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 Experion^TM 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 Creator^TM SMART^TM 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 GeneElute^TM 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 [3] were assembled into 107 contigs and 519 singletons, using the CLC Genomics Workbench 4.9 with the following parameters: Mismatch cost = 2, insertion cost = 3, deletion cost = 3, length fraction = 0.5, similarity = 0.9, alignment = global, conflict resolution = vote (A, C, G, and T), word size = automatic, non-specific matches = random and minimum contig length = 200. ESTs were then accessed for their coding potential using the AUGUSTUS gene prediction software [18] with B. malayi and C. elegans serving as model organisms. This tool employs a Generalized Hidden Markov Model (GHMM), defined by probability distributions for portions of genomic sequences thus identifying an optimal parse for a given genomic sequence and divides the sequence into states based on statistical models.

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 (Table 1). Assembly of the RN raw reads downloaded from GenBank resulted in 107 contigs and 519 singletons (626 unigenes). Comparison of our ESTs to those assembled from GenBank identified 617 entries out of which 553 were unique. The alignment lengths for homology varied from 32 to 754 bp, with similarities of greater than 77%. We therefore observed 209/626 (33.4%) unique GenBank ESTs with matches to our 410/20,596 (1.9%) sequence reads.

The most highly represented activities under each of these categories were ATP binding (Figure 1), oxidation-reduction (Figure 2), and integral to membrane (Figure 3) for molecular function, biological process, and cellular components respectively. Among the several contigs generated, 10,656 (51.7%) and 3079 (14.9%) coded for genes using B. malayi and C. elegans, as reference organisms respectively. More contigs coded for genes with B. malayi serving as a reference organism probably because of the parasitic nature of B. malayi as opposed to the free living nature of C. elegans. Sixteen contigs were predicted to code for genes both by C. elegans and B. malayi (Table 2). These contigs could further be studied because they may probably be involved in

Table 1. Reniform nematode transcriptome assembly.

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 (Figure 4). Similarly, when C. elegans was used as a model organism, 149 TBlastX hits and 99 ESTs with GO IDs matched with ESTs coding for genes (Figure 5). This analysis suggests that, majority of contigs predicted to code for genes have not been fully annotated within the reniform transcriptome. A full list of contigs with GOs and functions is available.

Table 2. Sixteen common EST contigs among C. elegans and B. malayi predicted by AUGUSTUS with annotations.

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%) (Figure 6). The complete list of Blastn analysis between RN ESTs and these nematodes is available

Fourteen plant parasitic nematode genes critical to modification of plant cell walls were identified within our RN ESTs (Table 3). These had expected values (E) of <1.0e−10 to regions of homology with parasitic genes in other nematodes (Table 4). The nematode parasitism genes identified include 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. The nematode parasitome consists mostly of secreted gene products critical in plant parasitism [19]. Among these are expansin and xylanase which are cellwall modifying proteins. Expansins digest cell walls by

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 [22]. The RNMSTG1_10661 contig had homology to the hexosaminidase gene (Cbr-hex-3) within B. malayi, suggesting the presence of this enzyme in the RN genome. Chitinases belong to glycosyl hydrolases family (EC 3.2.1.14), known to be involved in hydrolysis of β-1,4-N-acetyl-D-glucosamine linkages in chitin polymers. They catalyze reactions by hydrolyzing glycosidic bonds between two carbohydrate molecules, or a carbohydrate and a non-carbohydrate molecule, and have been identified in filarial nematodes [23,24]. The RNMSTG1_5271 contig had homology to a chitinase gene (Bm1_51425A) in B. malayi, indicating the involvement of this enzyme probably in hydrolysis of glycosidic bonds in chitin during RN parasitism. SXPRAL-2, 14-3-3, and RanBPM-like family proteins participate in regulation of cell-cycle, calcium ion binding, defense regulation mechanisms, and may act as molecular chaperones [20]. Within the RN ESTs, the ftt-2 gene belonging to 14-3-3 protein class was identified in the RNMSTG1_4096 contig, and had homologs in C. briggsae, C. elegans, C. brenneri, C. remanei, and Ancylostoma

Table 3. Candidate parasitism genes identified within the RN ESTs.

Continued

caninum. Xylanase is a component of hemicellulose, which functions in cleaving β-1,4-linkage of xylopyranose subunits [25]. The presence of xylanase in the RN ESTs, specifically in the RNMSTG1_6909 contig with homology to RS-xyl1 gene in Radopholus similis may suggest the importance of this enzyme in RN genome for the breakdown of xylan during parasitism. Glutathione peroxidases (GpX) plays roles in protecting animal parasitic nematodes through removal of hydrogen peroxide defensively released by plants in response to nematode attack [26]. This enzyme complex also catalyzes the conversion of peroxidised fatty acids to alcohols. The RNMSTG1_2532 contig had matches to gxp2 gene in G. rostochiensis while RNMSTG1_6876 contig had significant identity to gxp1, clle4.1, and Bm1_40465 genes in G. rostochiensis, C. elegans, and B. malayi, respectively, suggesting the importance of glutathione peroxidase in the RN for parasitism. The RNMSTG1_17025 contig encoded a pectate lyase gene (pel-1) gene previously cloned in M. incognita. Pectate lyases are commonly associated with proteomes of bacterial and fungal pathogens [27], and disrupt the glycosidic bonds in the primary cell wall and in middle lamella [28]. These enzymes act as pathogenicity factors, because of their disruptive nature to host cell walls [29]. A qPCR and RNAi analysis in H. glycines targeting pectate lyase revealed an increase in male: female ratios after J2 nematodes were soaked in a solution containing double-stranded (ds) RNA [30]. In another study involving M. incognita, two cDNAs (Mi-pel-1 and Mi-pel-2) encoding pectate lyase have been isolated from the esophageal gland-cell through subtractive cDNA libraries [31]. The β-1,4-endoglucanase gene identified in our RN ESTs, showed high homology to the endoglucanases of M. incognita and R. similis respectively. Cellulases belong to the glycosyl hydrolase family 5 and similarity of these genes to bacterial homologs suggests that, they may have been acquired through horizontal gene transfer (HGT) [1]. These genes have also been identified in H. glycines, G. rostochiensis, M. incognita, B. xylophilus, P. penetrans, and Heterodera spp. Respectively [32-40]. In invertebrates including nematodes, sperms serve as signals involved in meiosis during the arrested oocytes developmental stage [41]. The MSP has been identified as a 14.1-kD peptide using MALDI-TOF mass spectroscopy [42], and known to suppress Heterodera glycines reproduction in transgenic soybeans plants expressing specific siRNAs to the MSP gene [43]. The RNMSTG1_4729 and RNMSTG1_7752

Table 4. Candidate parasitism genes identified within the RN ESTs and their E-values.

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 [44], embryo nourishment and digestion [45], molting [46], and reproduction [47]. These enzymes can serve as targets for nematode control [48]. The RNMSTG1_10134, RNMSTG1_101, and RNMSTG1_16353 contigs had matches to lap-1, amp-1, and Bm1_08285 genes in C. elegans, H. glycines, and B. malayi, respectively. C-type lectin, a pathogen recognition protein was identified within the RN dataset. The RNMSTG1_1637 contig showed high homology to the clec-180 gene. In C. elegans silencing of this gene through RNAi, resulted in decrease in cyst nematode numbers [49]. The RNMSTG1_13256 contig encoded for the chitin synthase gene which has been previously silenced in M. artiellia hindering the formation and survival of the eggs in this nematode [50]. Chitin is an important component in the fungal cell wall, cuticle of insects [51], and nematodes [52]. This structure is composed of β-1,4 linked N-acetylglucosamine residue chains. FMRFamide-like peptides or FLPs identified in our ESTs are a class of neuropeptides with specific roles in sensory and motor activities in nematodes, and also serve as neurotransmitters or neuromodulators within the nervous system. Recently, two of these genes flp-12 and flp-16 have been cloned and characterized in H. avenae [53]. These genes have been proposed to serve as critical targets for plant parasitic nematode management. The C-terminal signature (Arg-Phe-NH2) of these peptides is highly conserved across C. elegans, A. suum, and M. incognita. Three of the reniform contigs coded for this class of genes. Calreticulins are a class of calcium-binding proteins, which are conserved in both plants and animals. These proteins are localized in the endoplasmic reticulum and primarily act as chaperones [54]. Other functions of calreticulins include transportation of proteins from the nucleus [54], mRNA degradation [55], cell adhesion [56], calcium regulation, cell cycle, endocytosis, exocytosis, secretion, and cell growth and differentiation [57]. Calreticulins are abundant in the esophageal secretions of nematodes especially at the nematode feeding sites (NFS). An example is Mi-CRT gene synthesized in the esophageal glands of M. incognita [58]. Three RN contigs were homologous with calreticulins of M. incognita and B. malayi respectively.

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