Figure 1.
Pathogenicity of North Carolina isolates on tomato, coronatine production and characterization of their AvrPto and AvrPtoB proteins.
A) The North Carolina isolates NC-C3 and NC-W201 caused bacterial speck disease on leaves of Rio Grande-PtoS (pto/pto) plants but not on RG-PtoR (Pto/Pto) indicating they are Pst race 0 strains like DC3000. Photos were taken 4 days after vacuum-infiltration of 1×104 CFU/mL of the strains indicated. B) Both NC-C3 and NC-W201 caused necrotic specks surrounded by diffuse yellow chlorosis indicative of coronatine production similar to DC3000 on leaves of RG-PtoS. The strains were cultured on mannitol-glutamate (MG) media to induce coronatine production prior to inoculation. DC3000Δcfa (CUCPB5502) is a coronatine-deficient mutant used as a control. Photos were taken 5 days after inoculation. C) Phylogenetic tree of the AvrPto proteins from the North Carolina strains and other Pst strains. D) Phylogenetic tree of the AvrPtoB proteins from the North Carolina strains and other Pst strains. The NC-C3 and NC-W201 sequences were identical and are therefore grouped together. Parameters for the analysis were neighbor-joining, 10,000 bootstrap replications and p-distance with AvrPphF used as an out-group. The bar indicates the average number of amino acid substitutions/distance. See Figure S1 for an alignment of the AvrPto and AvrPtoB amino acid sequences.
Table 1.
Tomato heirloom varieties used to screen for MAMP responsiveness and resistance to bacterial speck caused by NC-C3, a Pseudomonas syringae pv. tomato race 0 isolate.
Figure 2.
Response of heirloom tomatoes to NC-C3 in the field.
Disease severity of heirloom tomatoes inoculated with race 0 Pst isolate NC-C3 under field conditions (summer 2012). RG-PtoR has the Pto gene and was included as a resistant control. RG-PtoS and Moneymaker lack Pto and were included as susceptible controls. Inoculated plants were scored at 7 dpi using a 0–5 scale as described in Materials and Methods. Mean percent disease index (PDI; see Methods) was calculated from the disease scores of each genotype by totaling the score of 18 plants (three replicates each of 6 plants per line) and expressing the value as a percentage (%). Means marked with the same letter are not statistically different at a 5% probability level based on least significant difference separation.
Figure 3.
Natural variation in heirloom tomatoes for reactive oxygen species (ROS) production upon exposure to flg22, flgII-28, and csp22.
A) ROS production was measured over a period of 30 minutes in leaves from 4-week old heirloom tomatoes after exposure to 100 nM flg22. B) ROS production was measured over a period of 40 minutes in leaves from 4-week old heirloom tomatoes after exposure to 100 nM flgII-28. C) ROS production was measured over a period of 20 minutes in 4-week old heirloom tomato leaves after exposure to 500 nM csp22. The data are shown as mean ± SE from 8 leaf discs analyzed in triplicate with the experiments being repeated three times. Means marked with the same letter are not statistically different at a 5% probability level based on least significant difference separation.
Figure 4.
A genetic approach to characterize csp22 responsiveness.
Leaf discs from RG-PtoS and LA2109 were treated with 100 nM flg22 (A) or 1 µM csp22 (B) and ROS production measured every 2 minutes over the time course shown. The curves are the average of 2 plants from independent experiments, and are representative of the trend observed with several other plants. C) To identify csp22 low-responding F2 plants, the sum of the ROS curves (as shown in A) was determined for flg22 and csp22, and the ratio of csp22-to-flg22 response was determined (csp22/flg22). The numbers at the bottom indicate individual F2 plants. A threshold value of 0.25 was set as the upper limit for defining low responsiveness. Of 147 F2 plants tested, 41 were classified as low csp22 responders. A chi-square test for fitting to a 3∶1 was performed and Χ2 = 0.58.