As a control, three groups of six plants each were inoculated with either PBS or gomesin (50 μM) or used as sentinel (noninoculated). Thirty days after inoculation, tobacco plants were inspected for leaf lesions, a typical symptom developed in X. fastidiosa-infected tobacco plants. Results correspond to two independent experiments, resulting in
a total of 36 analyzed plants for each group. Cell viabilities of the bacterial suspensions used in the inoculations were assessed by plating a sample on 2% agar PW broth medium following incubation at 28 °C for 7 days. Differences between groups were analyzed using Students’s t-test and considered statistically significant if the P-value was <0.05. It has been shown that gomesin is an AMP that presents strong activity against a huge number of microorganisms (Silva et al.,
2000) disrupting CX-5461 concentration the microbial membrane (Miranda et al., 2009). We have LEE011 datasheet verified that gomesin is effective against X. fastidiosa 9a5c, a virulent strain against citrus plants (Li et al., 1999). The MBC of gomesin was determined by viability assays to be 4.5–9 μg mL−1, which corresponds to 200–400 μM (Table 1). Both the virulent strain 9a5c and the avirulent strain J1a12 (Koide et al., 2004) exhibited identical susceptibility to gomesin and to conventional antibiotics ampicillin, tetracycline and streptomycin, suggesting that the avirulent phenotype of the strain J1a12 is probably
not due to a lower resistance to antimicrobial agents, eventually encountered in the plant host or the insect vector. To evaluate the gene expression profile upon exposure to a sublethal concentration of AMP, the virulent strain 9a5c of X. fastidiosa was treated Dichloromethane dehalogenase for 60 min with 50 μM of gomesin (four- to eight-fold below the determined MBC for gomesin). Total RNA from treated and untreated cells was isolated and labeled for hybridization to DNA microarrays. As summarized in Table 2, gomesin treatment modulated the expression of 159 CDS of X. fastidiosa, among which 143 were upregulated and only 16 were downregulated (see Supporting Information, Table S1 for the full list of modulated CDS). Transcript levels of a subset of nine X. fastidiosa CDS were analyzed by RT-qPCR (Table 2), and the results for seven CDS (XF1127, XF1164, XF2367, XF0371, XF0589, XF0833 and XF1984) are in agreement with microarray experiment data. When X. fastidiosa was exposed to the same sublethal concentration of gomesin for shorter periods of time (15 or 30 min), no change in the gene expression profile was observed (data not shown). The CDS differentially expressed upon gomesin exposure belong to diverse functional categories (Fig. 1). Nevertheless, as typically observed in genome-wide transcriptome approaches, the majority encode hypothetical proteins (Fig. 1).