Function alterations of the ethylene receptors Never ripe and LeETR4 affect the susceptibility of tomato plants to the fungal pathogen Verticillium dahliae

Previous experimental evidence derived from pathogenicity tests of Verticillium dahliae on ethylene insensitive Arabidopsis thaliana plants (etr) showed increased tolerance of the etr mutants compared to the wild type control (Col-0). To investigate the role of the LeETR4 gene, which encodes an ethylene receptor in tomato, a Tobacco rattle virus (TRV) based VIGS (virus induced gene silencing) system was employed. A mixture of Agrobacterium tumefaciens cultures (carrying TRV RNA1, RNA2 containing an LeETR4 gene fragment) was vacuum infiltrated into tomato leaves of cv. Ailsa Craig. The efficiency of the VIGS system was confirmed by silencing the LePDS gene (responsible for carotenoid biosynthesis) resulting in a photo-bleaching phenotype. Plants infiltrated with a mixture of A. tumefaciens with TRV RNA1 and RNA2 were used as control. The LeETR4 expression level was assessed by Real-time PCR (RT-PCR). Silenced (TRV:LeETR4) and mutant Never ripe (Nr - ethylene receptors deactivated) plants were inoculated with V. dahliae (10 super(7) spores/ml) and disease symptoms were scored for 30 days. Both TRV:LeETR4 and Nr plants showed significantly less symptoms compared to the control plants. In addition, quantification of V. dahliae by RT-PCR revealed that the fungal biomass in the Nr and TRV:LeETR4 plants was significantly less than that in the control plants. These findings suggest a key role of ethylene perception in the interaction of tomato with V. dahliae.