*4.5. N-Acyl Homoserine Lactone (AHL) Lactonases Activity Assay*

The relative activity of mutant proteins was measured using a pH-sensitive colorimetric assay previously described by K Tang [9]. The test system consisted of a reaction buffer, morpholinepropanesulfonic acid (MOPS), a pH indicator, bromothymol blue (BTB), AHL substrate C6-HSL and the enzyme undergoing measurement. When AHL molecules were degraded to donate protons, the pH was weakly altered. Color changes due to BTB were measured using a microplate reader.

### *4.6. Site-Directed Mutagenesis of Moml*

To study multi-site mutant proteins, we constructed site-directed single mutant via site-directed mutagenesis [43]. Mutation sites and primers [44] are listed in Table S1. The mutated genes were amplified using Primer STAR GXL DNA polymerase (Takara) and cyclized after phosphorylation. Recombinant plasmids were expressed in *E. coli* BL21(DE3) and mutant proteins were purified as previously described. The enzyme activity of each mutant protein was measured as described above.

### *4.7. Kinetic Assay of Moml and Mutant Proteins Activities*

The catalytic activities of MomL and mutant proteins were measured by a pH sensitive colorimetric assay [45]. Briefly, 3.5nM enzyme, C6-HSL/3OC10-HSL (0.156 to 5 mM) were added to a MOPS (5 mM, pH 7.1)/BTB (1 mM) system of total 100 uL. Due to the pH-sensitive dye (BTB) mediated color change, OD630 was continuously measured using a microplate absorbance reader [9]. Initial rates were calculated and a GraphPad Prism software was used for calculating *Km* and *k*cat values. A standard curve using HCl was constructed to reflect the relationship between the absorbance change and the proton concentration, the value of OD630 would decrease by 0.193 after adding with 100 nmol of HCl.

#### *4.8. E*ff*ects of MomL and Mutant Proteins on the Pathogenicity of Pcc*

*Pcc* was cultured to the exponential phase and inoculated in 5 mL of LB medium containing equal amounts of enzymes (MomL, MomLI144V and MomLV149A), and ddH2O was added as a positive control. The cultures were grown on a shaker (170 rpm) at 28 ◦C for 24 h. Extracellular pectate lyase activity was determined using a DNS assay. First, 0.2% polygalacturonic acid reaction buffer (0.2% polygalacturonic acid; 0.2 M NaCl in 0.05 M sodium acetate buffer at pH 5.2) was prepared. Then, to obtain culture samples for enzyme assays, cultures were centrifuged at 4000 rpm for 5 min, and the supernatants were filter-sterilized through a 0.22 μm filter at 4 ◦C or on ice. Two hundred microliters of enzyme and 400 μL of 0.2% polygalacturonic acid reaction buffer were blended and immersed in a tube maintained at a constant temperature of 48 ◦C for 30 min. Four hundred microliters of DNS were added to the 600 μL reaction system, and the mixture was incubated in boiling water for 5 min and centrifuged at 12,000 rpm for 1 min; the precipitate was then discarded. The supernatant was diluted three times, and the absorbance was measured at 492 nm.

#### *4.9. Pcc Survival Rate Assay*

*Pcc* was cultured as mentioned above. A bacterial suspension was diluted with fresh LB and plated on LB agar; after 15 h of growth at 28 ◦C, colonies were counted. The colonies on *Pcc* plates were set to 100%.

#### *4.10. Pcc Infection Experiment*

Leaves were selected from the same Chinese cabbage, and 10 μL of *Pcc* bacterial suspension with enzyme (MomL and mutant proteins) were added to a cut surface. The inoculated leaves were incubated in sterile dishes at 28 ◦C for 24–48 h. *Pcc* alone was used as the positive control.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/5/300/s1. Figure S1: The detection of gel electrophoresis of *momL* fragment with series of epPCR condition. Figure S2: The protein activity test. Table S1: Mutation sites and mutation primers of MomL. Table S2: The mutation rate comparison of epPCR products with different conditions.

**Author Contributions:** Conceptualization, J.W. and Y.W.; Data curation, Y.Z., J.Z., T.F., H.L., X.W. and Q.S.; Formal analysis, J.W. and J.L.; Methodology, J.W. and J.L.; Software, J.L.; Supervision, X.Z. and Y.W.; Writing—original draft, J.W.; Writing—review and editing, X.Z. and Y.W.

**Funding:** This work was supported by the Fundamental Research Funds for the Central Universities (No. 201941009), the National Natural Science Foundation of China (No. 31870023, 31571970 and 41506160), the Young Elite Scientists Sponsorship Program by CAST (No. YESS20160009).

**Conflicts of Interest:** The authors declare no competing interests.

#### **References**


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