Use of MSAP for the identification of genes involved in the response of tomato to the infection by the geminivirus tomato leaf curl sardinia virus

Plants have developed several genetically regulated defense strategies against a wide range of biotic stresses (viruses, fungi, bacteria, nematodes). There is experimental evidence that DNA methylation and post-transcriptional gene silencing (PTGS) are strongly associated and have evolved as important plant defense mechanisms against invasion of foreign nucleic acids, including viruses. Recent studies have shown that PTGS is activated in plants infected by the geminivirus Tomato yellow leaf curl Sardinia virus (TYLCSV). To compare the DNA methylation profiles of tomato genome in plants infected by TYLCSV vs mock-inoculated plants, a methylation-sensitive amplified polymorphism (MSAP) methodology was applied. The pattern of methylation was assayed at different stages of infection (1, 7 and 14 days) following inoculation by the natural vector, the whitefly Bemisia tabaci. A total of 34 polymorphic fragments were identified of which ten were sequenced. For all, sequences similarities could be found to either expressed or genomic tomato sequences present in the public databases, mostly to genes involved in defence and stress responses. One sequence is presumably part of a regulatory element. RT quantitative PCR performed on six sequences (genes) displayed changes in their expression during infection. Our data demonstrate that TYLCSV induces changes in the methylation status of tomato genome and that MSAP analysis represents a powerful tool for identifying genes involved in a plant-virus interaction.