Hamlin trees verified as being HLB-free and ACP-friendly were purchased and placed in the green house. One branch cage placed in the upper part of each tree (three replicates) was filled with 75ACP from an infected population while other trees had cages with clean 75ACPs placed serving as control. The insects were allowed to feed on the trees for a week and then the insects were killed by spraying with topical insecticide. The ACPs were tested for CLas and the trees were subsequently returned to the greenhouse. The leaf samples (12 for each biological replicate and treatment) were collected from the untreated and infected plants and flash-frozen in liquid nitrogen and stored for further analysis.

The genes from Liberibacter asiaticus (non-culturable bacteria) were identified (LasP₂₃₅ and Effector 3), codon optimized and cloned in pUC57 by GenScript. The effectors were then amplified and cloned in pET28(a) vector between NdeI and BamHI sites and transformed in E. coli BL21 [BL21(DE3)pLysS]. The positive clones were inoculated overnight in LB with Kanamycin. The overnight culture (1%) was grown until the Optical density (OD) reached 0.6 and then induced with IPTG at 30°C for overnight. The cells were harvested next day and resuspended in protein isolation buffer (20-mM Tris-Cl, pH 7.4, 150-mM NaCl, and 10% glycerol). The cell suspension was sonicated and centrifuged at 14,000 rpm, 4°C, 30 min. The supernatant was collected and the inclusion bodies were treated with 9M urea. Following the treatment with urea, the cell suspension was centrifuged and supernatant was collected and refolded. The refolded protein from the inclusion bodies and the soluble fractions were purified using TALON metal affinity resin (Joshi and Puri, 2005).

Fresh leaf tissue, from five Hamlin trees (Citrus sinensis L. Osbeck) was pulverized in liquid nitrogen using a pestle and mortar and the resulting fine powder stirred with 1.5 volumes of extraction buffer [50-mM HEPES pH 7.5, 5-mM EDTA, 5-mM EGTA, 10-mM dithiothreitol (DTT), 10% glycerol, 7.5% polyvinylpolypyrolidone (PVPP), and a protease inhibitor cocktail, Complete™, Boehringer Mannheim]. The slurry was subsequently mixed on a reciprocating shaker (100 oscillations per min) for 10 min, at 4°C, followed by centrifugation 15,000 g for 30 min at 4°C. The supernatant was removed and immediately flash-frozen in liquid nitrogen and stored at −80°C until needed (Roy et al., 2011).

The purified refolded effector proteins were incubated with total protein (15 μg) isolated from healthy and infected citrus leaf extract for 2 h at 4°C. The effector–protein complex was incubated with TALON metal affinity resin at 4°C overnight. The resin was washed with column buffer (50-mM Tris-Cl, pH 7.4, 150 mM, 10% glycerol) and eluted with imidazole (250 mM). The eluted protein complex was sent for LC–MS/MS analysis to identify the citrus targets (Zhang et al., 2017). The spectra were searched against the Uniprot database, and taxonomy was set to C. sinensis. The only peptides that were ranked 1 were selected and finally those targets were selected for further analysis that had a 95% confidence (Karpievitch et al., 2012).

The superoxide dismutase (SOD) assay was quantified based on its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) by superoxide radical and assayed following (SOD Kit; catalog No.: 7,500-100-K) with some modifications. The reaction mixture (3 ml) contained 13 mM methionine, 75-mM NBT, 2-mM riboflavin,100-mM EDTA, and 0.3-ml leaf extracts. The volume was made up to 3 ml using 50-mM phosphate buffer with the addition of riboflavin at the very end. Once the reaction mixture was made, they were mixed well and incubated below two 15-W fluorescent tubes with a photon flux density of around 40 mmol m⁻² s⁻¹ for 10 min. Once the reaction is completed, the tubes were covered with a black cloth and the absorbance was measured at 560 nm. The non-irradiated mixture served as control and the absorbance so measured is inversely proportional to the amount of enzyme added. The SOD activity is defined as the amount of enzyme that caused 50% inhibition of the enzymatic reaction in the absence of the enzyme (Basu et al., 2010). The presented data were an average of three biological replicates, and two of them were of technical replicates.

The protease assay was performed using a fluorescence based (BODiPY) EnzChek protease assay kit. The analysis of aspartyl protease activity was done by incubating it with no other proteins in sodium citrate buffer (50 mm, pH 4.5). To perform the inhibitory effect of LasP₂₃₅ on the protease activity the renatured aspartyl protease was preincubated with increasing concentrations of LasP₂₃₅ at 4°C for 2 h in sodium citrate buffer. Following incubation, BODiPY-labeled casein substrate was added, and the reaction was monitored by measuring fluorescence in Tecan Infinite 200 PRO microplate reader at 485 ± 12.5 nm excitation/530 ± 15 nm emission filter. The assays were conducted in triplicates (Leippe et al., 2011; Coria et al., 2016).

The inhibitory effect of recombinant LasP₂₃₅ on recombinant glycosyl hydrolase was assayed using the β-Glucosidase Activity Assay Kit (MAK129, Sigma). The enzymatic reactions were carried out in K-Phosphate buffer (100 mM, pH 6.5) with p-nitrophenyl-β-D-glucopyranoside (β-NPG) for 20 min at 37°C. The final absorbance of the hydrolyzed product was measured at 405 nm (Henriques et al., 2017).

This assay was performed using aldehyde dehydrogenase (ALDH) Activity Abcam Assay Kit with modifications. In short, the purified ALDH was incubated with increasing concentration of substrate (acetaldehyde) for 1 h. The absorbance was measured at 450 nm and expressed in terms of NADH standard as mU/ml (Ouyang et al., 2019). The presented data were an average of three biological replicates, and two of them were of technical replicates.

The trypsin inhibition assay was done in triplicate and the result was expressed as a means of three replicates. In short, the residual trypsin activity was measured by monitoring the change in absorbance at 247 nm in presence of increasing concentration of recombinant purified Kunitz Trypsin inhibitor (KTI) when incubated with p-toluene-sulfonyl-l-Arg methyl ester (Sigma; Nehir El et al., 2015). The presented data were an average of three biological replicates, and two of them were of technical replicates.

Agrobacterium tumefaciens LBA4404 transformant cells carrying Las_P235, Effector 3 and the targets from citrus plants (Aspartyl protease, glycosyl hydrolase, SOD, KTI protein, lectin etc.), respectively, are cloned in pR101 vector and cultured overnight in LB medium with 50 μg ml⁻¹ of rifampicin and 50 μg ml⁻¹ kanamycin, and resuspended in 10-mM MgCl₂, 10-mM MES. The culture was diluted to an optical density of 0.5 (OD 600 nm). For each effector–target interaction, three leaves of 4 Nicotiana benthamiana plants overexpressing GFP1-9 were infiltrated with the A. tumefaciens suspension containing the effector and the target plasmids, respectively. The agro-infiltrated leaves were analyzed for protein localization at 3 dpi under a microscope (Olympus BX51-P) equipped with a UV light source. The agroinfiltrated plants were kept in a greenhouse for 24 h and the interaction was visualized using Illumatool lighting system (LT-9500; Lightools Research) with a 488-nm excitation filter (blue) and a colored glass 520-nm long pass filter. The photographs were taken by Photometric CoolSNAP HQ camera (Cabantous and Waldo, 2006; Liu et al., 2018).

The leaf disks from agro-infiltrated tobacco plants were incubated at 25°C on a shaker for 30 min in dark in 1 ml of K-phosphate buffer (20 mM, pH 6.0) containing 500-μM XTT. The increase in absorbance was measured at 470 nm in a spectrophotometer (Ramegowda et al., 2012). The presented data were an average of three biological replicates, and two of them were of technical replicates.

The lipid binding activity of recombinant LTP-6X His protein overexpressed and purified from E. coli was mixed with of 2-p-toluidinonaphthalene-6-sulphonate (TNS) at 25°C. The results were recorded at excitation 320 nm and the emission at 437 nm. The inhibitory action of LasP₂₃₅ on lipid transfer protein (LTP) was assessed using increasing concentration of LasP₂₃₅ and the results were measured. The purified GFP was used as a control (Melnikova et al., 2020). The minimum inhibitory concentration (MIC) of the LTP was performed using broth microdilution technique. The assay was carried out using 5 × 10⁵ colony forming units (CFU ml⁻¹) in MHB. The MIC was defined as the lowest concentration of the protein required to inhibit the visible growth of bacterial strains used (Ebbensgaard et al., 2015). The presented data were an average of three replicates, and two of them were of technical replicates.

Agrobacterium tumefaciens LBA4404 transformant cells carrying Effector 3 and the targets from citrus plants KTI protein cloned in pR101 vector was cultured overnight in LB medium with 50 μg ml⁻¹ of rifampicin and 50 μg ml⁻¹ kanamycin and resuspended in 10-mM MgCl₂, 10-mM MES. The culture was diluted to an optical density of 0.5 (OD 600 nm). For the assay, three leaves of N. benthamiana plants previously treated with paraquat (PQ; 100 μM) were infiltrated with the A. tumefaciens suspension containing the effector alone, Kunitz alone and the mixture of effector 3 and Kunitz, respectively (Pitino et al., 2016, 2018) and incubated for 48 h. The leaf disks were prepared by punching the leaf disks with a cork puncher. The punctured leaf disks were placed in water (50 ml) for 5 min to mitigate the error of measuring ion leakage due to injury inflicted on the leaves due to puncturing. The water was removed and the leaf discs were incubated with 5 ml. the conductivity was measured after 3 h using Mi180 bench meter and this value is referred to as A. The leaf disks with the bathing solution were then incubated at 95°C for 25 min and then cooled to room temperature to enable complete ion leakage. The conductivity was measured again, and this value is referred to as B. The ion leakage is subsequently expressed as (A/B) × 100. All experiments were carried out in three biological replicates with five leaf disks for each sample (Wu et al., 2017; Hatsugai et al., 2018).

The 3D structures of the two CLas proteins (LasP₂₃₅ and Effector 3) and the two citrus proteins (LTP and KTI) were predicted using I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). We then used HADDOCK version 2.2 webserver to predict interaction interfaces of LasP₂₃₅-LTP and Kunitz-E3 complexes (http://milou.science.uu.nl/services/HADDOCK2.2/). The selected complexes of LasP₂₃₅-LTP and Kunitz-E3 were further refined using molecular dynamics (MD) simulations of these complexes in the presence of water.

For the MD simulations, the TIP3P water model was used with CHARMM modifications (Albaugh et al., 2016). The water molecules were rigidified with SETTLE (Miyamoto and Kollman, 1993) and the molecular bond-lengths were constrained with P-LINC Lennard–Jones interactions (Wennberg et al., 2013) were evaluated using a group-based cutoff, truncated at 1 nm without a smoothing function. The Coulomb interactions were calculated using the smooth particle–mesh Ewald method (Cerutti et al., 2009; Kratz et al., 2016; Boateng, 2020) with a Fourier grid spacing of 0.12 nm (Fischer et al., 2015). The simulation in the NpT ensemble was achieved by semi-isotropic coupling at 1 bar with coupling constants of 4 ps (Aoki and Yonezawa, 1992; Blumer et al., 2020) and temperature-coupling the simulation system using velocity Langevin dynamics with a coupling constant of 1 ps (Washio et al., 2018). The integration time step was 2 fs. The non-bonded pair-list was updated every 20 fs (Walser et al., 2002).

Three targets of LasP₂₃₅, i.e., SOD, AP, and GH17, that are validated by in planta split GFP assay, are citrus PR or defense proteins with enzymatic activities. As described in the “Methods,” the citrus target proteins were expressed in E. coli, extracted from the inclusion body, and purified by affinity purification schemes. After purification, the proteins were re-folded. The identity of the proteins was confirmed by western blot analysis (Supplementary Figure 2) and Mass spec analysis (data are not shown). Therefore, before conducting in vitro inhibition assays, it was necessary to determine the enzymatic activities of the recombinant enzymes to confirm that they retained the native fold and function. We then determined the inhibitory activity of LasP₂₃₅ on them by measuring IC₅₀ (the concentration required for 50% reduction in enzymatic activity). The SOD, which is unique to plants, prevents damage caused by the ROS burst upon pathogen infection (Miller, 2012). While it facilitates the direct killing of the pathogen and induction of plant defense genes, excessive ROS is damaging to the plant. The SOD, produced in mitochondrion, peroxisome, and chloroplast, converts oxygen radical to molecular oxygen and hydrogen peroxide. The latter, also potentially phytotoxic, is subsequently converted by plant catalase into molecular oxygen and water. The SOD is also involved in regulating ROS signaling leading to the induction of defense genes (Wang et al., 2018). As shown in Table 1, LasP₂₃₅ inhibits the activity of the citrus SOD. The Citrus AP belongs to the A1 family of atypical aspartate proteases, primarily located in apoplast and chloroplast. It has been shown that an atypical aspartate protease, expressed in the apoplast, confers constitutive disease resistance 1 (CDR1) in Arabidopsis probably by producing a peptide ligand through cleavage and subsequent induction of SA signaling and expression of PR genes (Varghese et al., 1994; Simões et al., 2007). The results of enzyme assay show that LasP₂₃₅ inhibits the activity of the citrus AP. The GH17, a citrus (β1-3) glucanase, is another direct interactor of LasP₂₃₅. Typically, GH17 glucanases are known to provide disease resistance against fungi by hydrolyzing fungal chitins (Hrmova and Fincher, 2001). However, GH17 also has a role in immune defense in general in that it regulates the formation of callose (a β1-3 glucan polysaccharide), which is an essential component of papillae, an ultrastructure formed at the site of pathogen penetration. Apart from callose, the papillae also contain ROS and antimicrobial peptide thionin and thus provide the first line of defense against pathogen invasion. In papillae-mediated immunity, callose may be involved in two different mechanisms of plant defense against pathogens. First, the callose deposition in papillae may block pathogen spread. Second, the hydrolyzed products of callose by GH17 may serve as ligands for the PRRs and may induce SA signaling leading to plant immune defense. Thus, the GH17-mediated hydrolysis of callose may either support pathogen spread or induce SA signaling. As evident from Table 1, LasP₂₃₅ inhibits the glucanase activity of the citrus GH17. Therefore, it may induce SA signaling and help CLas to suppress citrus immune defense. The citrus LTP is the non-enzyme direct interactor of LasP₂₃₅. The plant LTPs possess (i) lipid binding property, which is critical to lipid homeostasis and membrane dynamics and (ii) bactericidal activity as a component of immune defense (Liu et al., 2015; Finkina et al., 2016; Salminen et al., 2016; Shenkarev et al., 2017). Table 1 shows that LasP₂₃₅ can block both lipid-binding and antimicrobial activities. Table 1 shows inhibitory activities of Effector 3 on two citrus target proteins: ALDH, which converts aldehydes into carboxylic acid using NADPH/NADH as a co-factor (Jimenez-Lopez et al., 2016) and KTI, which inhibits protease activity of PCD-inducing trypsin (Li et al., 2008).

In planta assays for monitoring ROS production, bacterial clearance, and PCD induction were performed in tobacco to examine the inhibitory effect LasP₂₃₅ and Effector 3 on their citrus target proteins. Paraquat was used for inducing the production of ROS in tobacco. The ROS level was monitored using a ROS/Superoxide detection assay (Zhou et al., 2018). In this experiment, the ROS level induced by agrobacterium carrying an empty vector (i.e., no gene) plus PQ was normalized to 100%. Note that, the infiltration of agrobacterium carrying citrus SOD reduced the ROS level significantly below 100%. However, as shown in Figure 3A (left), the simultaneous addition of agrobacteria carrying LasP₂₃₅, and citrus SOD showed the elevation in the ROS level proving in planta inhibition of citrus SOD by LasP₂₃₅. In planta bactericidal activity of citrus LTP was monitored by qPCR that showed the reduction of bacterial load in tobacco infected with P. syringae pv. DC3000. As shown in Figure 3A (right), agrobacterium carrying citrus LTP (0.4 × 10⁸ CFU ml⁻¹) reduced the bacterial load to 37%. Increasing agrobacterium carrying citrus LTP by 10 times (i.e., 0.4 × 10⁹ CFU ml⁻¹) led to the 75% reduction in the bacterial load. The addition of agrobacterium carrying LasP₂₃₅ (0.4 × 10⁸ CFU ml⁻¹) or 10 times of that increased the bacterial load. This proves that LasP₂₃₅ is able to block in planta the bactericidal activity of the citrus LTP. Supplementary Table 2 shows the Ct values of a Pst gene (a measure of bacterial load) at different LasP₂₃₅ concentrations. In planta studies were conducted in tobacco to examine the effect of (Effector 3–Lectin/Ef–Tu) interactions. As shown in Figure 3B (left), the infiltration of agrobacterium carrying Effector 3 induced ROS at a high level (85%). The ROS level due to agrobacterium carrying an empty vector plus PQ was set to 100%. Infiltration of agrobacterium carrying citrus Lectin or Ef–Tu had negligible effect on the ROS level. The co-infiltration of Effector 3 plus lectin or EF–Tu had very little effect on the ROS level induced by Effector 3 alone. However, combination of lectin and Ef–Tu was able to reduce the ROS level induced by Effector 3. In this regard, it is important to note that some bacteria, such as Porphyromonas gingivalis, Mycobacterium tuberculosis, Helicobacter pylori, and Bacillus anthracis, utilize ROS to support their growth and to establish infection (Paiva and Bozza, 2014) whereas plant lectin and Ef–Tu tend to inhibit ROS production or ROS-mediated signaling (Wang and Bouwmeester, 2017). It appears that pathogenic CLas may use Effector 3 to maintain ROS level that is beneficial to pathogen growth and infection by inhibiting anti-ROS citrus lectins and Ef–Tu. The PQ was also used to induce PCD via ROS in tobacco. The PCD was monitored by electrolyte leakage (Kacprzyk et al., 2016), which was set to 100% as induced by agrobacterium carrying an empty vector plus PQ. Infiltration of the agrobacterium carrying Effector 3 induced ~50% electrolyte leakage, which, as shown in Figure 3B (Right), was reduced on infiltration of agrobacterium carrying citrus KTI. The co-infiltration of Agrobacteria carrying citrus KTI and Effector 3 elevated PCD thereby confirming that Effector 3 is an inhibitor of the citrus KTI.