The reniform nematode (RN), Rotylenchulus reniformis, is an agriculturally important pest with a broad host range that results in a large economic impact in tropical, subtropical and in warm temperate zones. In an initial effort to understand the transcriptome and gene expression in RN, we present EST results that reveal numerous putative parasitism-related genes some of which play roles in plant cell wall modification. The characterized contigs included 8362 (40.6%) matches to unique proteins. Coding contigs predicted were 10,656 (51.7%) or 3079 (14.9%), that was similar to those identified in Brugia malayi and Caenorhabditis elegans as reference organisms respectively. Specific transcripts studied in more detail include putative plant parasitism genes, prominent among them were several plant cell wall modification genes. Contigs matching 14 parasitism genes found in sedentary endoparasitic nematodes included expansins, hexosaminidase, glycosyl hydrolases family, 14-3-3 protein, xylanases, glutathione peroxidase, pectate lyase, β-1,4-endoglucanase, major sperm protein, aminopeptidase, c-type lectin, chitin synthase, FMR famide-like peptide, and calreticulin. These genes function in suppression of host defenses and development of feeding sites.
Rotylenchulus reniformis, commonly referred to as reniform nematode (RN), is a semi-endoparasitic nematode with a broad host range of over 300 plant species. Infections of RN begin when the female penetrates the root cortex via specialized nematode feeding cells that are regulated by nematode parasitism genes expressed within the esophageal glands, and delivered into the feeding cell through the stylet [1,2]. One major approach employed in expression profiling involving nematode parasitism is transcriptome sequencing [
The objective of this study was to generate sufficient coverage ESTs to permit identification of genes that are expressed at elevated levels in cDNA libraries prepared from eggs and vermiform life stages of the RN to identify candidate parasitism genes.
Eggs of RNs cultured on roots of greenhouse-grown Micro-Tom tomato plants, were extracted from the roots and surface disinfested by immersing them in 5% bleach with shaking for 4 minutes in a beaker. The egg-containing solution was then poured through a sterilized 325-mesh sieve nested on a 500-mesh sieve autoclaved using a dry cycle (120˚C for 1 hr). The trapped eggs on the 500-mesh sieve were rinsed immediately with ~300 mL of sterilized distilled water to wash off the bleach for about 5 minutes, and then transferred into sterilized beakers containing 10 mL of sterilized distilled water. One mL of the solution containing the sterilized eggs was placed onto agar plates, these were sealed with Parafilm, and covered with aluminum foil, and placed in an incubator set at 25˚C for 2 to 4 days for the eggs to hatch into Juvenile 1 (J1), and permit growth up to Juvenile 2 (J2) stage on the 8th day.
Total RNA was extracted from eggs and vermiform stages of pooled nematodes using the PicoPure RNA kit (LifeTechnologies, Grand Island, NY) and treated with RNase-free DNase (Qiagen) following the manufacturer’s instructions. The quality and concentration of the RNA was assessed using the ExperionTM RNA StdSen Analysis Kit (Bio-Rad Laboratories, Inc., Hercules, CA), and Nanodrop 100 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE).
These libraries were constructed using the CreatorTM SMARTTM cDNA library kit (Clonetech, CA, USA) using Long Distance PCR (LD PCR) according to the manufacturer’s instructions. Amplified products were purified using a Sigma Aldrich GeneEluteTM PCR CleanUp kit (Sigma-Aldrich, St. Louis, MO). Concentrations of these libraries were assessed using a Nanodrop 100 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) and TKO 100 fluorometer (Hoefer Scientific Instruments, San Francisco).
Purified constructed cDNA libraries (5 µg) were used in high-throughput sequencing at the Advanced Centre for Genome Technology, University of Oklahoma. The raw reads generated were assembled using the SeqMan Lasergene software (DNASTAR Inc., Madison WI, USA) after removal of primer sequences, poly (A/T) tails, and ribosomal sequences. All EST sequences generated were submitted to GenBank at NCBI under the short read archive (SRA) with accession numbers SRX098224 and SRX098225. BLAST 2.2.21 was downloaded from NCBI ftp://ftp.ncbi.nlm.nih.gov/blast/) and used in creating a local Blast database for initially comparing the RN sequence reads to reniform ESTs available in GenBank with expected value (E) of <1.0e−10. Downloaded reniform ESTs in GenBank [
The universal Gene Ontology annotation, visualization, and analysis tool, (Blast2go) http://blast2go.org was employed for annotation of the functions and identification of protein-coding genes within our ESTs. The gene ontology (GO) classification scheme was used in categorizing transcripts by their putative function. These analyses were categorized into molecular function, biological process, and cellular components. Candidate parasitism genes were identified by restricting BlastX search with expected value (E) of <1.0e−10. Potential homologs in other nematodes within the reniform ESTs were through comparisons with NCBI EST others database using TBlastX. Further, comparisons of our sequences were with ESTs of C. elegans, B. malayi, Meloidogyne incognita, and Pristionchus pacificus with E-values of <1.0e−10.
Over 50,000 sequence reads were generated from the RN transcriptome, resulting in more than 4 Mb (4,781,676 bases) of data that were assembled into 20,596 contigs (
The most highly represented activities under each of these categories were ATP binding (
house-keeping roles. TBlastX analysis produced 11,588 matches (56.3%) with assigned functions. Majority of hits were observed with B. malayi, (620/11,588 or 5.4%). Further comparative analysis between RN ESTs predicted to code for genes by AUGUSTUS using B. malayi as the model organism (10,656 ESTs) with BlastX hits (11,588) and ESTs with GO IDs (8362) resulted in 179 and 135 matches, respectively (
Comparative analysis between RN ESTs and M. incognita revealed that 10.5% of RN sequences matched those of M. incognita. The RN ESTs also had 0.8% matches to both B. malayi and C. elegans ESTs, although more EST contigs were predicted to code for genes (Figures 4 and 5). These low percentages could be as a result of phylogenetic distance of these nematodes from the RN, and also the nature of some of the ESTs which may not be of full length. The least organism having matches to the RN ESTs was P. pacificus (0.7%) (
Fourteen plant parasitic nematode genes critical to modification of plant cell walls were identified within our RN ESTs (
cleaving the non-covalent bonds and thus enhancing the activities of cell wall-degrading, carbohydrate-active enzymes (CAZymes) [20,21]. A second class of enzyme identified within the RN ESTs was hexosaminidase (glycosyl hydrolases), which is highly conserved across certain domains of bacteria and human. The β-Hexosaminidases (EC 3.2.1.52), has a specific role in removal of terminal protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) residues found in glycoproteins and glycolipids [
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caninum. Xylanase is a component of hemicellulose, which functions in cleaving β-1,4-linkage of xylopyranose subunits [
contigs encoded MSP genes suggesting the importance of the MSP in RN reproduction. Aminopeptidases are known to mediate processes such as neuropeptide and signal transduction [
This study has generated a number of novel transcripts with hitherto unknown function that will need further characterization. The nematode parasitism gene identified here could serve as RNAi targets for investigating plant resistance against RNs and be further explored through functional analysis for effective design of management strategies
This work was supported by grants to RVK and/or to GCS: USDA-CSREES Grant # 2004-38814-15160, USDA ALAX-011-706 and NSF/PGRP award #DBI 0703470 and Agricultural Experiment Station. This is Journal article # 656 of Alabama A & M Agricultural Experiment Station.
ATP: Adenosine Triphosphate;
EST: Expressed Sequence Tag;
GO: Gene Ontology;
LD PCR: Long Distance Polymerase Chain Reaction;
MSP: Major Sperm Protein;
NFS: Nematode Feeding Site;
SRA: Short Read Achieve.