- To advance our understanding of the “ effectorome ” of P. - Our data provide valuable insights into nematode parasitism and contribute towards basic understating of the adaptation of P. - Full list of author information is available at the end of the article. - Members of the Pratylenchus genus are known as root lesion nematodes (RLNs) and belong to the third most important group of plant-parasitic nematodes (PPNs) [3]. - One of the common features of these migratory nematodes is their dynamic behavior within the host roots, i.e. - nematodes do not become sedentary and are able to migrate in and out of the roots, causing extensive damage as they do so.. - Pratylenchus penetrans is one of the most successful and economically devastating RLN species with a wide range distribution, associated with more than 400 hosts worldwide [4]. - Like other RLNs, it can enter the plant along the entire length of the root. - Once inside of the roots, nematodes migrate and feed almost exclusively from the cortical cells, where they cause mechanical damage, browning, and necrosis of the root tissue. - Infection of the roots by P. - penetrans often results in the release of phenolic compounds, oxidation of which has been associated with the browning of the root tissues [4].. - An important feature of all PPNs is the presence of a repertoire of secreted proteins (known as effectors), which are critical components determining the outcome of the plant-nematode interactions. - The majority of these effectors are synthesized in the esophageal glands of PPNs (one dorsal and two sub-ventral glands) and are ultimately secreted through the nematode stylet into plant tissues [8]. - During infection, PPNs can deploy dozens of different effectors that are capable of manipu- lating and suppressing key molecular pathways of the plant in order to complete their life cycle. - Although a growing number of nematode effectors have been analyzed [9], different parasitism strategies, host range, variability, and composition of the effector repertoire complicate their identification and characterization.. - A significant proportion of those predicted proteins have no homologues, and their functions are unknown, indicating a distinct complement of the P. - A high proportion of the predicted secreted proteins of P.. - [12], a large proportion of the reads. - Some of the key features for the identification of can- didate effectors, which are expected to be secreted by the classical ER-Golgi secretory pathway, are the pres- ence of a signal peptide and absence of a transmembrane domain. - Of the 11,514 transcripts with at least one read in the gland cell library encode putatively se- creted proteins. - 8 (Fragments Per Kilobase of Transcript per Million mapped reads), with the exception of the most highly represented bin due to a low n (FPKM >. - In some of the most highly represented bins, 80–100%. - Assuming representation in the library is a function of expression in the gland cells, these observations support the important secretory function of the nema- tode esophageal glands. - Remarkably of the genes validated previously were in this set of 230 transcripts with FPKM >. - 8 in the gland- cell library (Fig. - Most of the 21 previously validated genes ranked among the top of this list, when ranked by representation in the gland cell library (Additional file 1: Table S1). - A detailed examination of the remaining 209 transcripts revealed an extensive overlap with genes previously re- ported to be involved in parasitism of other PPNs (Fig. - It is noteworthy that the majority of the 230 transcripts encode novel proteins lacking any functional annotations or similar sequences in the NR database (BLAST e-value <. - 8), such as transcripts encoding an esophageal gland protein of the root-knot nematode (FPKM = 7.92), a chorismate mutase (FPKM = 6.7), among other candidate effectors. - Out of the 68 candidates tested,. - Following these results, we also validated the pres- ence of the CAA [A|G|T|C] TG [T|G] C motif previ- ously identified in the promoter region of some P.. - This motif was found in the promoter region of 12 genes (Additional file 5: Table S3), representing 40% of the total number of new candidate effectors.. - 8, with the exception of the most highly represented bin due to a low number of transcripts (FPKM. - penetrans of the same iso- late (Vieira and Nemchinov, unpublished), confirmed their nematode origin (0 = e-value <. - As most of the effectors found here were also novel, we quantified the levels of proline, cysteine and glycine resi- dues. - Twelve of the new candidate effectors were rich in proline, while others presented a high content of glycine residues (>. - 10% of the full protein sequence. - 2 Characterization of the most abundant transcripts encoding putative secreted proteins collected from the esophageal gland library of Pratylenchus penetrans. - penetrans, coupled with available genome and transcriptome sequences for many phylo- genetically well-positioned species, provides an oppor- tunity to investigate the evolutionary history of the effector repertoire. - Due to the high variability of the esophageal gland size among different specimens and nematode stages, both dorsal and subventral glands were labelled as esophageal glands. - We then searched for the presence of putative homologs of the entire P. - As evidence of the former, and despite the inclusion of three other Pratylenchus species in the analysis and the application of a relatively relaxed similarity threshold, 41% of the putative effectors (and 65% of the pioneer effectors) were unique to P. - Five putative effectors were common to three of the four Pratylenchus species, possibly indicative of disparate gene loss in the Pratylenchidae and in root- knot nematodes (given the monophyletic relationship with Pratylenchidae). - The simplest possible explanation could be an ancient origin of the gene, in the last common ances- tor of these species and subsequent convergent loss in sedentary endoparasitic root-knot and cyst nematodes.. - All of the selected genes could be detected within the nematode-infected roots and their expression varied (Fig. - penetrans, we took advantage of the transcriptome data previously generated from different plants, i.e., two dif- ferent cultivars of alfalfa and soybean, infected with the same isolate (Fig. - 100) during infection independently of the host genotype (e.g., a catalase, an. - Table 1 Characterization of candidate gene effector genes specifically localized in the esophageal glands of Pratylenchus penetrans.. - In this work, we conducted a comprehensive screening of potential effector proteins of the agriculturally im- portant nematode species P. - Comparative analyses of the gland cell transcriptome confirmed most of the previously described cases of gland-cell expression and, most importantly, drastically expanded the size of the roster of gland-cell expressed genes to double its previous value. - Of the new candidates identified, only four genes had a predicted functional domain or known annotation.. - Among them, transcripts encoding a second member of the SXP/RAL-2 family were validated to be gland-specific.. - Members of this gene family are characterized by the pres- ence of the DUF148 protein domain and can be found across different clades of nematodes [36]. - RAL-2 transcripts showed distinct localization in the sub- ventral esophageal glands of the root-knot nematode M.. - incognita [37] or in the epidermis and amphidial sheet cells of the cyst nematode G. - The bio- logical functions of the different members of this family are still lacking. - The secretion of these proteases has been linked to diverse functions, including host tissue penetration, modification of the host environment, destruction of plant defense pro- teins and digestion [43]. - Protease inhibitors on the other hand may be involved in the protection against degrad- ation by host proteases or manipulation of the host re- sponses. - always evidence of absence, the consistency of the pat- terns of presence and absence allowed us to inferred plausible evolutionary histories that shaped the effector repertoires of not just P. - species in the phylogeny. - pene- trans, thus, indicating substantial de novo gene gain (i.e., not neofunctionalization of the existing genetic pool).. - 5 Comparative analyses of the full set of candidate effector genes of Pratylenchus penetrans suggest a large-scale effector birth for this species. - Top panel corresponds to a schematic phylogeny of the phylum Nematoda based on 86 highly conserved CEGMA genes among plant- parasitic nematodes with different parasitism strategies and the free-living nematode Caenorhabditis elegans (Ce). - At the opposite end of the spectrum, many non-pioneer ef- fectors (i.e. - b Heat maps representing the expression profile of the full set of 53 candidate effectors identified so far for P. - Mi Mi-GST-1 [45] and MiPFN3 [46], play important roles in parasitism of the root-knot nematode M. - The cyst nematode effector HsIPT, which also lacks a signal peptide, is involved in the activation of the host cell cycle of the syncytium cells [47].. - pene- trans, that some of these proteins may be part of the molecular machinery of the esophageal glands rather than effectors per se. - penetrans is one of the most successful spe- cies in the genus and is able to infect a large number of plants, high diversity of its effectors may potentially cre- ate a strong molecular basis for this broad host range.. - One of the common features of host infection by RLNs is the massive damage induced in the root cortex as a consequence of migration and feeding activity by these nematodes. - Notable, 26 out of the 30 (86%) new can- didate effectors identified in this study represent genes without known domains, which demonstrates the lack of knowledge about this group of nematodes and warrants further investigation. - Future efforts should focus on the identification and characterization of the host targets of these effectors to determine their biological roles and provide crucial information for application of genetic engineering strategies for the control of PPNs. - Further- more, the results reported in this study may also con- tribute towards basic understanding of the adaptation of P. - The paired-end library totaled raw reads and the quality of the library was assessed with the pro- gram FASTQC (https://www.bioinformatics.babraham.. - The vast majority of the reads were greater than 144 bp in length. - The median quality value ranged from 32 to 40 over the length of the entire read with an overall per average sequence quality of 40. - Illumina RNA-seq reads of the esophageal gland cells were initially trimmed and mapped to the 23,715 tran- scripts generated for the same P. - Interpro were performed for the predicted proteins of the set of 230 transcripts using BLAST2GO [54] with default parameters. - 4.0 was used to confirm the presence/absence of protein signal peptide in the genome of the predicted proteins [56], and transmem- brane domains were predicted using TMHMM server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/).. - The quantity and quality of the. - penetrans [14], ap- proximately 600 nucleotides of the 5′ sequence from the start codon were manually extracted based on BLASTn coordinates against the draft genome of the same nema- tode isolate (Vieira and Nemchinov, unpublished). - These promoter regions were screened for the presence of the motif consensus CAA [A|G|T|C] TG [T|G]C.. - >1e − 10 ) against the draft genome of the same P. - and the draft genome of the burrowing nematode Rado- pholus similis [27]. - The abundance of the full set of candidate effectors were estimated as FPKM values and presented as heatmaps using previ- ously generated transcriptome data for P. - Summary of BLAST hit analyses of the top 230 most abundant transcripts of Pratylenchus penetrans encoding putative secreted proteins (FPKM >. - The 600 nt of the upstream region of the start codon was queried for the presence of the CAA[AG|T|C]TG[T|G] C motif previously found associated with gland cell expression of other candidate effectors of P. - TRM performed the preparation of nematodes and micro-aspiration of the gland cells. - Observations on the invasion and endoparasitic behavior of the root lesion nematode Pratylenchus penetrans. - The genome of the yellow potato cyst nematode, Globodera rostochiensis, revals insights into the basis of parasitism and virulence. - STATAWAARS: a promoter motif associated with spatial expression in the major effector-producing tissues of the plant-parasitic nematode Bursaphelenchus xylophilus. - Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans. - Analysis of the transcriptome of the root lesion nematode Pratylenchus coffeae generated by 454 sequencing technology. - Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita. - The genome of the soybean nematode (Heterodera glycines) reveals complex patterns of duplications involved in the evolution of parasitism genes. - SXP/RAL-2 proteins of the potato cyst nematode Globodera rostochiensis: secreted proteins of the hypodermis and amphids. - Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential. - Ultrastructure of esophageal glands and their secretory granules in the root-knot nematode Meloidogyne incognita.. - Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes. - The control of the false discovery rate in multiple testing under dependency
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