Use of solid‐phase subtractive hybridization for the identification of parasitism gene candidates from the root‐knot nematode Meloidogyne incognita

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Abstract
SUMMARY A solid-phase subtractive strategy was used to clone parasitism gene candidates (PGCs) expressed in the oesophageal gland cells of Meloidogyne incognita. Nematode intestinal first-strand cDNA was synthesized directly on magnetic beads and used to enrich for gland-specific sequences by high stringency hybridization to gland-cell mRNA. A gland-specific cDNA library was created from the nonhybridizing gland-cell mRNA by long-distance reverse transcription polymerase chain reaction. Subtraction of the gland cDNA library (1000 clones) with previously cloned M. incognita parasitism genes removed 89 cDNA clones and promoted efficient identification of new PGCs. Sequencing of 711 cDNA clones from the subtracted library revealed that deduced protein sequences of 67 cDNAs were preceded by a signal peptide for secretion, a key criterion for parasitism genes. In situ hybridization with probes from the cDNA clones encoding signal peptides showed that seven cDNA clones were specifically expressed in the subventral gland cells and four in the dorsal gland cell of M. incognita. BLASTP analyses revealed the predicted proteins of five cDNAs to be novel sequences. The six PGCs with similarities to known proteins included a pectate lyase, three beta-1,4-endoglucanases and two chorismate mutases. This subtractive protocol provides an efficient and reliable approach for identifying PGCs encoding oesophageal gland cell secretory proteins that may have a role in M. incognita parasitism of plants.