Mining the secretome of the root‐knot nematode Meloidogyne chitwoodi for candidate parasitism genes

Abstract

Parasite proteins secreted at the interface of nematode and host are believed to play an essential role in parasitism. Here, we present an efficient pipeline of bio-informatic algorithms and laboratory experiments to identify candidate parasitism genes within nematode secretomes, i.e. the repertoire of secreted proteins in an organism. We performed our approach on 12 218 expressed sequence tags (ESTs) originating from three life stages of the plant parasitic nematode Meloidogyne chitwoodi--a molecularly unexplored root-knot nematode species. The ESTs from M. chitwoodi were assembled into 5880 contigs and open reading frames translated from the consensus sequences were searched for features of putative signal peptides for protein secretion and trans-membrane regions, resulting in the identification of 398 secretome members. The products of parasitism genes are secreted by a range of organs, including the oesophageal, amphidial and rectal glands, the intestine, and the hypodermis. To localize the site of expression in M. chitwoodi, we subjected the most abundant secretome members to in situ hybridization microscopy. We found hybridization of one tag in the dorsal oesophageal gland, seven in the two subventral oesophageal glands, two in the intestine and one tag hybridized to the tail tip in the proximity of the phasmids. Four sequences showed similarity to putative parasitism genes from other nematode species, whereas seven represented pioneering sequences. Our approach presents an efficient method to identify candidate parasitism genes, which does not require sophisticated cDNA isolation and selection protocols, and can therefore be used as a powerful starting point for the molecular investigation of parasites.

Transcriptomic Analysis of Nematode Infestation

“…Several studies using the microaspiration approach identified over 50 different putative secreted parasitism genes from the esophageal glands of root-knot and cyst nematodes (Davis et al 2008 ). In contrast, Roze et al (2008) isolated only eight secreted gland expressed genes after screening more than 12,000 expressed sequence tags (ESTs) generated from whole nematodes (representing three developmental stages). Clearly, a targeted approach can uncover more putative genes involved in parasitism; however, it can be time consuming and require specialized equipment and expertise.…”

Degradation of the Plant Cell Wall by Nematodes

“…Functional assays of the Gr-EXP1 protein in plant tissues confirmed its expansin-like activity (Qin et al, 2004), representing the first report of an expansin-like protein outside of the plant kingdom. More recently, EST analyses have identified putative expansin-like sequences in several phytoparasitic nematode species including both sedentary and migratory endoparasites (Abad et al, 2008;Haegeman et al, 2009bHaegeman et al, , 2010Kikuchi et al, 2007Kikuchi et al, , 2009Opperman et al, 2008;Roze et al, 2008). The presence of expansin domains in the absence of a CBM has been reported in B. xylophilus and other phytoparasitic nematode species, providing evidence for hypotheses on the evolution of the expansin gene family in nematodes ).…”

Characterization of the venom allergen—like protein (vap-1) and the fatty acid and retinol binding protein (far-1) genes in Meloidogyne hispanica

“…These include the cell wall structure, manipulation of cell fate, protein synthesis and alteration of signaling pathways. Functional tests, using RNAi, have supported the putative role of some of these nematode effectors in pathogenesis (Bellafiore et al 2008;Roze et al 2008;Bellafiore and Briggs 2010).…”

“…FAR-1 induces susceptibility to RKN through the manipulation of jasmonate-dependent defence response (Iberkleid et al 2013). FAR-1 has also been identified in H. schachtii, M. chitwoodi, M. hapla, M. incognita, P. coffeae and Radopholus similis (Vanholme et al 2006;Bellafiore et al 2008;Jacob et al 2008;Opperman et al 2008;Roze et al 2008;Haegeman et al 2009Haegeman et al , 2011.…”

“…Although, the VAPs are part of a family of effectors considered to be conserved in all parasitic nematodes of plants and animals, its function is still unknown (Haegeman et al 2012). They have been identified and characterized from the RKNs M. incognita, M. hapla and M. chitwoodi, the cyst nematodes Heterodera glycines, H. schachtii and Globodera pallida, the root-lesion nematode Pratylenchus coffeae and the pinewood nematode Bursaphelenchus xylophilus (Ding et al 2000;Gao et al 2001;Vanholme et al 2006;Wang et al 2007;Opperman et al 2008;Roze et al 2008;Jones et al 2009;Kang et al 2010Kang et al , 2012Haegeman et al 2011Haegeman et al , 2012. Various allergen proteins were described as being highly transcribed during plant nematode parasitism (Ding et al 2000;Gao et al 2001;Wang et al 2007).…”