Nine genes involved in different plant defense pathways were selected: SOD (superoxide dismutase), CAT (catalase), APX (peroxidase ascorbate) and POX (peroxidase), NtPR1a (pathogenesis-related protein 1a), NtNPR3 (pathogenesis-related protein 3) and NtCOI1 (coronatine-insensitive 1) (Chen et al., Selleck CH5424802 1993; Shoji et al., 2008). The actin gene was used as an internal control. Gene-specific primers of these genes are shown in Supporting Information

Table S1. Results were expressed as mean±SD. P-value <0.05 was considered statistically significant. All statistical analyses were performed using spss 11.5 for Windows. Initial results indicated that after a 4-day treatment with Trichokonins, tobacco plants achieved the highest resistance to TMV (data not shown). Therefore, a 4-day treatment was used in the following experiments. Trichokonins of various concentrations (50, 100 and 200 nM) were used to analyze their ability to induce

tobacco C59 wnt resistance against TMV infection. Six days after inoculation with TMV, the number and diameter of lesions were measured. Trichokonin treatment led to a remarkable reduction in the number of lesions that appeared in the tobacco leaves compared with the control plants (Fig. 1a). The lesion number in tobacco pretreated with 50, 100 and 200 nM Trichokonins was 15%, 54% and 35% less, respectively, compared with the control. These results indicated that tobacco resistance against TMV was significantly improved after Trichokonins treatment, and that 100 nM Trichokonins was the most effective concentration (Fig. 1a). After treatment with 100 nM Trichokonins, the final lesion diameter in the inoculated leaves was 2.25±0.61 mm on average, which was much smaller than that of the control plants (5.22±0.79 mm) (Fig. 1b). The

final lesion area of Trichokonin-treated PLEKHB2 tobacco was about 28.9% in average, which was 1.5-fold less than that in the control plants (41.4%) (Fig. 1c). Together, these results indicated that Trichokonin treatment induced tobacco resistance against TMV infection. Production of reactive oxygen species and accumulation of phenolic compounds are early responses in plant–pathogen or elicitor recognition (Hutcheson, 1998). We tested the ability of Trichokonins to elicit these responses. Compared with the control plants, higher levels of O2− and H2O2 were produced in tobacco leaves after tobacco plants were cultured in 100 nM Trichokonin solution for 4 days (Fig. 2a and c). In addition, 100 nM Trichokonins resulted in the production of O2− and H2O2 around the application area on leaves instantaneously (Fig. 2b and d). These results showed that Trichokonins induced the production of O2− and H2O2 locally and systemically in tobacco plants. Furthermore, the autofluorescence of phenolic compounds was tested.