The calamondin and ‘Taoyecheng’ sweet orange were grown in a greenhouse at 25 ± 1°C in Wuhan, China. The leaves of calamondin and ‘Taoyecheng’ sweet orange were utilized in Xcc inoculation experiments and transient overexpression analyses. The Xcc strain was routinely cultured at 28°C in an Lysogeny Broth (LB) solid culture medium.

The transcriptome data of CBC-resistant ‘Meiwa’ kumquat (F. crassifolia) and CBC-susceptible ‘Mexican’ lime (C. aurantifolia) responding to Xcc infection at 24 hpi, 48 hpi and 72 hpi were obtained through CitrusKB database (http://bioinfo.deinfo.uepg.br/citrus/) ( Supplementary Table S1 ).

In addition, the young leaves of calamondin were selected for Xcc in vivo inoculation. Xcc was cultured overnight at 28 °C. The activated bacterial solution was diluted to OD=0.6 (10⁸ cfu/ml) with sterile water and further diluted to 10⁴ cfu/ml for infiltration inoculation. Sterile water inoculation was used as a blank control. Subsequently, leaf samples were collected at different time points 1 dpi, 3 dpi and 5 dpi for transcriptome analysis and differentially expressed genes (DEGs) analysis. ( Supplementary Table S2 ).

The whole MES proteins of ‘Hong Kong’ kumquat (F. hindsii) were downloaded from the CPBD database (http://citrus.hzau.edu.cn/index.php, accessed on 13 May 2024), and 20 MES proteins of A. thaliana were obtained from the TAIR database (https://www.arabidopsis.org/, accessed on 13 May 2024). Four candidate MES proteins from calamondin were cloned and sequenced; the primers were listed in Supplementary Table S3 . Then, the phylogenetic relationships of the MES proteins were constructed using the neighbor-joining method by MEGA 11.0 software with the following parameters: Poisson model, pairwise deletion, and 1000 bootstrap replications. The phylogenetic trees were visualized using the iTOL web package. All the gene accession numbers and protein sequences are listed in Supplementary Tables S4 .

The structure of MES proteins from F. hindsii was analyzed using the MEME Suite 5.5.2 online program (Multiple Em for Motif Elicitation 5.5.2, http://alternate.meme-suite.org/tools/meme, accessed on) and TBtools (https://github.com/CJChen/TBtools, accessed on). The 2000-bp promoter sequences of the upstream regions of MES proteins were obtained. Cis-acting elements were predicted via PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on), and the responsive regulatory elements were analyzed via TBtools.

Due to CBC mainly infects young leaves, stems and fruits of citrus, young and mature leaves, stem and fruit were used for tissue-specific analysis of four methylesterase family genes.

Young leaves of calamondin were selected for plant hormone response analysis. 500 µM SA, 100 mg/L IAA, 5 mg/L GA₃, 200µM mg/L methyl Jasmonate, and 100 mM abscisic acid (ABA) were used to spray the calamondin leaves. Hormone-treated leaf samples were collected 1, 2, and 3 days after spraying, and untreated leaf samples were used as a control.

The total RNA of calamondin leaves was extracted using the RNA prep Pure Plant Kit (Tiangen, Beijing, China), and the cDNA was obtained by the Monad MonScriptTMRTIII Super Mix with dsDNase (Two-Step) (Monad, Suzhou, China). The Monad MonAmpTM SYBR^® Green qPCR Mix (Vazyme, Nanjing, China) and Applied Biosystems PCR-7500 (ABI, USA) were used for amplification and the qRT-PCR analysis. The primers used for the qRT-PCR are shown in Supplementary Table S3 . Three biological replicates were conducted.

The CDS sequences of CmMES1.1 (441 bp), CmMES17.3 (885 bp), CmMES10.2 (642 bp) and CmMES1.5 (804 bp) from calamondin were inserted into the PK7203-RFP vector to generate a fusion protein of the target gene with RFP. First, PK7203-RFP vector was digested by Sall-HF enzyme, and then the coding sequence of each MES gene was cloned into the vector by homologous recombination cloning technology. The overexpression vector includes an enhanced green fluorescent protein (EGFP) activated by minimal CaMV 35S promoter as a screening marker, and an MES protein fusion mRFP promoted by minimal CaMV 35S promoter for subcellular localization. Please refer to Supplementary Figure S1 for detailed map. The specific steps were as follows: first, the single colony of each Agrobacterium strain EHA105 was cultured on LB medium at 28 °C overnight; then resuspended with infiltration buffer (10 mM 2-(N-morpholino) ethanesulfonic acid, pH 5.85; 10 mM MgCl₂; 30 mg/L acetosyringone) to OD=0.6; finally the infiltration buffer was injected into the leaves of Nicotiana benthamiana, which was then cultured in the dark for 12 hours and 16-h/8-h light/dark photocycle for 2 days. The fluorescence was observed through a laser scanning confocal microscope (TCS-SP8 SR, Leica, Wetzlar, Germany). The primers used for the construction of the overexpression vector are shown in Supplementary Table S3 .

The above four EHA105 strains for subcellular localization analysis were used in transient transformation assay, with the leaves of calamondin and ‘Taoyecheng’ sweet orange (C.sinensis) as explants. The optimized transient transformation method was used to obtain overexpressed calamondin leaves. The specific steps were as follows: first, the single colony of each Agrobacterium strain EHA105 was grown at 28 °C overnight; then resuspended with infiltration buffer (10 mM 2-(N-morpholino) ethanesulfonic acid, pH 5.85; 10 mM MgCl₂; 30 mg/L acetosyringone) to OD=1.0-1.5; finally injected the infiltration buffer into the young calamondin leaves and ‘Taoyecheng’ sweet orange with a needleless syringe, and injected repeatedly for three times at one-hour intervals and cultured in the dark for 2 days. Finally, the instantaneous transformation of Agrobacterium was determined by green fluorescence observation (Supplementary Figures S2).

To determine the CBC resistance ability of the four key MES genes, leaves were picked from the transiently transformed calamondin and ‘Taoyecheng’ sweet orange leaves for in vitro Xcc inoculation. 0.1 mL Xcc (10⁸ cfu/ml) was infiltrated into the calamondin leaves, and 5 µL Xcc (10⁸ cfu/ml) was dripped onto each puncture site made with a pin (0.5 mm in diameter) on’Taoyecheng’sweet orange leaves. The leaves were then cultured in an incubator at 28°C, with 80% relative humidity and a 16-h/8-h light/dark photocycle. CBC symptom and resistance degree were evaluated at 5 and 15 days post-inoculation (dpi) of calamondin and ‘Taoyecheng’ sweet orange leaves, respectively. Lesion area was analyzed by ImageJ software, and all experiments were repeated at least three times.

Approximately 100 mg of frozen calamondin leaves were ground and extracted with 1 ml ice-cold 50% aqueous acetonitrile (vol/vol). Subsequently, the samples were sonicated for 3 min and extracted for 30 min at 4°C. The supernatant was obtained after centrifugation (10 min, 12,000 rpm, 4°C) and purified using C18 reversed-phase. After this solid phase extraction, the samples were blown dry by nitrogen and dissolved in 200 μl of 30% acetonitrile (vol/vol). Ultra-efficient liquid chromatography (Vanquish, UPLC, Thermo, USA) and high-resolution mass spectrometry (Q Exactive, Thermo, USA) were used to determine the plant hormone levels. The analytical conditions were as follows: column: Waters HSS T3 (50×2.1 mm, 1.8 μm); mobile phase: phase A is ultra-pure water (containing 0.1% acetic acid) and Phase B is acetonitrile (containing 0.1% acetic acid). The data were collected using the Q Exactive high-resolution mass spectrometry system from Thermo Fisher Scientific and processed using TraceFinder Software. Triplicates were conducted for determine each plant hormone.

All data analyses were performed using GraphPad Prism 6.0 (GraphPad, USA), the results were presented as means ± standard deviation (SD), and comparisons were made using ANOVAs with Duncan’s multiple range test.

The transcriptome data of CBC-resistant ‘Meiwa’ kumquat (F. crassifolia) and calamondin (Citrofortunella microcarpa), and CBC-susceptible ‘Mexican’ lime (C. aurantifolia) in response to Xcc infection were re-analyzed with Tbtools. Eight differentially expressed MES genes were screened from CBC-resistant calamondin, including MES1.1, MES1.4, MES17.3, MES17.1, MES10.2, MES1.5, MES1.3 and MES11.5. Among them, MES1.1, MES17.3, MES10.2 and MES1.5 were shared by CBC-resistant ‘Meiwa’ kumquat and calamondin and CBC-susceptible ‘Mexican’ lime. Compared with the control, the expression of MES1.1, MES10.2 and MES1.5 were down-regulated at 24 hpi after Xcc inoculation in CBC-susceptible ‘Mexican’ lime, but up-regulated at 24 hpi hpi, 48 hpi and 72 hpi after Xcc inoculation in CBC-resistant ‘Meiwa’ kumquat and up-regulated at 1 dpi and/or 3 dpi and/or 5dpi after Xcc inoculation in CBC-resistant calamondin. The expression of MES17.3 was down-regulated in all three citrus species. The expression level of MES17.3 was decreased by 7.5 and 3.1 times at 48 hpi and 72 hpi after Xcc inoculation in CBC-susceptible ‘Mexican’ lime, respectively ( Figure 1A ). However, it was decreased by 16.8, 54.2 and 26.9 times at 24 hpi, 48 hpi and 72 hpi in CBC-resistant ‘Meiwa’ kumquat and 1.8, 1.5 and 8.5 times at 1 dpi, 3 dpi and 5 dpi in CBC-resistant calamondin ( Figures 1B, C ). These results indicated that MES family genes played an important role in CBC resistance, among which MES1.1, MES17.3, MES10.2 and MES1.5 might be key regulatory genes.

The expression patterns of four MES genes (CmMES1.1, CmMES17.3, CmMES10.2, and CmMES1.5) that responded to Xcc were further analyzed by qRT-PCR in calamondin. Young leaves of calamondin were infiltrated with low (10⁴ cfu/ml) and high (10⁸ cfu/ml) concentrations of Xcc suspension and analyzed at four stages (1 dpi, 3 dpi, 5 dpi, and 7 dpi) after Xcc inoculation. Compared with sterile water inoculation used as a blank control, the leaves of calamondin inoculated with a low concentration of Xcc showed no obvious symptoms within 7 dpi, while the leaves inoculated with a high concentration of Xcc had obvious pustule symptoms after 5 dpi and appeared hypersensitive necrotic after 7 dpi ( Figure 2A ). When inoculated with a high concentration of Xcc, the expression levels of CmMES1.1, CmMES10.2 and CmMES1.5 were all significantly down-regulated at an early stage (1 dpi and 3 dpi) and up-regulated at a later stage (5 dpi and/or 7 dpi) ( Figure 2B ). However, the expression level of CmMES17.3 was significantly down-regulated at the whole stage, particularly at the later stage ( Figure 2B ). The expression levels of the four MES genes were only slightly regulated by low concentration Xcc infection. For example, all the four MES genes were slightly up or down regulated at the early stage (1 dpi and 3 dpi). Although CmMES1.1 and CmMES10.2 were significantly up-regulated at 5 dpi, CmMES17.3 and CmMES1.5 were significantly down regulated at 5 dpi and 7 dpi, their up-regulation and down-regulation levels were significantly lower than those with high concentration Xcc infection. These results indicated that the function of these MES genes might be strongly activated at the later stage upon Xcc infection associated with the development of the symptoms.

To explore the phylogenetic relationships of kumquat MES proteins, we constructed a phylogenetic tree based on the amino acid sequences of 12 kumquat MES proteins and 20 A.thaliana MES proteins. In the kumquat (F. hindsii) genome, Sjg142100, Sjg239980, and Sjg214930 contained two spliceosomes, Sjg072760 had three spliceosomes, and the rest had only one spliceosome. Based on comparison analysis with A.thaliana MES proteins, the kumquat MES family genes were mainly divided into six groups, namely group AtMES1 (FhMES1.1-FhMES1.5), group AtMES10 (FhMES10.1 and FhMES10.2), group AtMES11 (FhMES11.1-FhMES11.5), group AtMES14 (FhMES14), group AtMES17 (FhMES17.1-FhMES17.3) and group Sjg190120 ( Figure 3 ). In addition, four candidate CBC resistance-related MES genes from calamondin were cloned and sequenced, and they belonged to FhMES1.1, FhMES17.3, FhMES10.2 and FhMES1.5 ( Supplementary Figure S3 ). Gene structure analysis showed that the kumquat MES family genes contained 2 - 6 exons. Promoter element analysis showed that the promoters of kumquat MES family genes contained a large number of plant hormone-responsive elements (SA, auxin, methyl jasmonate, ABA (abscisic acid), and gibberellin), stress (low temperature and drought) and defense-related response elements ( Figure 3 ).

Therefore, the MES genes might be involved in response of plant hormones including SA, IAA, JA, ABA or GA.

The stems, leaves, and fruits of calamondin at different growth stages were used to analyze the tissue-specific expression patterns of CmMES1.1, CmMES17.3, CmMES10.2, and CmMES1.5. The results indicated that CmMES1.1 was mainly expressed in fruits. However, the expression level was higher in the young tissues of leaves and stems than in mature tissues. CmMES17.3 was mainly expressed in stems, especially in mature stems. CmMES10.2 and CmMES1.5 were highly expressed in young tissues, particularly in young fruits ( Figure 4A ). Furthermore, the four MES genes were regulated by different plant hormones. CmMES1.1 and CmMES1.5 were down-regulated by SA treatment and up-regulated by JA treatment, meanwhile, CmMES1.5 was also significantly up-regulated by ABA treatment. CmMES17.3 was mainly up-regulated by JA treatment and CmMES10.2 was mainly up-regulated by ABA treatment ( Figure 4B ). These results revealed that the four MES genes might prone to express in young tissues and be involved in different plant hormone pathways.

To examine the subcellular localization of the four MES proteins, the positive control (RFP), CmMES1.5-RFP, CmMES1.5-RFP, CmMES10.2-RFP, and CmMES17.3-RFP were introduced into N. benthamiana leaves by Agrobacterium-mediated transient transformation. Three days later, fluorescence of all four MES genes fused to RFP could be observed in the cytoplasm ( Figure 5 ). The result indicated that the four MES proteins might function in the cytoplasm.

Transient overexpression in leaves of calamondin and ‘Taoyecheng’ sweet orange by infiltration were employed to verify the functions of the four MES genes. Green fluorescence observation showed that four key MES genes were successfully overexpressed in the leaves ( Figures 6A, B ). The quantitative analysis results showed that the expression levels of the four methylesterase genes were significantly increased in the transiently overexpressed citrus leaves compared with the control (Supplementary Figures S4). To identify the function of four key MES genes associated with CBC-resistance, transiently overexpressed leaves were in vitro inoculated with high concentrations (10⁸ cfu/ml) of Xcc suspension. The results of phenotypic observation and disease area statistics showed that overexpression of CmMES17.3 and CmMES10.2 increased the disease area, while overexpression of CmMES1.1 and CmMES1.5 decreased the disease area compared with the controls ( Figures 6B, C ). These results indicated that CmMES17.3 and CmMES10.2 might be negative regulatory factors for CBC resistance, while CmMES1.1 and CmMES1.5 might be positive regulatory factors.

The transiently overexpressed calamondin leaves were further employed to calculate the contents of SA, JA and IAA. The results showed that overexpression of CmMES1.1 and CmMES1.5 significantly increased the content of SA, and CmMES17.3 increased the content of IAA, however, CmMES10.2 increased the JA level ( Figure 6D ).