Bacterial strains, plasmids and growth conditions

The bacterial strains and plasmids used in this study are listed in Supplement F1 (Table S1). The X. citri subsp. citri strains were cultivated in nutrient-rich broth (NB) media or NB with 1.5% agar (NA) at 28 °C [24]. Strains of Escherichia coli were cultured in LB media at 37 °C. Antibiotics were applied at the following concentrations: kanamycin (Km) at 50 µg mL-1, spectinomycin (Sp) at 50 µg mL-1, and gentamycin (Gm) at 10 µg mL-1.

Mutant construction and complementation analysis

Based on the genome sequence of X. citri subsp. citri strain 29 − 1 (No. CP004399), the specific primers 12301.F and 12301.R were designed to amplify a 280 bp DNA fragment upstream of the PKC gene. A 468 bp DNA fragment containing 105 bp PKC gene 3′ terminus was amplified using the primer set 12302.F/12302.R (Supplement F1, Table S2). The two fragments were individually cloned into the suicide vector pKMS1, which generated pKMS-XAC1230 (Supplement F1, Table S1). The recombinant plasmid was introduced into X. citri subsp. citri 29 − 1 to create deletion mutants by two steps of homologous recombination [24]. After selection on NA plates supplemented with 10% sucrose, the colonies sensitive to kanamycin were identified by PCR with the primer pair 12301.F/12302.R. This produced a 748 bp PCR product with a deletion of the 1062 bp coding sequence. The deletion of targeted fragments was confirmed by sequencing the PCR products obtained from mutants.

The primer pair CXAC1230.F/CXAC1230.R was used to amplify a 1447 bp DNA fragment that contained the PKC gene and its promoter region (Supplement F1, Table S2). The PCR product was cloned into the vector pBBR1MCS-5 to obtain the construct pBB-XAC1230. The pBB-XAC1230 construct was transformed into the deletion mutant for complementation analysis. Complementation with the PKC mutants was additionally studied by designing specific primers to combine with CXAC1230.F to generate constructs expressing 1–100, 1–180, and 1–255 amino acids of the PKC protein. The corresponding generated constructs M1 − 255, M1 − 180, and M1 − 100 were transformed into the deletion mutant to evaluate their ability to restore pathogenicity.

Expression of the PKC gene under stress conditions

The approximately 500 bp DNA sequence upstream of the PKC gene was retrieved from the 29 − 1 genome (No. CP004399). The putative promoter sequence was predicted using Softberry (http://www.softberry.com/). According to the promoter location, the primer pair PXAC1230.F/PXAC1230.R was used to amplify a 280 bp DNA fragment composed of the promoter region. The PCR product was inserted into the pRG960 vector for fusion with GUS at the PstI and BamHI sites (Supplement F1, Table S2). The generated PXAC1230-GUS construct was electrotransformed into 29 − 1 to analyze the activity of its promoter.

The 29 − 1 and 29 − 1/PXAC1230-GUS strains were cultured in NB broth at 28 °C for 36 h and sub-cultured (1:100) in 5 mL of fresh NB broth until their OD600 ≈ 1.0. The cultures were supplied with 1% NaCl, 5% sorbitol, 0.008% SDS, and 0.2 mM CuSO4. At 2 h post-incubation, the 29 − 1 and 29 − 1/PXAC1230-GUS cells were subjected to a real-time quantitative reverse transcription PCR (qRT-PCR) analysis to evaluate the levels of transcripts of both PKC and gusA. The experiments were repeated three times.

Pathogenicity, hypersensitive response, and replication assays in planta

The X. citri subsp. citri strains were cultured in NB broth for 48 h and sub-cultured (1:100) in 5 mL of fresh NB broth until the OD600 reached 1.0. The cultured cells were suspended in sterile distilled water to a final concentration of 107 CFU mL− 1 (OD600 = 0.03). The bacterial suspensions were infiltrated into grapefruit (Citrus × paradisi) leaves with a needleless syringe for pathogenicity assay. Disease symptoms were scored and documented as images at 5 d post-inoculation [25]. For the growth assay in planta, 0.8-cm-diameter leaf discs were collected at 4 d post-infiltration. The discs were completely ground in 1 mL of sterile ddH2O. Serial dilutions of the suspension were spread on NA plates, and the individual colonies were recorded to determine the CFUs per cm2 leaf. The SD was calculated based on the colony counts of four replicates. The HR response was analyzed by infiltrating the cell suspension into tomato (Solanum esculentum) leaves. The reaction was viewed 24 h post-inoculation. The tests for pathogenicity and the HR were repeated three times, and the replication assay in planta was repeated four times.

Analysis of the expression of the gene for T3SS

The expression of the T3SS gene was analyzed by separately electrotransforming PhrpG-GUS and PhrpX-GUS into mutant and complemented strains. The wild type 29 − 1, mutant, and complemented strains that harbored PhrpG-GUS or PhrpX-GUS were cultured in NB broth until the OD600 reached 1.0. After centrifugation (5000 × g for 10 min), the cells were re-suspended with the hrp-inducing medium XVM2 [26]. A 2 µL volume of cells was spotted onto 1.5% agar XVM2 plates supplied with 20 µg/ml p-nitrophenol-β-D-glucuronide (X-gluc), and the activity of GUS was determined via colony color at 3 d post-inoculation. The levels of gusA transcripts were quantified by incubating the cells in XVM2 liquid media for 6 h and then subjected to qRT-PCR. The levels of the transcripts of hrpG, hrpX, hrpD6, and hrcV in wild type 29 − 1, deletion mutant, and complemented strain were studied after cell suspensions (107 CFU mL− 1) were infiltrated into citrus leaves. At 2 d post-inoculation, qRT-PCR was conducted to detect the levels of transcripts of the four hrp genes.

Real-time quantitative reverse transcription PCR

RNA was extracted from the X. citri subsp. citri cells using an RNAprep Pure Kit for Cells/Bacteria (Tiangen Biotech, Beijing, China). A Plant RNA Kit (Omega Bio-Tek, Norcross, GA, USA) was used to isolate RNA from the grapefruit leaves. A total of 2 µg RNA was reverse-transcribed into single-stranded cDNA synthesized with HiScript QRT SuperMix (Vazyme, Nanjing, China). All the primers used for qRT-PCR are listed in Supplement F1 (Table S2). The PCR thermal cycling conditions were as follows: 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 20 s. The level of expression of gyrA was evaluated as the internal control. All the experiments included three biological replicates, and each had three technical replicates.

Identification of the differentially expressed metabolites using UPLC-MS/MS

The metabolomic analysis was conducted by Shanghai BioTree Biotech Co., Ltd. (Shanghai, China). The X. citri subsp. citri cells were cultured in NB broth and resuspended in XVM2 liquid for 6 h of incubation. The cells were then centrifuged and suspended in 1 mL of extract solution (acetonitrile: methanol: water, 2:2:1 [v:v:v]) to extract the metabolites. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analyses were performed using an ultra-high performance liquid chromatography (UHPLC) system (Vanquish, Thermo Fisher Scientific, Waltham, MA, USA) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled to a Q-Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher Scientific). The MS/MS spectra were acquired in the information-dependent acquisition (IDA) mode using acquisition software (Xcalibur, Thermo Fisher Scientific). In this mode, the ESI source conditions were set as follows: sheath gas flow rate, 50 Arb; Aux gas flow rate, 10 Arb; capillary temperature, 320 ℃; full MS resolution, 60,000; MS/MS resolution, 7,500; collision energy, 10/30/60 in NCE mode; spray voltage, 3.5 kV (positive ion) or -3.2 kV (negative ion).

The raw data were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration. An in-house MS2 database (BiotreeDB) was then applied to annotate the metabolites. The p- and fold-change values were set to 0.05 and 2.0, respectively, to screen for the differentially accumulated metabolites. In addition, commercial databases, including KEGG (http://www.genome.jp/kegg/) and MetaboAnalyst (http://www.metaboanalyst.ca/), were used for the pathway enrichment analysis.

Quantification and statistical analysis

The statistical analyses were conducted using GraphPad Prism (GraphPad Software, San Diego, CA, USA), including two sample Student’s t-tests and a one-way analysis of variance (ANOVA) with a Tukey’s multiple comparison test at the 95% level. The values are presented as the mean ± SD.