Molecular Cloning of a cDNA Encoding an Amphid-Secreted Putative Avirulence Protein from the Root-Knot NematodeMeloidogyne incognita

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Abstract
Amplified fragment length polymorphism fingerprinting of three pairs of Meloidogyne incognita near-isogenic lines (NILs) was used to identify markers differential between nematode genotypes avirulent or virulent against the tomato Mi resistance gene. One of these sequences, present only in the avirulent lines, was used as a probe to screen a cDNA library from second-stage juveniles (J2s) and allowed cloning of a cDNA encoding a secretory protein. The putative full-length cDNA, named map-1, encoded a 458 amino acid (aa) protein containing a predictive N-terminal secretion signal peptide. The MAP-1 sequence did not show any significant similarity to proteins deposited in databases. The internal part of the protein, however, was characterized by highly conserved repetitive motives of 58 or 13 aa. Reverse transcription polymerase chain reaction (RT-PCR) experiments confirmed that map-1 expression was different between avirulent and virulent NILs. In PCR reactions, map-1-related sequences were amplified only in nematode populations belonging to the three species against which the Mi gene confers resistance: M. arenaria, M. incognita, and M. javanica. Polyclonal antibodies raised against a synthetic peptide deduced from the MAP-1 sequence strongly labeled J2 amphidial secretions in immunofluorescence microscopy assays, suggesting that MAP-1 may be involved in the early steps of recognition between (resistant) plants and (avirulent) nematodes.