## 3 Results  
### X. citri Triggers Persister Cell Formation under Antibiotic Stress  
In order to study persistence in bacteria, it is necessary to increase its proportion in a given population. As a proof of concept, we applied the same method previously utilized in _E. coli_[18; 43; 44]. The basic principle behind this technique is that if persister cells are formed by given stress, they can withstand a subsequent treatment with another antibiotic and are killed to a lesser extent than control cells (those not receiving the persister-inducing stress).  
To initiate the persister study with _X. citri_, we used the antibiotic tetracycline, which blocks translation, and therefore induces a persistence state by blocking protein synthesis [43]. Then, a ciprofloxacin treatment was followed to kill non-persister cells. Therefore, _X. citri_ cultures were grown in NBY media, and when they reached the mid-exponential phase, they were exposed to 10 \(\times\) MIC tetracycline (10 \(\mu\)g/mL), for 15 to 60 min, centrifuged, and the pellet was resuspended in NBY supplemented with 100 \(\times\) MIC ciprofloxacin (50 \(\mu\)g/mL). Indeed, there was an increase in cell survival among those cultures that were previously exposed to tetracycline before the ciprofloxacin treatment (Figure 1A). After only 15 min of tetracycline pretreatment, the amount of surviving cells was almost 10-fold higher than the control culture (only ciprofloxacin treatment, without tetracycline), after 3 h ciprofloxacin challenge, and from 30 to 60 min, the survivor ratio was slightly higher (Figure 1A).  
We sought to determine how these persister cells would behave after prolonged ciprofloxacin treatment. The experiment was performed as before (Figure 1A), but at this time, a 45 min tetracycline incubation was used. Afterward, the ciprofloxacin challenge was carried out for 6 h, and the colony-forming unit (CFUs) was obtained for both the tetracycline pretreated and the control cultures (Figure 1B). After 6 h of ciprofloxacin treatment, no survivors were recovered in the control culture, while the tetracycline pretreated culture presented \(10^{4}\) CFU/mL. A characteristic biphasic killing curve was seen, an expected result since persisters are more tolerant to antibiotic treatments. Therefore, we conclude that the inhibition of protein translation can induce persister cell formation in the plant-pathogen _X. citri_.  
Figure 1: Persister cell induction by inhibiting translation and biphasic killing curve. (**A**) Exponentially growing _X. citri_ cultures pretreated with the translation inhibitor tetracycline for 15, 30, 45 and 60 min, followed by ciprofloxacin treatment (50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, 3 h, to kill non-persisters). Samples were serially diluted for colony-forming unit (CFU) counting to determine fold changes (relative to control without tetracycline). Results are shown as the mean of biological replicates. The symbols * (\(p<0.05\)) and *** (\(p<0.001\)) indicate significant differences using Student’s \(t\)-test. (**B**) Time course of cell death during ciprofloxacin treatment (50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) in persisters (after tetracycline pretreatment) and exponential cells (without tetracycline pretreatment).