## 2 Results and Discussion
### N-Acyl-l-homoserine Lactone (AHL) Accumulation in the Cross Feeding Assay and Extract Assay  
To determine the effect of Rsh\({}_{\text{US6-1}}\) on the AHL accumulation, cross-feeding and extract assays were performed. The results of the cross-feeding and extract assays were consistent (Figure 2). In the cross-feeding assay, strains were grown as biofilm and only US6-1 showed AHL accumulation. In the extract assay, US6-1 accumulated AHL signals in static cultures while no AHL was detected in \(\Delta\)_rsh_ during the whole growth period. To verify that the _rsh\({}_{\text{US6-1}}\)_ deletion was responsible for the absence of AHL, a _rsh\({}_{\text{US6-1}}\)_ complementation strain \(\Delta\)_rsh_ (pCM62-rsh) was created. \(\Delta\)_rsh_ (pCM62-rsh) restored the same phenotype as the wild-type strain, suggesting that Rsh\({}_{\text{US6-1}}\) was required for the AHL accumulation. In the extract assay, the AHL molecules in the extract of culture would diffuse through the soft agar to the reporter strain _A. tumefaciens_ A136, and activate the _tral-lacZ_ fusion. Therefore, we could determine the quantity of AHL molecules by the diffusion area indicated by the diameter of the blue coloration in the presence of 5-bromo-4-chloro-3-indolyl-\(\beta\)-D-galactopyranoside (X-Gal) [37]. The relationship between the growth of US6-1 and AHL accumulation was analyzed carefully. The bacteria cells grew exponentially for about 48 h and then entered the stationary phase. Similarly, the AHL accumulation was maximized at 48 h and then declined, indicating US6-1 could degrade AHL signals.  
Figure 2: AHL accumulation in US6-1, _Arsh and \(\Delta\)rsh_ (pCM62-rsh). (**a**) AHL accumulation assay by cross-feeding. P5Y3 agar plate was covered with 50 \(\upmu\)L X-gal. The AHL reporter strain A136 and the tested strains were streaked side by side on the agar plates. Plates were incubated for 24 h, when the activation of the reporter was recorded. Strain A136 versus A136 was used as negative control. One representative experiment out of three independent biological replicates is shown. (**b**) Time course of population density of US6-1 (filled squares), \(\Delta\)_rsh_ (filled circles) and \(\Delta\)_rsh_ (pCM62-rsh) (filled triangles) and AHL accumulation of US6-1 (open squares) and \(\Delta\)_rsh_ (pCM62-rsh) (open triangles) in the extract assay. Fresh colonies of strains were first inoculated into P5Y3 broth at the shaking speed of 200 rpm to an OD\({}_{\text{600}}\) value of 1. Then 250 \(\upmu\)L of this seed culture was re-inoculated into 50 mL of fresh P5Y3 broth and incubated statically. The growth curve was drawn from measuring the OD\({}_{\text{600}}\) values. AHL signals in cultures were then extracted by ethyl acetate (EA). The extracts were spotted onto LB soft agar plates plus X-gal mixed with A136. The plates were incubated for 12 h at 30 \({}^{\circ}\)C and the diameters of blue stains which represented the quantity of AHL signals in bacterial cultures were measured. No AHL was detected in \(\Delta\)_rsh_. Values shown are the average of biological triplicate experiments with standard deviations marked with error bars.