. HG-eng1 and GR-eng1 both contained eight introns and structural domains of 2151 and 2492bp, respectively. HG-eng2 and GR-eng2 both contained seven introns and structural domains of 2324 and 2388bp, respectively. No significant similarity in intron sequence or size was observed between HG-eng1 and HG-eng2, whereas the opposite was true between GR-eng1 and GR-eng2. Intron positions among all four cyst nematode cellulase genes were conserved identically in relation to the predicted amino acid sequence. HG-eng1, GR-eng1, and GR-eng2 had several introns demarcated by 5'-GCellipsisAG-3' in the splice sites, and all four nematode cellulase genes had the polyadenylation and cleavage signal sequence 5'-GAUAAA-3'-both rare occurences in eukaryotic genes. The 5'- flanking regions of each nematode cellulase gene, however, had signature sequences typical of eukaryotic promoter regions, including a TATA box, bHLH-type binding sites, and putative silencer, repressor, and enhancer elements. Database searches and subsequent phylogenetic comparison of the catalytic domain of the nematode cellulases placed the nematode genes in one group, with Family 5, subfamily 2, glycosyl hydrolases from Scotobacteria and Bacilliaceae as the most homologous groups. The overall amino acid sequence identity among the four nematode cellulases was from 71 to 83%, and the amino acid sequence identity to bacterial Family 5 cellulases ranged from 33 to 44%. The eukaryotic organization of the four cyst nematode cellulases suggests that they share a common ancestor, and their strong homology to prokaryotic glycosyl hydrolases may be indicative of an ancient horizontal gene transfer.
Polyclonal sera specific to beta-1,4-endoglucanases (cellulases) synthesized in the subventral esophageal gland cells of the soybean cyst nematode, Heterodera glycines, were used to provide the first identification of a nematode esophageal gland protein that is secreted into host plant tissue. Sera generated to proteins encoded by Hg-eng-1 and Hg-eng-2 (endoglucanases) did not cross-react with soybean root proteins on Western blots (immunoblots) or in immunofluorescence microscopy of noninoculated (control) soybean root sections. In cross sections of soybean roots at 24 h after inoculation of roots with second-stage juveniles of H. glycines, HG-ENG-1 was localized within the nematode's subventral gland cells and was not detected in root tissue. HG-ENG-2 was localized within the subventral gland cells and was secreted from the juvenile's stylet into root cortical tissue at 24 h after inoculation of roots with second-stage juveniles of H. glycines. HG-ENG-2 was localized along the juvenile's migratory path through the root cortex.
Two beta-1,4-endoglucanases (EGases), Hg-eng-1 and Hg-eng-2, were recently cloned from the soybean cyst nematode, Heterodera glycines, and their expression was shown in the subventral esophageal glands of hatched second-stage juveniles (J2). We examined the expression of these EGases in the subventral glands of all post-embryonic life stages of H. glycines by in situ hybridization and immunolocalization. The first detectable accumulation of EGase mRNAs occurred in the subventral glands of unhatched J2. EGase transcripts remained detectable in J2 after hatching and during subsequent root invasion. However, in late parasitic J2 and third-stage juveniles (J3), the percentage of individuals that showed EGase transcripts decreased. In female fourth-stage juveniles and adult females, EGase transcripts were no longer detected in the subventral glands. EGase hybridization signal reappeared in unhatched males coiled within the J3 cuticle, and transcripts were also present in the subventral glands of migratory adult males. Immunofluorescence labeling showed that EGase translation products are most abundantly present in the subventral glands of preparasitic J2, migratory parasitic J2, and adult males. The presence of EGases predominantly in the migratory stages suggests that the enzymes are used by the nematodes to soften the walls of root cells during penetration and intracellular migration.
A beta-1,4-endoglucanase encoding cDNA (EGases, E.C. 3.2.1.4), named Mi-eng-1, was cloned from Meloidogyne incognita second-stage juveniles (J2). The deduced amino acid sequence contains a catalytic domain and a cellulose-binding domain separated by a linker. In M. incognita, the gene is transcribed in the migratory J2, in males, and in the sedentary adult females. In pre-parasitic J2, endoglucanase transcripts are located in the cytoplasm of the subventral esophageal glands. The presence of beta-1,4-endoglucanase transcripts in adult females could be related to the expression of the gene in esophageal glands at this stage. However, cellulase activity within the egg matrix of adult females suggests that the endoglucanase may also be synthesized in the rectal glands and involved in the extrusion of the eggs onto the root surface. The maximum identity of the predicted MI-ENG-1 catalytic domain with the recently cloned cyst nematode beta-1,4-endoglucanases is 52.5%. In contrast to cyst nematodes, M. incognita pre-parasitic J2 were not found to express a beta-1,4-endoglucanase devoid of a cellulose-binding domain.
The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
Cyst nematodes and root-knot nematodes elaborately transform cells within the vascular cylinders of plant roots into enlarged, multinucleate, and metabolically active feeding cells. The giant cells of root-knot nematodes are formed by repeated karyokinesis uncoupled from cytokinesis, whereas the syncytia formed by cyst nematodes arise from coordinated cell wall dissolution and the coalescing of cell cytoplasm of adjacent cells. Both giant cells and syncytia undergo extensive cell wall architectural modifications, including thickening and the formation of numerous ingrowths that increase the plasmalemma surface area for solute uptake. The origin of enzymes involved in these cell wall modifications has been the subject of debate for several decades. Immunolocalization of endo-beta-1,4-glucanases (EGases) secreted from cyst nematodes was observed in root cortical tissue during the intracellular migration of the nematodes, but secretion of cyst nematode EGases into developing syncytia was not detected. We have identified five EGase genes from tobacco that are upregulated within plant roots upon infection by both root-knot and cyst nematodes. In situ localization of tobacco EGase transcripts demonstrated that their expression was specifically and developmentally upregulated within giant cells, syncytia, root tips, and lateral root primordia. These data confirm that cell wall modifications within plant-parasitic-nematode feeding cells arise from cell wall-modifying enzymes of plant, rather than nematode, origin.
The ability of nematodes to live on plant hosts involves multiple parasitism genes. The most pronounced morphological adaptations of nematodes for plant parasitism include a hollow, protrusible stylet (feeding spear) connected to three enlarged esophageal gland cells that express products that are secreted into plant tissues through the stylet. Reverse genetic and expressed sequence tag (EST) approaches are being used to discover the parasitism genes expressed in nematode esophageal gland cells. Some genes cloned from root-knot (Meloidogyne spp.) and cyst (Heterodera and Globodera spp.) nematodes have homologues reported in genomic analyses of Caenorhabditis elegans and animal-parasitic nematodes. To date, however, the candidate parasitism genes endogenous to the esophageal glands of plant nematodes (such as the ss-1,4-endoglucanases) have their greatest similarity to microbial genes, prompting speculation that genes for plant parasitism by nematodes may have been acquired by horizontal gene transfer.
Expansins are cell wall proteins that induce pH-dependent wall extension and stress relaxation in a characteristic and unique manner. Two families of expansins are known, named alpha- and beta-expansins, and they comprise large multigene families whose members show diverse organ-, tissue- and cell-specific expression patterns. Other genes that bear distant sequence similarity to expansins are also represented in the sequence databases, but their biological and biochemical functions have not yet been uncovered. Expansin appears to weaken glucan-glucan binding, but its detailed mechanism of action is not well established. The biological roles of expansins are diverse, but can be related to the action of expansins to loosen cell walls, for example during cell enlargement, fruit softening, pollen tube and root hair growth, and abscission. Expansin-like proteins have also been identified in bacteria and fungi, where they may aid microbial invasion of the plant body.
Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.
Cyst nematodes and root-knot nematodes elaborately transform cells within the vascular cylinders of plant roots into enlarged, multinucleate, and metabolically active feeding cells. The giant cells of root-knot nematodes are formed by repeated karyokinesis uncoupled from cytokinesis, whereas the syncytia formed by cyst nematodes arise from coordinated cell wall dissolution and the coalescing of cell cytoplasm of adjacent cells. Both giant cells and syncytia undergo extensive cell wall architectural modifications, including thickening and the formation of numerous ingrowths that increase the plasmalemma surface area for solute uptake. The origin of enzymes involved in these cell wall modifications has been the subject of debate for several decades. Immunolocalization of endo-β-1,4-glucanases (EGases) secreted from cyst nematodes was observed in root cortical tissue during the intracellular migration of the nematodes, but secretion of cyst nematode EGases into developing syncytia was not detected. We have identified five EGase genes from tobacco that are upregulated within plant roots upon infection by both root-knot and cyst nematodes. In situ localization of tobacco EGase transcripts demonstrated that their expression was specifically and developmentally upregulated within giant cells, syncytia, root tips, and lateral root primordia. These data confirm that cell wall modifications within plant-parasitic-nematode feeding cells arise from cell wall–modifying enzymes of plant, rather than nematode, origin.
Cellulose-binding domains (CBDs) are discrete protein modules found in a large number of carbohydrolases and a few nonhydrolytic proteins. To date, almost 200 sequences can be classified in 13 different families with distinctly different properties. CBDs vary in size from 4 to 20 kDa and occur at different positions within the polypeptides; N-terminal, C-terminal and internal. They have a moderately high and specific affinity for insoluble or soluble cellulosics with dissociation constants in the low micromolar range. Some CBDs bind irreversibly to cellulose and can be used for applications involving immobilization, others bind reversibly and are more useful for separations and purifications. Dependent on the CBD used, desorption from the matrix can be promoted under various different conditions including denaturants (urea, high pH), water, or specific competitive ligands (e.g. cellobiose). Family I and IV CBDs bind reversibly to cellulose in contrast to family II and III CBDs which are in general, irreversibly bound. The binding of family II CBDs (CBDCex) to crystalline cellulose is characterized by a large favourable increase in entropy indicating that dehydration of the sorbent and the protein are the major driving forces for binding. In contrast, binding of family IV CBDs (CBDN1) to amorphous or soluble cellulosics is driven by a favourable change in enthalpy which is partially offset by an unfavourable entropy change. Hydrogen bond formation and van der Waals interactions are the main driving forces for binding. CBDs with affinity for crystalline cellulose are useful tags for classical column affinity chromatography. The affinity of CBDN1 for soluble cellulosics makes it suitable for use in large-scale aqueous two-phase affinity partitioning systems.
Most cellulolytic enzymes consist of distinct catalytic and cellulose-binding domains (CBDs). Similar domain structures are also found in enzymes degrading other insoluble carbohydrates such as raw starch and chitin. Such binding domains improve the binding and facilitate the activity of the catalytic domain on the insoluble but not on soluble substrates. Based on their amino acid sequence similarities, the CBDs have been divided into several different families. Structure determination and subsequent mutagenesis studies have revealed that CBDs rely on several aromatic amino acids for binding to the cellulose surfaces. The CBDs binding to crystalline cellulose have different topologies but share similar rigid backbone structures for correct positioning of the side chains required for the substrate recognition and binding. CBDs represent ideal affinity tags for specific immobilisation of various other proteins to cellulose. Furthermore, improved understanding and control of their action will be important for the improvement of the biotechnological value of cellulolytic enzymes.
Polymerase chain reaction (PCR) with degenerate primers was performed on cDNA from Pratylenchus penetrans to amplify a conserved region in β-1,4-endoglucanase genes. Two distinct genomic DNA fragments that encoded partial β-1,4-endoglucanase sequences were isolated. Two corresponding full-length cDNAs, designated Pp-eng-1 and Pp-eng-2, were obtained by 3′ and 5′ rapid amplification of cDNA ends. Pp-eng-1 and Pp-eng-2 consisted of 1521 nucleotides encoding 455 amino acids and 1179 nucleotides encoding 328 amino acids, respectively. The two cloned genes encoded family 5 β-1,4-endoglucanases. The predicted protein PP-ENG-1 contained a cellulose-binding domain (CBD), and the predicted protein PP-ENG-2 lacked a CBD.
Bands that have cellulolytic activity are visualized after polyacrylamide gel electrophoresis by laying the slab gel on top of a thin sheet of 2% agar containing 0.1% carboxymethylcellulose. After a suitable incubation time, zones of carboxymethylcellulose hydrolysis are revealed by staining the agar replica with Congo red.
The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RT-PCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues.
Dynamic programming algorithms to determine similar regions of two sequences are useful for analyzing biosequence data. This paper presents a time-efficient algorithm that produces k best “non-intersecting” local alignments for any chosen k. The algorithm's main strength is that it needs only O(M + N + K) space, where M and N are the lengths of the given sequences and K is the total length of the computed alignments.
SUMMARY The soybean cyst nematode, Heterodera glycines, produces beta-1,4-endoglucanases (cellulases) that are secreted during infection of soybean. The gene structures of three, hg-eng-4, hg-eng-5 and hg-eng-6, of the six beta-1,4-endoglucanase genes, all family 5 glycosyl hydrolases previously identified from H. glycines, are presented here. Furthermore, we present the detailed expression analyses of beta-1,4-endoglucanase genes as well as the biochemical properties of four H. glycines endoglucanase enzymes. Two of the endoglucanases, HG-ENG-5 and HG-ENG-6, differed significantly in their amino acid sequence of the catalytic domains and their gene structure from that of the other four beta-1,4-endoglucanases. Quantitative real-time RT-PCR revealed distinct developmental expression differences among the hg-eng family members during the early stages of parasitism and relatively low expression levels in late parasitic stages, with the exception of the adult male stage for some eng genes. Recombinant HG-ENGs degraded carboxymethylcellulose and optimum enzyme activity ranged from pH 5.5 for HG-ENG-5 to pH 8 for HG-ENG-6. EDTA, Ca(2+), Co(2+), Mg(2+) and Fe(2+) did not affect enzyme activity of any ENG protein, whereas Zn(2+), Cu(2+) and Mn(2+) inhibited enzyme activity from 23% to 73% in some cases. In tests with 12 different polysaccharide substrates, enzyme activity was restricted to beta-1,4 linkages with all ENG proteins tested. Only HG-ENG-5 and HG-ENG-6 had relatively high activity on xylan and slightly degraded microcrystalline cellulose. Together, these data reveal distinct differences in expression and biochemistry of cyst nematode parasitism genes and proteins, respectively, and cast light on the intricate interactions between a parasitic animal and its plant host.
Two beta-1,4-endoglucanase (EGase) cDNAs were isolated from Globodera tabacum, the tobacco cyst nematode, and have been designated as GT-eng-1 and GT-eng-2. GT-eng-1 and GT-eng-2 encode precursor proteins with a predicted secretion signal sequence, cellulolytic catalytic domain, and a linker domain. The protein product GT-ENG-1 contains an additional 95 amino acid carboxy terminal sequence with strong similarity to type II cellulose binding domains. Riboprobes and polyclonal antibodies raised to recombinant cyst nematode EGases were used to follow expression patterns of EGase transcripts and proteins throughout the nematode life cycle. EGase transcripts and proteins were specifically detected within the subventral esophageal gland cells of G. tabacum second-stage juveniles (J2) within eggs prior to hatching, in preparasitic J2, and in parasitic J2 that had invaded tobacco roots. EGase transcripts and proteins were not detected in G. tabacum after the molt to the sedentary J3, J4, and adult female life stages. Interestingly, EGase transcription and translation resumed in the subventral esophageal glands of late J4 males. It is hypothesized that secreted EGases play a major role to facilitate intracellular migration of G. tabacum within tobacco roots.
The nucleotide sequence of engD, an endo-beta-1,4-glucanase gene from Clostridium cellulovorans was determined (Genbank Accession No. M37434). The COOH-terminal part of the gene product, EngD, contained a Thr-Thr-Pro repeated sequence followed by a region that has homology to the exoglucanase of Cellulomonas fimi. EngD and EngB, another C. cellulovorans endoglucanase, show 75% amino acid sequence homology at their NH2-termini, in contrast to their carboxyterminal domains which show no homology. EngD had endoglucanase activity on carboxymethylcellulose (CMC), cellobiosidase activity on p-nitrophenyl-cellobioside (p-NPC), and partial hydrolytic activity on crystalline cellulose (Avicel), while EngB showed hydrolytic activity against only CMC. Chimeric proteins between EngB and EngD were constructed by exchanging the non-homologous COOH-terminal regions. Chimeric proteins that contained the NH2-terminus of EngD retained cellobiosidase activity but chimeras with the EngB NH2-terminus showed no cellobiosidase activity. Hydrolysis of crystalline cellulose (Avicelase activity) was observed only with the enzyme containing the EngD NH2-terminus and EngD COOH-terminus.
The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)
The complete nucleotide sequence of the gene coding for one of the carboxymethylcellulases (CMCase), expressed by Pseudomonas fluorescens subsp. cellulosa, has been determined. The structural gene consists of an open reading frame, commencing with an ATG start codon, of 2886 base pairs followed by a TAA stop codon. The gene was shown to code for a signal peptide which closely resembles the signal peptides of other secreted proteins. Unlike most Pseudomonas genes, the CMCase sequence does not have a high G + C (51%) content and there is no marked preference for codons ending in G or C. Upstream of the structural gene there are no sequences which bear a strong resemblance to consensus Escherichia coli promoters. A sequence is present, however, which exhibits homology to the consensus DNA sequence that binds the catabolic activator protein (CAP). Bal31 deletions of the structural gene revealed the extent by which the gene could be modified and still encode a functional CMCase. Subclones of the cellulase gene have been constructed in pUC18 and pUC19. One of the resultant plasmids, pJHS1 directs a 20-fold increase in CMCase synthesis, when compared to the original construct, pJHH2. Analysis of cells harbouring pJHS1 showed the cellulase polypeptide to have a molecular weight of 106000. This is in close agreement with the predicted size of the enzyme deduced from the nucleotide sequence data.
Multidimensional, multinuclear nuclear magnetic resonance spectroscopy combined with dynamical simulated annealing has been used to determine the structure of a 110 amino acid cellulose-binding domain (CBD) from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi (CBDcex). An experimental data set comprising 1795 interproton NOE-derived restraints, 50 phi, 34 chi 1, and 106 hydrogen bond restraints was used to calculate 20 final structures. The calculated structures have an average root-mean-square (rms) deviation about the mean structure of 0.41 A for backbone atoms and 0.67 A for all heavy atoms when fitted over the secondary structural elements. Chromatography, ultracentrifugation, and 15N NMR relaxation experiments demonstrate that CBDcex is a dimer in solution. While attempts to measure NOEs across the dimer interface were unsuccessful, a computational strategy was employed to generate dimer structures consistent with the derived data set. The results from the dimer calculations indicate that, while the monomer topologies produced in the context of the dimer can be variable, the relative positioning of secondary structural elements and side chains present in the monomer are restored upon dimer formation. CBDcex forms an extensive beta-sheet structure with a beta-barrel fold. Titration with cellohexaose, [beta-D-glucopyranosyl-(1,4)]5-D-glucose, establishes that Trp 54 and 72 participate in cellulose binding. Analysis of the structure shows that these residues are adjacent in space and exposed to solvent. Together with other proximate hydrophilic residues, these residues form a carbohydrate-binding cleft, which appears to be a feature common to all CBDs of the same family.
Cellulose is the major polysaccharide of plants where it plays a predominantly structural role. A variety of highly specialized
microorganisms have evolved to produce enzymes that either synergistically or in complexes can carry out the complete hydrolysis
of cellulose. The structure of the major cellobiohydrolase, CBHI, of the potent cellulolytic fungus Trichoderma reesei has
been determined and refined to 1.8 angstrom resolution. The molecule contains a 40 angstrom long active site tunnel that may
account for many of the previously poorly understood macroscopic properties of the enzyme and its interaction with solid cellulose.
The active site residues were identified by solving the structure of the enzyme complexed with an oligosaccharide, o-iodobenzyl-1-thio-beta-cellobioside.
The three-dimensional structure is very similar to a family of bacterial beta-glucanases with the main-chain topology of the
plant legume lectins.
The crystal structure of the catalytic domain of the thermostable endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose has been solved by multiple isomorphous replacement and refined at 2.4 A resolution to an R-factor of 0.18 (Rfree = 0.24). E1cd is a member of the 4/7 superfamily of hydrolases, and as expected, its structure is an (alpha/beta)8 barrel, which constitutes a prototype for family 5-subfamily 1 cellulases. The cellotetraose molecule binds in a manner consistent with the expected Michaelis complex for the glycosylation half-reaction and reveals that all eight residues conserved in family 5 enzymes are involved in recognition of the glycosyl group attacked during cleavage. Whereas only three residues are conserved in the whole 4/7 superfamily (the Asn/Glu duo and the Glu from which the name is derived), structural comparisons show that all eight residues conserved in family 5 have functional equivalents in the other 4/7 superfamily members, strengthening the case that mechanistic details are conserved throughout the superfamily. On the basis of the structure, a detailed sequence of physical steps of the cleavage mechanism is proposed. A close approach of two key glutamate residues provides an elegant mechanism for the shift in the pKa of the acid/base for the glycosylation and deglycosylation half-reactions. Finally, purely structural based comparisons are used to show that significant differences exist in structural similarity scores resulting from different methods and suggest that caution should be exercised in interpreting such results in terms of implied evolutional relationships.
A cDNA encoding a secretory cellulose-binding protein was cloned from the root-knot nematode (Meloidogyne incognita) with RNA fingerprinting. The putative full-length cDNA, named Mi-cpb-1, encoded a 203 amino acid protein containing an N-terminal secretion signal peptide. The C-terminal sequence of the putative MI-CBP-1 was similar to a bacterial-type cellulose-binding domain, whereas the N-terminal sequence did not show significant similarity to any proteins in data bases. Recombinant MI-CBP-1 lacked cellulase activity, but bound to cellulose and plant cell walls. In Southern blot hybridization, Mi-cbp-1 hybridized with genomic DNA from M. incognita, M. arenaria, and M. javanica, but not M. hapla, Heterodera glycines, or Caenorhabditis elegans. Polyclonal antibodies raised against recombinant MI-CBP-1 strongly labeled secretory granules in subventral gland cells of second-stage juveniles in indirect immunofluorescence microscopy. Enzyme-linked immunosorbent assay detection of MI-CBP-1 in stylet secretions of second-stage juveniles with the polyclonal antibodies indicated MI-CBP-1 could be secreted through the nematodes' stylet, suggesting that the cellulose-binding protein may have a role in pathogenesis.
Cellulose-binding domains (CBDs) are discrete protein modules found in a large number of carbohydrolases and a few nonhydrolytic proteins. To date, almost 200 sequences can be classified in 13 different families with distinctly different properties. CBDs vary in size from 4 to 20 kDa and occur at different positions within the polypeptides; N-terminal, C-terminal and internal. They have a moderately high and specific affinity for insoluble or soluble cellulosics with dissociation constants in the low micromolar range. Some CBDs bind irreversibly to cellulose and can be used for applications involving immobilization, others bind reversibly and are more useful for separations and purifications. Dependent on the CBD used, desorption from the matrix can be promoted under various different conditions including denaturants (urea, high pH), water, or specific competitive ligands (e.g. cellobiose). Family I and IV CBDs bind reversibly to cellulose in contrast to family II and III CBDs which are in general, irreversibly bound. The binding of family II CBDs (CBD(Cex)) to crystalline cellulose is characterized by a large favourable increase in entropy indicating that dehydration of the sorbent and the protein are the major driving forces for binding. In contrast, binding of family IV CBDs (CBD(N1)) to amorphous or soluble cellulosics is driven by a favourable change in enthalpy which is partially offset by an unfavourable entropy change. Hydrogen bond formation and van der Waals interactions are the main driving forces for binding. CBDs with affinity for crystalline cellulose are useful tags for classical column affinity chromatography. The affinity of CBD(N1) for soluble cellulosics makes it suitable for use in large-scale aqueous two-phase affinity partitioning systems.
A method for the objective evaluation of the hearing threshold using cortical evoked response audiometry was developed. The method results in a kind of objective audiogram, visualizing the significance of an auditory evoked potential (AEP) in a scheme similar to a conventional audiogram. In the present implementation of the method, four frequencies are tested quasi-simultaneously (500, 1000, 2000 and 3000 Hz; intensity steps of 5 dB). The significance of an evoked potential is assessed by means of the Rayleigh test, which is applied to the phase values derived from certain time windows of the single-trial epochs. A retrospective analysis of 1, 920 threshold estimations in 240 subjects suggested that the detection threshold (lowest stimulus intensity yielding a significant response) was, on the average, 7.5 dB above the electrophysiological threshold (intensity where the AEP amplitude vanishes). The grand-average amplitude-intensity characteristic was approximated by the function a(1 - exp(-I/b)), with a = 6.25 microV, b = 22.3 dB and I representing the intensity (in decibels) relative to the electrophysiological threshold.
We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequences. The method performs significantly better than previous prediction schemes, and can easily be applied to genome-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal signal-anchor sequences is also possible, though with lower precision. Predictions can be made on a publicly available WWW server: http://www.cbs.dtu.dk/services/SignalP/.
A beta-1,4-endoglucanase named MI-ENG1, homologous to the family 5 glycoside hydrolases, was previously isolated from the plant parasitic root-knot nematode Meloidogyne incognita. We describe here the detection of the enzyme in the nematode homogenate and secretion and its complete biochemical characterization. This study is the first comparison of the enzymatic properties of an animal glycoside hydrolase with plant and microbial enzymes. MI-ENG1 shares many enzymatic properties with known endoglucanases from plants, free-living or rumen-associated microorganisms and phytopathogens. In spite of the presence of a cellulose-binding domain at the C-terminus, the ability of MI-ENG1 to bind cellulose could not be demonstrated, whatever the experimental conditions used. The biochemical characterization of the enzyme is a first step towards the understanding of the molecular events taking place during the plant-nematode interaction.
Cloning parasitism genes encoding secretory proteins expressed in the esophageal gland cells is the key to understanding the molecular basis of nematode parasitism of plants. Suppression subtractive hybridization (SSH) with the microaspirated contents from Heterodera glycines esophageal gland cells and intestinal region was used to isolate genes expressed preferentially in the gland cells of parasitic stages. Twenty-three unique cDNA sequences from a SSH cDNA library were identified and hybridized to the genomic DNA of H. glycines in Southern blots. Full-length cDNAs of 21 clones were obtained by screening a gland-cell long-distance polymerase chain reaction cDNA library. Deduced proteins of 10 clones were preceded by a signal peptide for secretion, and PSORT II computer analysis predicted eight proteins as extracellular, one as nuclear, and one as plasmalemma localized. In situ hybridization showed that four of the predicted extracellular clones were expressed specifically in the dorsal gland cell, one in the subventral gland cells, and three in the intestine in H. glycines. The predicted nuclear clone and the plasmalemma-localized clone were expressed in the subventral gland cells and the dorsal gland cell, respectively. SSH is an efficient method for cloning putative parasitism genes encoding esophageal gland cell secretory proteins that may have a role in H. glycines parasitism of soybean.
Cellulose is one of the most abundant polymers in nature. Different living systems evolved simultaneously, using structurally similar proteins to synthesize and metabolize polysaccharides. In the growing plant, cell wall loosening, together with cellulose biosynthesis, enables turgor-driven cell expansion. It has been postulated that endo-1,4-beta-glucanases (EGases) play a central role in these complex activities. Similarly, microorganisms use a consortium of lytic enzymes to convert cellulose into soluble sugars. Most, if not all, cellulases have a modular structure with two or more separate independent functional domains. Binding to cellulose is mediated by a cellulose-binding domain (CBD), whereas the catalytic domain mediates hydrolysis. Today, EGases and CBDs are known to exist in a wide range of species and it is evident that both possess immense potential in modifying polysaccharide materials in-vivo and in-vitro. The hydrolytic function is utilized for polysaccharide degradation in microbial systems and cell wall biogenesis in plants. The CBDs exerts activity that can be utilized for effective degradation of crystalline cellulose, plant cell wall relaxation, expansion and cell wall biosynthesis. Applications range from modulating the architecture of individual cells to an entire organism. These genes, when expressed under specific promoters and appropriate trafficking signals can be used to alter the nutritional value and texture of agricultural crop and their final products. EGases and CBDs may also find applications in the modification of physical and chemical properties of composite materials to create new materials possessing improved properties.
The feeding sites induced by sedentary root-endoparasitic nematodes have long fascinated researchers. Nematode feeding sites are constructed from plant cells, modified by the nematode to feed itself. Powerful new techniques are allowing us to begin to elucidate the molecular mechanisms that produce the ultrastructural features in nematode feeding cells. Many plant genes that are expressed in feeding sites produced by different nematodes have been identified in several plant species. Nematode-responsive plant genes can now be grouped in categories related to plant developmental pathways and their roles in the making of a feeding site can be illuminated. The black box of how nematodes bring about such elaborate cell differentiation in the plant is also starting to open. Although the information is far from complete, the groundwork is set so that the functions of the plant and nematode genes in feeding site development can begin to be assessed.
A chitinase full-length cDNA (designated Hg-chi-1) was isolated from a Heterodera glycines oesophageal gland cell-specific long-distance PCR cDNA library. The cDNA hybridised to genomic DNA of H. glycines in Southern blots. The Hg-chi-1 cDNA contained an open reading frame encoding 350 amino acids with the first 23 amino acids being a putative signal peptide for secretion. Hg-CHI-1 contained a chitinase 18 family catalytic domain, and chitinolytic activity of recombinant Hg-CHI-1 was confirmed in glycol-chitin substrate gel electrophoresis. In situ mRNA hybridisation analyses showed that transcripts of Hg-chi-1 accumulated specifically in the subventral oesophageal gland cells of parasitic stages of H. glycines, but Hg-chi-1 expression was not detected in eggs or hatched pre-parasitic second-stage juveniles, suggesting that this chitinase does not have a role in egg hatching of H. glycines. The biological function of Hg-CHI-1 in H. glycines remains to be determined.
Parasitism genes expressed in the esophageal gland cells of phytonematodes encode secretions that control the complex process of plant parasitism. In the soybean cyst nematode, Heterodera glycines, the parasitome, i.e., the secreted products of parasitism genes, facilitate nematode migration in soybean roots and mediate the modification of root cells into elaborate feeding cells required to support the growth and development of the nematode. With very few exceptions, the identities of these secretions are unknown, and the mechanisms of cyst nematode parasitism, therefore, remain obscure. The most direct and efficient approach for cloning parasitism genes and rapidly advancing our understanding of the molecular interactions during nematode parasitism of plants is to create gland cell-specific cDNA libraries using cytoplasm microaspirated from the esophageal gland cells of various parasitic stages. By combining expressed sequence tag analysis of a gland cell cDNA library with high throughput in situ expression localization of clones encoding secretory proteins, we obtained the first comprehensive parasitome profile for a parasitic nematode. We identified 51 new H. glycines gland-expressed candidate parasitism genes, of which 38 genes constitute completely novel sequences. Individual parasitome members showed distinct gland cell expression patterns throughout the parasitic cycle. The parasitome complexity discovered paints a more elaborate picture of host cellular events under specific control by the nematode parasite than previously hypothesized.
Expansin proteins, which have so far been identified only in plants, rapidly induce extension of plant cell walls by weakening the non-covalent interactions that help to maintain their integrity. Here we show that an animal, the plant-parasitic roundworm Globodera rostochiensis, can also produce a functional expansin, which it uses to loosen cell walls when invading its host plant. As this nematode is known to be able to disrupt covalent bonds in plant cell walls, its accompanying ability to loosen non-covalent bonds challenges the prevailing view that animals are genetically poorly equipped to degrade plant cell walls.
Most phytoparasitic nematodes infect plant roots and some species have evolved sophisticated interactive relationships with host cells to sustain a sedentary parasitic habit. The recent isolation of multiple 'parasitism genes' expressed specifically within the esophageal gland cells of sedentary phytonematodes suggests that an arsenal of different secreted parasitism proteins from the nematode might have direct effects on recipient host cells. These include cell wall modifications and potential interactions with signal transduction receptors in the extracellular space, as well as direct introduction of proteins into host cells that might influence cellular metabolism, the cell cycle, selective protein degradation, a localized defense response and regulatory activity within the host cell nucleus.
Nematode parasitism of plants Physiology and Biochemistry of Freeliving and Plant Parasitic Nematodes
- R S Hussey
- F M Grundler
Hussey, R.S., Grundler, F.M., 1998. Nematode parasitism of plants, in:
Perry, R.N., Wright, D.J. (Eds.), Physiology and Biochemistry of Freeliving and Plant Parasitic Nematodes. CAB International Press,
England, pp. 213–243.