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

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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.