The Type III Effector XopLXcc in Xanthomonas campestris pv. campestris Targets the Proton Pump Interactor 1 and Suppresses Innate Immunity in Arabidopsis

Xanthomonas campestris pathovar campestris (Xcc) is a significant phytopathogen causing black rot disease in crucifers. Xcc injects a variety of type III effectors (T3Es) into the host cell to assist infection or propagation. A number of T3Es inhibit plant immunity, but the biochemical basis for a vast majority of them remains unknown. Previous research has revealed that the evolutionarily conserved XopL-family effector XopLXcc inhibits plant immunity, although the underlying mechanisms remain incompletely elucidated. In this study, we identified proton pump interactor (PPI1) as a specific virulence target of XopLXcc in Arabidopsis. Notably, the C-terminus of PPI1 and the Leucine-rich repeat (LRR) domains of XopLXcc are pivotal for facilitating this interaction. Our findings indicate that PPI1 plays a role in the immune response of Arabidopsis to Xcc. These results propose a model in which XopLXcc binds to PPI1, disrupting the early defense responses activated in Arabidopsis during Xcc infection and providing valuable insights into potential strategies for regulating plasma membrane (PM) H+-ATPase activity during infection. These novel insights enhance our understanding of the pathogenic mechanisms of T3Es and contribute to the development of effective strategies for controlling bacterial diseases.


Introduction
Xanthomonas is a genus of Gram-negative phytopathogens that threatens >400 plant species worldwide.Most Xanthomonas species utilize the type III secretion system to directly inject type III effector proteins (T3Es) into plant cells [1].Once inside, T3SEs contribute to pathogenesis, where a few are required for full pathogen virulence, and promote pathogen propagation in the host.Some are perceived by pattern recognition receptors (PPRs) to suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI); and others are monitored by the proteins of the host to activate strong defense responses [1][2][3].
In plants, the plasma membrane (PM) H + -ATPase, the well-known PM H + pump, is a central regulator in plant physiology, which mediates not only growth and development but also adaptation to diverse environmental stimuli [23,24].In vivo, its activity is modulated by various signals, with the major regulators being 14-3-3 family proteins, which bind to the auto inhibitory domain in the C-terminus of the ATPase, thereby stimulating pump activity [25].Limited information exists regarding the regulation of PM H + ATPase by other effectors.Proton pump interactor 1 (PPI1) is a regulatory protein that interacts with the regulatory C-terminus of the Arabidopsis PM H + -ATPase at a site distinct from the 14-3-3 binding site, thereby stimulating its activity in vitro [24,26].The main part of PPI1 is localized at the endoplasmic reticulum, from which it might translocate to the PM for interaction with H + -ATPase in response to as-yet-unidentified signals [27].PPI1 is highly expressed in most plant organs [28] and has been documented in several species, including Arabidopsis [24], rice [29], potato, and tomato [30].Additionally, previous research has revealed that PPI1 in plants responds to multiple abiotic stresses, including cold, salt, drought, and Fe deficiency stress [30,31].However, its role in the plant immune response to pathogens remains unclear.
This study revealed that XopL Xcc enhances virulence and suppresses innate immunity by targeting the proton pump interactor 1 (PPI1), a potential player in Arabidopsis immune responses.Moreover, the C-terminus of PPI1 and the LRR domains of XopL Xcc play crucial roles in facilitating this interaction.These results led us to propose a model in which XopL Xcc binds to PPI1, disrupting the early defense responses activated in Arabidopsis during Xcc infection and providing valuable insights into potential strategies for regulating PM H + -ATPase activity during infection.These insights shed light on the virulence strategies employed by Xcc and offer the potential for the development of novel control strategies against Xcc infections.

Ectopic Expression of XopL Xcc Inhibited PTI to Promote Xcc 8004 Proliferation in Arabidopsis
The roles of XopL Xcc in the pathogenic processes of Xcc 8004 were investigated by constructing three independent transgenic lines (Line1, -2, and -3) that overexpressed 35S::XopL Xcc :GUS (Figure S1).Upon exposure to Xcc 8004, all three lines exhibited more severe disease symptoms (Figure 1A) and harbored significantly larger bacterial populations compared to the control plants (Figure 1B).The flg22-induced accumulation of callose deposition (Figure 2A,B) and oxidative burst (Figure 2C) were suppressed in these lines.Additionally, the impact of XopL Xcc on disease resistance in Arabidopsis was assessed by analyzing the expression of four established PTI-related genes, including FRK1.Subsequent to inoculation with Xcc 8004∆hrcV, their transcript levels in XopL Xcc transgenic plants were reduced by varying degrees (Figure 2D).In conclusion, these findings demonstrate that XopL Xcc suppresses plant PTI by inhibiting the expression of PTI-related genes, the generation of flg22-induced ROS, and callose deposition in Arabidopsis.

XopL Xcc Interacts with PPI1 in Planta and in Yeast
A yeast two-hybrid screen against a normalized Arabidopsis Col-0 cDNA library was conducted to identify XopL Xcc interactors (Figure S2, see Methods Section 4).The yeast strain Cub-XopL Xcc was utilized as bait, with the cDNA library serving as the prey.A total of 10 7 primary yeast transformants were screened, resulting in the identification of 30 potential candidates.From these, PPI1, comprising 612 amino acids and encoded by At4g27500, was chosen due to its consistent presence during the screening process.Both the truncated protein PPI1∆ 1-358aa (lacking the N-terminal domain from 1 to 358 amino acids) and the full-length PPI1-encoding cDNA interacted with XopL Xcc during the yeast two-hybrid point-point verification (Figure 3A).Conversely, no interactions were observed between XopL Xcc and PPI2 (a homolog of PPI1), PPI1∆ 1-358aa , and XopL Xcc ∆LRR (lacking LRR domains) (Figure 3A). of 10 7 primary yeast transformants were screened, resulting in the identification of 3 tential candidates.From these, PPI1, comprising 612 amino acids and encode At4g27500, was chosen due to its consistent presence during the screening process the truncated protein PPI1Δ 1-358aa (lacking the N-terminal domain from 1 to 358 amin ids) and the full-length PPI1-encoding cDNA interacted with XopLXcc during the two-hybrid point-point verification (Figure 3A).Conversely, no interactions wer served between XopLXcc and PPI2 (a homolog of PPI1), PPI1Δ 1-358aa , and XopLXcc (lacking LRR domains) (Figure 3A).
The BiFC assay revealed specific interactivity between cEYFP-PPI1Δ 1-358aa and nE XopLXcc in Arabidopsis.As expected, PPI1 and XopLXcc also interplayed with each ot planta (Figure 3B).Consistent with the observations in the yeast two-hybrid experim no interaction was evident between PPI2 and XopLXcc or between PPI1Δ 1-358aa and Xo ΔLRR in the BiFC assay (Figure 3B).Together, these findings demonstrate that Xo interacts with PPI1 in plant cells.Moreover, the data suggest that XopLXcc binds sp cally to the C-terminus of PPI1, highlighting the essential role of the LRR domain i diating this interactivity.

PPI1 Can Potentially Influence the Subcellular Localization of XopLXcc
Our prior research established the subcellular localization of XopLXcc to the cell brane and cytoplasm [20].However, the results from the BiFC analysis were intrigu they demonstrated a lack of interaction between XopLXcc and PPI1 in the plasma brane (PM) (Figure 3B).Following this observation, we conducted transient co-expre experiments involving XopLXcc-EYFP with either the empty vector pXSN (EV) or P Arabidopsis protoplasts (Figure 4C).Under consistent fluorescence excitation and d tion, significantly diminished fluorescence signals of EYFP at the PM were noted upo expression with PPI1 compared to EV (Figure 4A,B).These findings indicate that interaction may have modified subcellular localization.The BiFC assay revealed specific interactivity between cEYFP-PPI1∆ 1-358aa and nEYFP-XopL Xcc in Arabidopsis.As expected, PPI1 and XopL Xcc also interplayed with each other in planta (Figure 3B).Consistent with the observations in the yeast two-hybrid experiments, no interaction was evident between PPI2 and XopL Xcc or between PPI1∆ 1-358aa and XopL Xcc ∆LRR in the BiFC assay (Figure 3B).Together, these findings demonstrate that XopL Xcc interacts with PPI1 in plant cells.Moreover, the data suggest that XopL Xcc binds specifically to the C-terminus of PPI1, highlighting the essential role of the LRR domain in mediating this interactivity.

PPI1 Can Potentially Influence the Subcellular Localization of XopL Xcc
Our prior research established the subcellular localization of XopL Xcc to the cell membrane and cytoplasm [20].However, the results from the BiFC analysis were intriguing as they demonstrated a lack of interaction between XopL Xcc and PPI1 in the plasma membrane (PM) (Figure 3B).Following this observation, we conducted transient co-expression experiments involving XopL Xcc -EYFP with either the empty vector pXSN (EV) or PPI1 in Arabidopsis protoplasts (Figure 4C).

PPI1 Plays a Role in Arabidopsis Immune Response to Xcc
PPI1 encodes proton pump interactor 1, which can bind to the Arabidops ATPase (EC 3.6.3.6) and stimulate its activity [27].However, the function and sp naling mechanisms to which PPI1 responds to remain unknown.Inoculation 8004ΔhrcV or the flg22 peptide (a 22-amino-acid sequence from the N-terminal flagellin) led to a 3-7-fold increase in PPI1 expression in Arabidopsis Col-0 (Fig The role of PPI1 in the response of Arabidopsis to Xcc infection was further inves inoculating both the wild-type Col-0 and a PPI1 loss-of-function muta (SALK_042646C), with 10 6 CFU/mL of the Xcc 8004ΔhrcV mutant (see Methods As anticipated, ppi1-1 exhibited markedly enhanced ΔhrcV bacterial growth com the wild type (Figure 5C).A previous study observed that XopLXcc could amplify ogenicity of Δ17E (Xcc 8004 strain lacking 17 known T3Es, including XopLXcc, as in Table S2) in the Col-0 genotype [20].When the same experimental procedur plied to ppi1-1, no significant variations in bacterial growth were observed, as a (Figure 5D).These results indicate that PPI1 potentially plays a role in the im sponse of Arabidopsis to Xcc and support the hypothesis that PPI1 is a target of X

PPI1 Plays a Role in Arabidopsis Immune Response to Xcc
PPI1 encodes proton pump interactor 1, which can bind to the Arabidopsis PM H + -ATPase (EC 3.6.3.6) and stimulate its activity [27].However, the function and specific signaling mechanisms to which PPI1 responds to remain unknown.Inoculation with Xcc 8004∆hrcV or the flg22 peptide (a 22-amino-acid sequence from the N-terminal region of flagellin) led to a 3-7-fold increase in PPI1 expression in Arabidopsis Col-0 (Figure 5A,B).The role of PPI1 in the response of Arabidopsis to Xcc infection was further investigated by inoculating both the wild-type Col-0 and a PPI1 loss-of-function mutant, ppi1-1 (SALK_042646C), with 10 6 CFU/mL of the Xcc 8004∆hrcV mutant (see Methods Section 4).As anticipated, ppi1-1 exhibited markedly enhanced ∆hrcV bacterial growth compared to the wild type (Figure 5C).A previous study observed that XopL Xcc could amplify the pathogenicity of ∆17E (Xcc 8004 strain lacking 17 known T3Es, including XopL Xcc , as described in Table S2) in the Col-0 genotype [20].When the same experimental procedure was applied to ppi1-1, no significant variations in bacterial growth were observed, as anticipated (Figure 5D).These results indicate that PPI1 potentially plays a role in the immune response of Arabidopsis to Xcc and support the hypothesis that PPI1 is a target of XopL Xcc .

XopLXcc Suppresses Innate Immunity in Arabidopsis by Targeting PPI1
In a previous investigation, XopLXcc was demonstrated to inhibit the expression of four PTI-related genes in Arabidopsis protoplasts [20].Hence, PPI1 was transiently co-expressed with XopLXcc or empty vector in Col-0 protoplasts (Figure 6B).The results revealed that while PPI1 induced the expression of PTI-related genes by ~5-9 fold, XopLXcc was able to suppress this response by interacting with PPI1 (Figure 6A).
Next, XopLXcc was transiently expressed in Col-0 and ppi1-1 protoplasts (Figure 7E, F).Following treatment with flg22, the expression of the four PTI-related genes in ppi1-1 declined markedly compared to that of the wild-type Col-0 (Figure 7A-D).In Col-0, Xo-pLXcc suppressed the expression of PTI-related genes, which was attenuated in ppi1-1 (Figure 7A-D).These findings underscore the importance of the interaction between PPI1 and XopLXcc in the immune response of Arabidopsis to Xcc.
The responses of stomatal apertures to flg22 in different lines were examined to investigate the involvement of PPI1 in flg22 signaling and stomatal immunity.The stomatal apertures of XopLXcc-expressing lines resembled those of ppi1-1, exhibiting a marked increase compared to that in the control (Figure 8).In summary, these results led us to propose a model wherein XopLXcc binds to PPI1, disrupting the early defense responses activated in Arabidopsis during Xcc infection.

XopL Xcc Suppresses Innate Immunity in Arabidopsis by Targeting PPI1
In a previous investigation, XopL Xcc was demonstrated to inhibit the expression of four PTI-related genes in Arabidopsis protoplasts [20].Hence, PPI1 was transiently co-expressed with XopL Xcc or empty vector in Col-0 protoplasts (Figure 6B).The results revealed that while PPI1 induced the expression of PTI-related genes by ~5-9 fold, XopL Xcc was able to suppress this response by interacting with PPI1 (Figure 6A).Next, XopL Xcc was transiently expressed in Col-0 and ppi1-1 protoplasts (Figure 7E,F).Following treatment with flg22, the expression of the four PTI-related genes in ppi1-1 declined markedly compared to that of the wild-type Col-0 (Figure 7A-D).In Col-0, XopL Xcc suppressed the expression of PTI-related genes, which was attenuated in ppi1-1 (Figure 7A-D).These findings underscore the importance of the interaction between PPI1 and XopL Xcc in the immune response of Arabidopsis to Xcc.The responses of stomatal apertures to flg22 in different lines were examined to investigate the involvement of PPI1 in flg22 signaling and stomatal immunity.The stomatal apertures of XopL Xcc -expressing lines resembled those of ppi1-1, exhibiting a marked increase compared to that in the control (Figure 8).In summary, these results led us to propose a model wherein XopL Xcc binds to PPI1, disrupting the early defense responses activated in Arabidopsis during Xcc infection.

Discussion
Plant pathogenic bacteria commonly secrete T3Es into host cells to modula responses, facilitating infection, establishment, and proliferation [1].For instance, 8004, ~12 T3Es, such as XopL, XopD, XopN, XopAC, XopK, and others, inhibited t munity induced by flg22 in Arabidopsis [9].Nevertheless, the specific functions and of many T3Es remain primarily unclear [32].Given that Xcc 8004 is responsible for c economically damaging black rot diseases in multiple crop species [3,4], an urgen has arisen to identify the targets and illuminate the pathogenic mechanisms of T this specific pathogenic strain.
XopLXcc is a member of the XopL effector superfamily, which is widespread a Xanthomas species and serves as a core effector group [33].These effectors are cha ized by the presence of homologs with LRR domains and an XL box known for ligase activity.XopLXcv can suppress the expression of defense-related genes in thereby undermining their immune responses.The XL box is crucial for E3 ubiqui ase activity and influences plastid phenotypes [33,34].However, XopLXap (lacking box) retained the ability to suppress immune responses [22].XopL from X. euves (XopLXe) directly associates with microtubules and causes severe cell death in N. be iana [22].In this study, we observed that XopLXcc suppressed innate plant immun reducing the expression of PTI-related genes (Figure 2D) and the generation of flg duced callose deposition (Figure 2A,B), as well as reactive oxygen species (ROS) ( 2C) in transgenic Arabidopsis.These results are consistent with those of prior studi ducted using protoplasts or distinct transgenic platforms [16,20].
The identification of T3E targets is a fundamental question in plant patholog Our study revealed that XopLXcc could interact with both PPI1Δ 1-358aa and full-lengt through yeast two-hybrid and BiFC assays (Figure 3), indicating PPI1 as one of t mary targets of XopLXcc.Moreover, no interactivity was detected between PPI2 an pLXcc or PPI1Δ 1-358aa and XopLXccΔLRR, suggesting that XopLXcc engages explicitly w C-terminus of PPI1, with the LRR domains being crucial for this interaction.All p containing LRR domains are believed to facilitate protein-protein associations [36 ous invasive bacterial proteins were identified as containing multiple LRR domain Consequently, their absence could result in structural alterations that impact protein tion.
Although the precise molecular mechanism remains elusive, our study indi

Discussion
Plant pathogenic bacteria commonly secrete T3Es into host cells to modulate host responses, facilitating infection, establishment, and proliferation [1].For instance, in Xcc 8004, ~12 T3Es, such as XopL, XopD, XopN, XopAC, XopK, and others, inhibited the immunity induced by flg22 in Arabidopsis [9].Nevertheless, the specific functions and targets of many T3Es remain primarily unclear [32].Given that Xcc 8004 is responsible for causing economically damaging black rot diseases in multiple crop species [3,4], an urgent need has arisen to identify the targets and illuminate the pathogenic mechanisms of T3Es in this specific pathogenic strain.
XopL Xcc is a member of the XopL effector superfamily, which is widespread among Xanthomas species and serves as a core effector group [33].These effectors are characterized by the presence of homologs with LRR domains and an XL box known for its E3 ligase activity.XopL Xcv can suppress the expression of defense-related genes in plants, thereby undermining their immune responses.The XL box is crucial for E3 ubiquitin ligase activity and influences plastid phenotypes [33,34].However, XopL Xap (lacking the XL box) retained the ability to suppress immune responses [22].XopL from X. euvesicatoria (XopL Xe ) directly associates with microtubules and causes severe cell death in N. benthamiana [22].In this study, we observed that XopL Xcc suppressed innate plant immunity by reducing the expression of PTI-related genes (Figure 2D) and the generation of flg22-induced callose deposition (Figure 2A,B), as well as reactive oxygen species (ROS) (Figure 2C) in transgenic Arabidopsis.These results are consistent with those of prior studies conducted using protoplasts or distinct transgenic platforms [16,20].
The identification of T3E targets is a fundamental question in plant pathology [35].Our study revealed that XopL Xcc could interact with both PPI1∆ 1-358aa and full-length PPI1 through yeast two-hybrid and BiFC assays (Figure 3), indicating PPI1 as one of the primary targets of XopL Xcc .Moreover, no interactivity was detected between PPI2 and XopL Xcc or PPI1∆ 1-358aa and XopL Xcc ∆LRR, suggesting that XopL Xcc engages explicitly with the C-terminus of PPI1, with the LRR domains being crucial for this interaction.All proteins containing LRR domains are believed to facilitate protein-protein associations [36].Various invasive bacterial proteins were identified as containing multiple LRR domains [37].Consequently, their absence could result in structural alterations that impact protein function.
Although the precise molecular mechanism remains elusive, our study indicates a potential role for PPI1 in the immune responses in Arabidopsis.Both ∆hrcV and flg22 could upregulate the expression of PPI1 in Col-0 (Figure 5A,B).Notably, the expression levels of the four PTI-related genes in ppi1-1 were significantly reduced (Figure 7A-D), aligning with the observation that ppi1-1 exhibited markedly higher ∆hrcV bacterial growth compared to the wild type (Figure 5C).In Arabidopsis protoplasts, PPI1 induced the expression of PTI-related genes, while XopL Xcc counteracted this response through its interaction with PPI1 (Figure 6A).Notably, in Col-0 protoplasts, XopL Xcc suppressed the expression of PTIrelated genes, which was mitigated in ppi1-1 (Figure 7A-D).Moreover, the stomatal aperture of XopL Xcc -expressing lines resembled those of ppi1-1 mutants, exhibiting a remarkable elevation compared to that of the control Col-0, which aligns with the phenotype observed in response to Xcc (Figures 1, 2, 5 and 8).Thus, these findings suggest a model in which XopL Xcc binds to PPI1, disrupting the early defense responses elicited in Arabidopsis during Xcc infection.
PPI1 consists of 612 amino acids and is predicted to encode three coiled-coil regions and a transmembrane domain, which might be recruited to the PM for interaction with H + -ATPase [27].Full-length PPI1 or its N-terminal domain could bind PM H + -ATPase at a site different from the known 14-3-3 binding locations and stimulate its activity [24].PM H + -ATPase, the well-known PM H + pump, is a central regulator in plant physiology, which mediates not only growth and development but also adaptation to diverse environmental stimuli [23,38,39].Its activation can trigger immune responses [40], while its mutants exhibit a defective PAMP-triggered production of ROS, altered MAPK activation, malfunctioning PAMP-triggered stomatal closure, and changed bacterial infection phenotypes [41].It is a crucial element in the defense mechanisms of plants against pathogen attack.However, it also functions as a target for pathogens that enable tissue invasion [42].In Xcc 8004, XopL Xcc did not interact with PPI1 at the PM (Figure 4), indicating a potential inhibition of PPI1 recruitment to the PM.This hindrance could disrupt the PPI1-PM H + -ATPase interactivity, ultimately affecting the activation of H + -ATPase and immune responses in plants.In contrast, XopL Xcc downregulated the salicylic acid (SA)-and PTI-related genes (Figure 6A) [22], aligning with the enhancement in PM H + -ATPase activity, which could cause SA accumulation and the expression of pathogenesis-related genes in tomatoes [40].In this context, the investigation of the possible disruption of the PPI1-H + -ATPase complex by XopL Xcc via ubiquitination, as well as the intricate spatial and temporal modulation of PM H + -ATPase activity during the initial stages of pathogen recognition, will be the emphasis of forthcoming research.
In conclusion, previous research has revealed that XopL Xcc interferes with the innate immunity of Arabidopsis by suppressing PTI and SA signaling, independent of MAPKs [16,20].However, the specific virulence targets and underlying mechanisms remain incompletely elucidated.In this study, we identified proton pump interactor PPI1 as a specific virulence target of XopL Xcc in Arabidopsis.Moreover, the C-terminus of PPI1 and the LRR domains of XopL Xcc are pivotal for facilitating this interactivity.This novel discovery marks the first identification of PPI1's role in conferring resistance to pathogen infection, providing valuable insights into potential strategies for regulating PM H + -ATPase activity during pathogen infection.These findings significantly enhance our understanding of the mechanisms employed by the T3Es of pathogenic bacteria and contribute to the development of effective strategies for controlling bacterial diseases.

Bacterial Strains and Growth Conditions
Xcc strains were cultured at 28 • C in a nutrient broth-yeast extract (NYG) medium.Escherichia coli and Agrobacterium tumefaciens strains were cultured in LB media at 37 • C and 28 • C, respectively.The antibiotics added were ampicillin (50 µg/mL), rifampicin (50 µg/mL), and kanamycin (50 µg/mL for E. coli and 25 µg/mL for Xcc and A. tumefaciens).

Vector Constructions
Full-length DNA fragments of XopL Xcc , PPI1, and PPI2 were amplified by employing FastPfu DNA polymerase (Beijing TransGen Biotech, Beijing, China) using the primers listed in Table S1.For transient expression in protoplasts, PCR products were cloned into the pXSN-HA vector [43].For constructing transgenic Arabidopsis plants, the PCR products were cloned into the 35S::GUS-pBI121 vector to generate the GUS-tagged constructs.

Plant Growth and Generation of the Transgenic Arabidopsis Plants
The Arabidopsis plants were grown in a mixture of vermiculite, perlite, and peat moss (1:1:2) in an environmentally controlled growth room at 22 • C and 70% relative humidity under a 12/12 h day/night light cycle.They were transformed with A. tumefaciens GV3101 carrying 35S::XopL xcc :GUS-pBI121 or 35S::GUS-pBI121 using the flower-dipping method [44].Transgenic lines were selected using 50 µg/mL kanamycin, and homozygous lines in the T 3 generation were identified.

Virulence Assays, Callose Deposition Assays, and Oxidative Burst Measurement
Virulence assays of Xcc strains were conducted utilizing mesophyll infiltration, as previously described [39].For the callose deposition assays, the leaves of six-week-old Arabidopsis plants were infused with 1 µM flg22.They were harvested 8 h after infiltration, washed with 95% ethanol, stained for callose with 0.1% aniline blue in 7 mM K 2 HPO 4 (pH 9.5), and then mounted in 50% glycerol.They were observed using an SZX16 fluorescence microscope (Olympus, Tokyo, Japan) under ultraviolet light, and the number of callose deposits in a 0.1 mm 2 microscopic field was counted in randomly coded samples from ten leaves by applying OpenCFU Version 1.0 software [45].
For oxidative burst measurement, the leaves of six-week-old Arabidopsis plants were cut into 1 mm-long strips and incubated in 200 µL of H 2 O in a 96-well plate for 12 h.Next, 1 µM flg22 in 200 µL of reaction buffer supplemented with 20 mM luminol and 1 µg of horseradish peroxidase (Sigma) was added.Luminescence was recorded for 45 min using a Synergy HT plate reader luminometer (Bio-Tek).

Transient Expression in Arabidopsis Protoplasts
Mesophyll protoplasts were prepared and transfected as previously described [33].Briefly, leaves from five-six-week-old plants were used for protoplast isolation.Enzyme solutions containing Cellulase R10 and Macerozyme R10 (Yakult, Tokyo, Japan) were utilized for leaf digestion.Plasmid DNA was purified by a HiSpeed plasmid Mini kit (QIAGEN, Dusseldorf, Germany) according to the manufacturer's instructions.

Gene Expression Analyses
Total RNA from the leaves or protoplasts was isolated using Trizol Reagent (Solarbio, Beijing, China).First-strand cDNA was synthesized from 500 ng of the total RNA utilizing a PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan) per the manufacturer's instructions.For real-time RT-qPCR, 20 ng of the cDNA was mixed with SYBR Premix Ex Taq (TaKaRa) and analyzed in triplicate by employing a LightCycler ® 480 Real-Time PCR System (Roche, Basel, Switzerland).Gene expression levels were normalized to those of the reference gene Atactin2.The sequences of the primers used are listed in Table S1.

Yeast Two-Hybrid Screening
Yeast two-hybrid assays were performed by following the Yeast Protocols Handbook.The leaves of four-week-old Arabidopsis plants were infiltrated with 10 6 CFU/mL Xcc 8004∆hrcV, and leaf samples were collected at 0 and 6 h.Total RNA was extracted using an RNeasy Plant Mini Kit (QIAGEN).Subsequently, reverse transcription was conducted using Switching Mechanism at 5 ′ End of RNA Template (SMART) technology.RT-PCR utilized the synthesized cDNA (sscDNA) as a template for dscDNA amplification.The products were purified, cleaved with SfiI, and ligated to the SfiI-digested pPR3N plasmid.Lastly, the Arabidopsis cDNA library was generated and employed to transform the Escherichia coli.
The entire XopL Xcc coding region was amplified and inserted in the pDHB1 vector to generate a fusion between the membrane protein Ost4 and the C-terminal half of ubiquitin (Cub), followed by the artificial transcription factor LexA-VP1 [46].The yeast strain NMY51 carrying the DHB1-XopL Xcc vector was transformed with the Arabidopsis cDNA library.Diploid cells were selected on a medium lacking Leu, Trp, and His supplemented with 10 mM 3-aminotriazole.Then, 2 × 10 7 transformants were screened, of which ~300 transformants that grew on the selective medium were obtained.Cells growing on the selective medium were further tested for lacZ reporter gene activity using a β-galactosidase assay.Direct interaction of two proteins was investigated by co-transformation of the yeast strain NMY51 with the respective plasmids; followed by the selection of transformants on a medium lacking Leu and Trp at 30 • C for 3 days; with the subsequent transfer to a medium lacking Leu, Trp, and His for growth selection; and testing of the lacZ activity in the interacting clones.To generate the PPI1 or PPI2 fusions with the N-terminal half of ubiquitin (NubG), as well as the XopL Xcc or XopL Xcc ∆LRR fusion with the C-terminal half of ubiquitin (Cub), the corresponding coding regions were amplified by PCR using the primers detailed in Table S1.They were inserted into SfiI sites of the pPR3N and pDHB1 vectors, respectively, and the sequence was verified.

Stomatal Aperture Measurement
The Arabidopsis plants were exposed to light for 2 h to ensure that most stomata were opened before treatment.Leaf peels were collected from the abaxial side of the leaves of five-week-old plants and floated in a buffer (10 mM MES [pH 6.15], 10 mM KCl, and 10 mM CaCl 2 ).After treatment with 100 nM flg22 or the mock solution for 1 h, the stomata were observed under a microscope (Olympus, Tokyo, Japan).The stomatal aperture was measured by applying ImageJ version 1.0 software.

Figure 1 .
Figure 1.XopLXcc promotes Xcc 8004 proliferation in Arabidopsis.(A)Disease symptoms.(B) Bacterial populations.Lines-1, -2, and -3 represent the three independent XopLXcc transgenic plants, and EV represents the control plants.The a-g labels on panel (B) represent significant differences (n = 30, p < 0.05; estimated by two-way ANOVA with Tukey's HSD test).The same letters mean no statistically significant differences.

Figure 1 .
Figure 1.XopL Xcc promotes Xcc 8004 proliferation in Arabidopsis.(A) Disease symptoms.(B) Bacterial populations.Lines-1, -2, and -3 represent the three independent XopL Xcc transgenic plants, and EV represents the control plants.The a-g labels on panel (B) represent significant differences (n = 30, p < 0.05; estimated by two-way ANOVA with Tukey's HSD test).The same letters mean no statistically significant differences.

Figure 1 .
Figure 1.XopLXcc promotes Xcc 8004 proliferation in Arabidopsis.(A)Disease symptoms.(B) Bacterial populations.Lines-1, -2, and -3 represent the three independent XopLXcc transgenic plants, and EV represents the control plants.The a-g labels on panel (B) represent significant differences (n = 30, p < 0.05; estimated by two-way ANOVA with Tukey's HSD test).The same letters mean no statistically significant differences.
Under consistent fluorescence excitation and detection, significantly diminished fluorescence signals of EYFP at the PM were noted upon co-expression with PPI1 compared to EV (Figure 4A,B).These findings indicate that their interaction may have modified subcellular localization.nt.J. Mol.Sci.2024, 25, 9175

Figure 4 .
Figure 4. PPI1 affected the subcellular localization of XopLXcc.(A) EYFP fluorescence w in Arabidopsis protoplasts co-expressing XopLXcc-EYFP with either the EV (the left panel) right panel).Row 1 displays cell fluorescence at a 100 µm scale, while Rows 2 and 3 are 10 µm scale.Both Rows 2 and 3 were subjected to identical experimental conditions, an sents two independent typical cells.(B) The fluorescence intensity of EYFP at the Arabid plast membrane.** p < 0.01 estimated by Student's t-test (n = 50).(C) The expression level and PPI1 in Arabidopsis protoplasts.The mRNA levels of all genes were normalized wi The a/b/c labels represent significant differences (n = 30, p < 0.05; estimated by two-wa with Tukey's HSD test).The same letters indicate no statistically relevant differences.

Figure 4 .
Figure 4. PPI1 affected the subcellular localization of XopL Xcc .(A) EYFP fluorescence was detected in Arabidopsis protoplasts co-expressing XopL Xcc -EYFP with either the EV (the left panel) or PPI1(the right panel).Row 1 displays cell fluorescence at a 100 µm scale, while Rows 2 and 3 are shown at a 10 µm scale.Both Rows 2 and 3 were subjected to identical experimental conditions, and each presents two independent typical cells.(B) The fluorescence intensity of EYFP at the Arabidopsis protoplast membrane.** p < 0.01 estimated by Student's t-test (n = 50).(C) The expression levels of XopL Xcc and PPI1 in Arabidopsis protoplasts.The mRNA levels of all genes were normalized with Atactin2.The a/b/c labels represent significant differences (n = 30, p < 0.05; estimated by two-way ANOVA with Tukey's HSD test).The same letters indicate no statistically relevant differences.

Figure 5 .
Figure 5. PPI1 influenced the resistance of Arabidopsis to Xcc. (A,B) The expression of PPI1 was induced by XccΔhrcV (A) and flg22 (B).Statistically significant differences at ** p < 0.01 were ascertained by Student's t-test (n = 20).(C,D) Bacterial growth was assessed at 0, 3, and 7 days postinfection.The a/b/c labels represent significant differences (n = 30, p < 0.05; estimated by two-way ANOVA with Tukey's HSD test).The same letters indicate no statistically relevant differences.

Figure 5 .
Figure 5. PPI1 influenced the resistance of Arabidopsis to Xcc. (A,B) The expression of PPI1 was induced by Xcc∆hrcV (A) and flg22 (B).Statistically significant differences at ** p < 0.01 were ascertained by Student's t-test (n = 20).(C,D) Bacterial growth was assessed at 0, 3, and 7 days post-infection.The a/b/c labels represent significant differences (n = 30, p < 0.05; estimated by two-way ANOVA with Tukey's HSD test).The same letters indicate no statistically relevant differences.

Figure 6 .
Figure 6.XopLXcc suppresses the expression of PTI-related genes induced by PPI1.(A) Th sion levels of PTI-related genes in XopLXcc or XopLXcc + PPI1-transfected protoplasts of A Col-0. (B) XopLXcc and PPI1 expression levels in protoplasts.The mRNA levels of all ge normalized to those of Atactin2.The a-g labels represent statistically significant variation < 0.05, two-way ANOVA with Tukey's HSD test).The same letters indicate no statisticall differences.

Figure 6 .
Figure 6.XopL Xcc suppresses the expression of PTI-related genes induced by PPI1.(A) The expression levels of PTI-related genes in XopL Xcc or XopL Xcc + PPI1-transfected protoplasts of Arabidopsis Col-0.(B) XopL Xcc and PPI1 expression levels in protoplasts.The mRNA levels of all genes were normalized to those of Atactin2.The a-g labels represent statistically significant variations (n = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters indicate no statistically relevant differences.

Figure 6 .
Figure 6.XopLXcc suppresses the expression of PTI-related genes induced by PPI sion levels of PTI-related genes in XopLXcc or XopLXcc + PPI1-transfected protopl Col-0.(B) XopLXcc and PPI1 expression levels in protoplasts.The mRNA levels normalized to those of Atactin2.The a-g labels represent statistically significant < 0.05, two-way ANOVA with Tukey's HSD test).The same letters indicate no st differences.

Figure 7 .
Figure 7. XopLXcc could not suppress the expression of PTI-related genes in ppi1.sion levels of PTI-related genes in XopLXcc-transfected protoplasts of Arabidopsis (E,F) The expression levels of XopLXcc in the protoplasts of Col-0 (E) and ppi1-1 (F) mRNA levels of all genes were normalized to those of Atactin2, and the relative were determined in protoplasts transfected with the control vector.The a/b/c labe tically significant variations (n = 5, p < 0.05, two-way ANOVA with Tukey's HS letters indicate no statistically relevant differences.

Figure 7 .
Figure 7. XopL Xcc could not suppress the expression of PTI-related genes in ppi1.(A-D) The expression levels of PTI-related genes in XopL Xcc -transfected protoplasts of Arabidopsis Col-0 and ppi1-1.(E,F)The expression levels of XopL Xcc in the protoplasts of Col-0 (E) and ppi1-1 (F), respectively.The mRNA levels of all genes were normalized to those of Atactin2, and the relative expression levels were determined in protoplasts transfected with the control vector.The a/b/c labels represent statistically significant variations (n = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters indicate no statistically relevant differences.

Figure 8 .
Figure 8.The ppi1 mutant and XopLXcc-transgenic plants exhibited abolished flg22-induced s closure.The a/b/c labels represent statistically significant variations (n = 50, p < 0.05, tw ANOVA with Tukey's HSD test).The same letters indicate no statistically relevant differenc