Comparison of two strains of Meloidogyne javanica differing in virulence on tomato with the resistance gene Mi, and identification of a polymorphism that correlates with avirulence

Two near-isogenic M. javanica strains that differ in their ability to reproduce on tomatoes carrying the resistance gene Mi (VW4 and VW5) were studied by AFLP-based DNA fingerprinting. The genomic AFLP analysis revealed that the two strains were similar at the DNA level. Therefore, expressed genes were compared by cDNA-AFLP, which identified two transcripts present in the avirulent strain VW4 but absent in the virulent strain VW5. One of these transcripts was polymorphic between the strains on DNA blots, and the corresponding gene was designated Cg-1.; Sequences homologous to Cg-1 were present in the root-knot nematode species M. incognita and M. arenaria, both of which are affected by Mi-mediated resistance, but Cg-1 was not present in M. hapla, a nematode unaffected by Mi. 3'RACE demonstrated that there were at least three different family members of Cg-1 expressed in the second stage juveniles of VW4 (Cg-1A, Cg-1B, and Cg-1C). Based on sequence comparisons, Cg-1C is most likely equivalent to Cg-1, with Cg-1A and C-1B corresponding to different family members. The RT-PCR and 3 'RACE data demonstrated that there were different family members of Cg-1, possibly involving alternatively spliced transcripts. 5'RACE identified two putative trans-splice sites, and together with the 3'RACE data, predicted two putative Cg-1 transcripts that are approximately 0.6 Kb and 1.2 Kb in size. RT-PCR analysis showed VW5 was missing at least of 750 nt of the Cg-1 transcript, which was consistent with the hypothesis that deletion or mutation of the Cg-1 transcript may have lead to virulence in VW5.; The Cg-1 transcript does not have a large open reading frame; therefore, it may be a non-coding RNA or encode a functional peptide. Attempts to identify the function of Cg-1 by RNA interference (RNAi) did not produce an observable phenotype. Agrobacterium rhizogenes -mediated root transformation and leaf transient assays were performed to test the expression of a construct called Cg-P1. The Cg-P1 construct contained part of the Cg-1 transcript and did not produce a detectable phenotype in either resistant or susceptible tomato. Although these functional analyses were inconclusive, Cg-1 has many novel attributes and serves as a useful marker that correlates with avirulence in M. javanica.