Functional Analysis of Type III Effectors in Xanthomonas campestris pv. campestris Reveals Distinct Roles in Modulating Arabidopsis Innate Immunity

Xanthomonas campestris pv. campestris (Xcc) is a significant phytopathogen causing black rot disease in crucifers. Its virulence relies heavily on the type III secretion system (T3SS), facilitating effector translocation into plant cells. The type III effectors (T3Es) disrupt cellular processes, promoting pathogen proliferation. However, only a few T3Es from Xcc have been thoroughly characterized. In this study, we further investigated two effectors using the T3Es-deficient mutant and the Arabidopsis protoplast system. XopE2Xcc triggers Arabidopsis immune responses via an unidentified activator of the salicylic acid (SA) signaling pathway, whereas XopLXcc suppresses the expression of genes associated with patterns-triggered immunity (PTI) and the SA signaling pathway. These two effectors exert opposing effects on Arabidopsis immune responses. Additionally, we examined the relationship between the specific domains and functions of these two effector proteins. Our findings demonstrate that the N-myristoylation motif and N-terminal domain are essential for the subcellular localization and virulence of XopE2Xcc and XopLXcc, respectively. These novel insights enhance our understanding of the pathogenic mechanisms of T3Es and contribute to developing effective strategies for controlling bacterial disease.


Introduction
The Gram-negative bacterial genus Xanthomonas comprises 27 species that collectively infect approximately 400 different host plants [1].Most Xanthomonas species utilize the type III secretion system (T3SS) to directly translocate type III effectors (T3Es) into host cells, where they interfere with key steps in plant immune responses.After long-term coevolution with host plants, the known T3Es exhibit diverse structures and functions, disrupting the plant immune system in various ways, including inhibiting patterns-triggered immunity (PTI) or effector-triggered immunity (ETI), ultimately leading to plant infection [2][3][4].Despite the identification of numerous T3Es, the virulence targets and molecular mechanisms remain incompletely understood.
XopE2 Xcc acts as an Avr protein, resulting in the avirulence of Xcc 8004 on the resistant host plant Chinese cabbage cv.Zhongbai-83 [8,18].A previous study revealed that XopE2 Xcc was regulated by hrpG and hrpX, and the product was translocated into plant cells in a T3SS-dependent manner [17].Similarly, XopL Xcc , a leucine-rich repeats protein encoded by the gene XC_4273, is crucial for Xcc 8004 pathogenicity.XopL Xcc , a virulence factor, suppresses PTI signaling in Arabidopsis [15].However, the underlying mechanisms of these T3Es remain incompletely understood.Further investigations are necessary to understand the pathogenicity of XopE2 Xcc and XopL Xcc .In this study, a T3Es-deficient mutant and a protoplast system were employed to investigate the function of the two effectors on the host plant Arabidopsis thaliana.These findings contribute to unveiling the molecular mechanisms of T3Es and aid in developing new strategies to control Xcc infections.

Bacterial Strains and Growth Conditions
Xcc strains were cultured at 28 • C in either a nutrient broth-yeast extract (NYG) medium or minimal medium (MMX).Antibiotics were added as necessary at the following concentrations: rifampicin (50 µg/mL), ampicillin (50 µg/mL), and kanamycin (50 µg/mL for E. coli and 25 µg/mL for Xcc).For growth curve analysis, Xcc strains were pre-cultured in 5 mL of NYG until reaching OD 600 = 1.0.Subsequently, for growth assessment in the MMX medium, pre-cultures were washed with MMX and inoculated in 300 µL of MMX at OD 600 = 0.1.For growth assessment in the NYG medium, pre-cultures were inoculated in 300 µL of NYG at OD 600 = 0.01.Optical density was measured every 12 h in MMX and every 4 h in NYG using an Automated Microbiology Growth Curve Analysis System.

Vector Constructions
Full-length DNA fragments of XopE2 Xcc (XC_2602) and XopL Xcc (XC_4273) were amplified from Xcc 8004.For protoplast transient expression, PCR products were cloned into the pXSN-HA vector [19] to generate hemagglutinin (HA)-tagged constructs.For subcellular localization, PCR products were fused to the yellow fluorescent protein (YFP) in the pA7-YFP vector [20] under the control of the Cauliflower mosaic virus (CaMV) 35S promoter to generate C-terminal EYFP-tagged constructs.Site-directed mutagenesis was employed to introduce the myristoylation mutation G2A in XopE2 Xcc .Plasmids were verified through DNA sequencing.Primer details are provided in Table S1.

Arabidopsis Protoplast Transient Expression
Arabidopsis thaliana Columbia (Col-0) was cultivated at 22 • C and 70% relative humidity.Protoplasts were prepared and transfected as previously described [21].Briefly, leaves from 5-to 6-week-old plants were used for protoplast isolation.Enzyme solutions containing Cellulase R10 and Macerozyme R10 (Yakult, Tokyo, Japan) were used for leaf digestion.Plasmid DNAs were purified using the HiSpeed Plasmid Mini Kit (QIAGEN, Dusseldorf, Germany) in accordance with the manufacturer's instructions.Subsequently, isolated protoplasts were transfected with 20 µg plasmid DNA using the polyethylene glycol (PEG)calcium method (20 µg per 1 × 10 6 cells).After a 6-h incubation period, protoplasts were exposed to the flg22 peptide (a 22-amino acid sequence from the N-terminal region of flagellin) for 1 h.Protoplasts transfected with an empty vector served as controls.

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

Virulence Assays
Virulence assays of Xcc strains on Col-0 were conducted using mesophyll infiltration as previously described [22].Briefly, Xcc 8004 was cultured in NYG medium with appropriate antibiotics for 1 d.Bacterial cells were diluted to a concentration of 10 6 CFU/mL in 10 mM MgCl 2 , and 10 µL of the mixture was injected into the leaves of 5-to 6-week-old Arabidopsis plants.Plant samples were maintained under humid conditions post-inoculation.Bacterial growth in planta was monitored by sampling leaf discs at different time points (days 0, 1, 3, 5, and 7 post-inoculation) and quantifying bacterial densities in log CFU/leaf.

Subcellular Localization
Subcellular localization of EYFP fusion proteins in plant cells was determined by tagging effector proteins to the N-terminus of the EYFP protein.Arabidopsis mesophyll protoplasts were transformed with purified plasmid DNA (20 µg per 2 × 10 5 cells).After 16 h of dark incubation at 23 • C, protoplasts were observed using a laser scanning confocal microscope (TCS SP8, Leica, Solms, Germany).YFP fluorescence was excited at 488 nm and detected in the 525-550 nm range.Autofluorescence of the mesophyll coat was visualized by excitation at 514 nm and detected in the 650-704 nm range.

Distinct Contributions of XopE2 Xcc and XopL Xcc to Xcc 8004 Virulence on Arabidopsis
We investigated the impact of XopE2 Xcc or XopL Xcc on Xcc 8004 pathogenicity in Arabidopsis.Growth curves analysis (Figure 1A,B) revealed similar cell densities among mutants ∆xopE2 Xcc , ∆xopL Xcc , and Xcc 8004 in both NYG and MMX mediums.Moreover, no significant differences were observed in the bacterial population or disease symptom production among ∆xopE2 Xcc , ∆xopL Xcc , and Xcc 8004 (Figure 1C,D).These results indicate that the deletion of XopE2 Xcc or XopL Xcc does not significantly affect Xcc 8004 pathogenicity in Arabidopsis.
Furthermore, we generated a mutant lacking 17 known T3Es, including XopE2 Xcc and XopL Xcc , designated as ∆17E (Table S2).Subsequently, XopE2 Xcc and XopL Xcc were cloned into the pLAFR3 vector and introduced into the mutant ∆17E strain.The growth curves of ∆17E, ∆17E(xopE2 Xcc ), ∆17E(xopL Xcc ), and Xcc 8004 were similar in both NYG and MMX mediums (Figure 2A,B).Following the same experimental procedure, these strains infiltrated into Arabidopsis leaves.∆17E caused slight yellowing around the inoculation site, whereas ∆17E(xopE2 Xcc ) exhibited significantly milder disease symptoms and low bacterial proliferation, indicating that XopE2 Xcc expression conferred resistance to Xcc 8004 infection in Arabidopsis.Conversely, the bacterial populations of ∆17E(xopL Xcc ) were significantly higher than those of ∆17E and ∆hrcV in Arabidopsis plants but lower than those of Xcc 8004 (Figure 2C).Notably, ∆17E(xopL Xcc ) induced severe disease symptoms, which were attributed to high bacterial titers (Figure 2D).These findings demonstrated the critical role of XopL Xcc in the virulence of Xcc 8004.

Effect of XopE2 Xcc and XopL Xcc on Defense Resistance in Arabidopsis
To investigate the impact of XopE2 Xcc and XopL Xcc on defense resistance in Arabidopsis, we analyzed the expression of four PTI-related genes and six salicylic acid (SA)-related genes using RT-qPCR.The expression levels of the PTI-related genes were significantly higher in XopE2 Xcc -expressing protoplasts than in the control group (Figure 3A-F).In contrast, the expression levels of these genes were significantly weaker in XopL Xcc -expressing protoplasts than those in the control.These results suggest that XopE2 Xcc can activate the expression of PTI-related genes, whereas XopL Xcc possesses the ability to inhibit their expression in Arabidopsis.Furthermore, we generated a mutant lacking 17 known T3Es, including XopE2Xcc and XopLXcc, designated as Δ17E (Table S2).Subsequently, XopE2Xcc and XopLXcc were cloned into the pLAFR3 vector and introduced into the mutant Δ17E strain.The growth curves of Δ17E, Δ17E(xopE2Xcc), Δ17E(xopLXcc), and Xcc 8004 were similar in both NYG and MMX mediums (Figure 2A,B).Following the same experimental procedure, these strains infiltrated into Arabidopsis leaves.Δ17E caused slight yellowing around the inoculation site, whereas Δ17E(xopE2Xcc) exhibited significantly milder disease symptoms and low bacterial proliferation, indicating that XopE2Xcc expression conferred resistance to Xcc 8004 infection in Arabidopsis.Conversely, the bacterial populations of Δ17E(xopLXcc) were significantly higher than those of Δ17E and ΔhrcV in Arabidopsis plants but lower than those of Xcc 8004 (Figure 2C).Notably, Δ17E(xopLXcc) induced severe disease symptoms, which were attributed to high bacterial titers (Figure 2D).These findings demonstrated the critical role of XopLXcc in the virulence of Xcc 8004.All SA-related genes were significantly upregulated in XopE2 Xcc -expressing protoplasts (Figure 3G-L).Notably, AtSID2 expression was upregulated approximately 7.1-fold in control protoplasts, whereas it increased by approximately 18.3-fold in XopE2 Xcc -expressing protoplasts.Conversely, XopL Xcc downregulated the expression of these genes.These findings indicate that XopE2 Xcc activates plant defense response-associated genes, including those involved in PTI and the SA signaling pathway.However, XopL Xcc exerts the opposite effect, suggesting contrasting impacts on defense resistance in protoplasts.
To further explore whether XopE2 Xcc directly induces the SA signaling pathway, we analyzed the expression level of defense response-associated genes in sid2 mutant protoplasts.All defense response-associated genes were induced in sid2 mutant protoplasts expressing XopE2 Xcc , albeit to a significantly lower extent than that in wild-type protoplasts (Figure 4), indicating that XopE2 Xcc expression in SA-deficient plants does not enhance the expression of these defense response-associated genes.Notably, the expression levels of three genes (AtEDS5, AtPAD4, and AtFMO1) in the SA signaling pathway were significantly increased in sid2 mutant protoplasts expressing XopE2 Xcc (Figure 4H-J).These results suggest that XopE2 Xcc boosts the expression of defense response-associated genes independently of SA accumulation, demonstrating a potential induction of upstream activators of the SA signaling pathway in Arabidopsis.

Effect of XopE2Xcc and XopLXcc on Defense Resistance in Arabidopsis
To investigate the impact of XopE2Xcc and XopLXcc on defense resistance in Arabidopsis, we analyzed the expression of four PTI-related genes and six salicylic acid (SA)-related genes using RT-qPCR.The expression levels of the PTI-related genes were significantly higher in XopE2Xcc-expressing protoplasts than in the control group (Figure 3A-F).In contrast, the expression levels of these genes were significantly weaker in XopLXcc-expressing protoplasts than those in the control.These results suggest that XopE2Xcc can activate the expression of PTI-related genes, whereas XopLXcc possesses the ability to inhibit their expression in Arabidopsis.(A-L), The expression levels of defense-related genes in XopE2Xcc or XopLXcc-transfected pro of Arabidopsis Col-0 were measured using real-time qRT-PCR, respectively.The mRNA leve genes were normalized with Atactin2, and the relative expression levels were determined in plasts transfected with the control vector.The a\b\c\d\e labels represent significant differe = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistica ence.
All SA-related genes were significantly upregulated in XopE2Xcc-expressing plasts (Figure 3G-L).Notably, AtSID2 expression was upregulated approximately 7 in control protoplasts, whereas it increased by approximately 18.3-fold in XopE pressing protoplasts.Conversely, XopLXcc downregulated the expression of these These findings indicate that XopE2Xcc activates plant defense response-associated including those involved in PTI and the SA signaling pathway.However, XopLXc the opposite effect, suggesting contrasting impacts on defense resistance in protop To further explore whether XopE2Xcc directly induces the SA signaling pathw analyzed the expression level of defense response-associated genes in sid2 mutant plasts.All defense response-associated genes were induced in sid2 mutant protopla pressing XopE2Xcc, albeit to a significantly lower extent than that in wild-type proto (Figure 4), indicating that XopE2Xcc expression in SA-deficient plants does not enha expression of these defense response-associated genes.Notably, the expression le three genes (AtEDS5, AtPAD4, and AtFMO1) in the SA signaling pathway were cantly increased in sid2 mutant protoplasts expressing XopE2Xcc (Figure 4H-J).Th sults suggest that XopE2Xcc boosts the expression of defense response-associated ge dependently of SA accumulation, demonstrating a potential induction of upstrea vators of the SA signaling pathway in Arabidopsis.(A-L), The expression levels of defense-related genes in XopE2 Xcc or XopL Xcc -transfected protoplasts of Arabidopsis Col-0 were measured using real-time qRT-PCR, respectively.The mRNA levels of all genes were normalized with Atactin2, and the relative expression levels were determined in protoplasts transfected with the control vector.The a\b\c\d\e labels represent significant differences (n = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistical difference.
Pathogens 2024, 13, 448 7 of 14 The expression levels of defense-associated genes in XopE2Xcc or XopLXcctransfected protoplasts of Arabidopsis sid2 mutant were measured using real-time qRT-PCR, respectively.The mRNA levels of all genes were normalized with Atactin2, and the relative expression levels were determined in protoplasts transfected with the control vector.The a\b\c\d labels represent significant differences (n = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistical difference.

Subcellular Localization and Function Analysis of Mutant XopE2Xcc(G2A) Protein
Myristoylation modification is crucial for anchoring proteins to cell membranes [23,24].XopE members possess a conserved putative myristoylation motif (Met-Gly-X-X-X-Ser/Thr-) at the N-terminus.It was reported that the covalent attachment of myristate to the Gly residue is an important mechanism for protein anchor [3,23,25].To assess the significance of the putative myristoylation site in XopE2Xcc for plasma membrane localization, we substituted the glycine (Gly2) residue in XopE2Xcc with Alanine (Ala), resulting in the mutant XopE2Xcc(G2A) protein fused with EYFP.These constructs were transiently expressed in Arabidopsis protoplasts.XopE2Xcc was localized to the plasma membrane in pro- Influence of XopE2 Xcc on the expression of defense response-associated genes in sid2 mutant protoplasts.(A-L), The expression levels of defense-associated genes in XopE2 Xcc or XopL Xcctransfected protoplasts of Arabidopsis sid2 mutant were measured using real-time qRT-PCR, respectively.The mRNA levels of all genes were normalized with Atactin2, and the relative expression levels were determined in protoplasts transfected with the control vector.The a\b\c\d labels represent significant differences (n = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistical difference.

Subcellular Localization and Function Analysis of Mutant XopE2 Xcc (G2A) Protein
Myristoylation modification is crucial for anchoring proteins to cell membranes [23,24].XopE members possess a conserved putative myristoylation motif (Met-Gly-X-X-X-Ser/Thr-) at the N-terminus.It was reported that the covalent attachment of myristate to the Gly residue is an important mechanism for protein anchor [3,23,25].To assess the significance of the putative myristoylation site in XopE2 Xcc for plasma membrane localization, we substituted the glycine (Gly2) residue in XopE2 Xcc with Alanine (Ala), resulting in the mutant XopE2 Xcc (G2A) protein fused with EYFP.These constructs were transiently expressed in Arabidopsis protoplasts.XopE2 Xcc was localized to the plasma membrane in protoplasts.Conversely, XopE2 Xcc (G2A) was detected in the cytoplasm surrounding chloroplasts and some undefined organelles, rather than the plasma membrane (Figure 5).Furthermore, the mutant XopE2 Xcc (G2A) protein failed to induce the expression of defense responseassociated genes (Figure 6).These findings indicate that the G2A mutation in XopE2 Xcc disrupts its plasma membrane localization and its ability to induce defense responses, highlighting the dependence of XopE2 Xcc function on myristoylation modification.

Subcellular Localization and Function Analysis of Various Mutant XopL Xcc Proteins
Full-length XopL Xcc cDNA and three mutants, XopL Xcc ∆ 1-194aa (lacking the N-terminal domain [NTD] and Leucine-rich repeat [LRR1 and LRR2]), XopL Xcc ∆ 1-138aa (lacking NTD and LRR1), and XopL Xcc ∆ 290-504aa (lacking the C-terminal domain), were constructed.The results indicated strong EYFP fluorescence at the plasma membrane and in the cytoplasm in XopL Xcc -expressing protoplasts.The subcellular localization of XopL Xcc ∆ 290-504aa was similar to that of XopL Xcc -expressing protoplasts, exhibiting membrane localization in protoplasts.XopL Xcc ∆ 1-138aa showed EYFP fluorescence in some undefined organelles and the plasma membrane.Interestingly, XopL Xcc ∆ 1-194aa exhibited subcellular localization surrounding chloroplasts, with no EYFP fluorescence observed in the protoplast membrane (Figure 7).These results demonstrate that the leucine-rich repeats (LRRs) of the N-terminal region of XopL Xcc play a critical role in protein localization in protoplasts.(A-H),The expression of response-associated genes in XopE2Xcc and XopE2Xcc (G2A) transfected protoplasts was measured using real-time qRT-PCR, respectively.The a\b\c\d\e labels represent significant differences (n = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistical difference.

Subcellular Localization and Function Analysis of Various Mutant XopLXcc Proteins
Full-length XopLXcc cDNA and three mutants, XopLXccΔ 1-194aa (lacking the N-terminal domain [NTD] and Leucine-rich repeat [LRR1 and LRR2]), XopLXccΔ 1-138aa (lacking NTD and LRR1), and XopLXccΔ 290-504aa (lacking the C-terminal domain), were constructed.The results indicated strong EYFP fluorescence at the plasma membrane and in the cytoplasm in XopLXcc-expressing protoplasts.The subcellular localization of XopLXccΔ 290-504aa was similar to that of XopLXcc-expressing protoplasts, exhibiting membrane localization in protoplasts.XopLXccΔ 1-138aa showed EYFP fluorescence in some undefined organelles and the plasma membrane.Interestingly, XopLXccΔ 1-194aa exhibited subcellular localization surrounding chloroplasts, with no EYFP fluorescence observed in the protoplast membrane  (A-H),The expression of response-associated genes in XopE2 and XopE2 Xcc (G2A) transfected protoplasts was measured using real-time qRT-PCR, respectively.The a\b\c\d\e labels represent significant differences (n = p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistical difference.
Pathogens 2024, 13, 448 9 of 14 (Figure 7).These results demonstrate that the leucine-rich repeats (LRRs) of the N-terminal region of XopLXcc play a critical role in protein localization in protoplasts.Furthermore, considering the varied localization of the N-terminal truncated versions and wild-type XopLXcc in Arabidopsis protoplasts, we evaluated the expression levels of defense-related genes.Notably, XopLXccΔ 1-138aa and XopLXccΔ 1-194aa significantly suppressed the expression of all four defense response-associated genes, while XopLXccΔ 290- 504aa only negatively affected two marker genes, At2g17740 and At5g57220 (Figure 8).The N-terminal deletion of XopLXcc altered the subcellular localization and function of this protein in plants, indicating that the N-terminal region of XopLXcc may play a critical role in suppressing plant immunity.Furthermore, considering the varied localization of the N-terminal truncated versions and wild-type XopL Xcc in Arabidopsis protoplasts, we evaluated the expression levels of defense-related genes.Notably, XopL Xcc ∆ 1-138aa and XopL Xcc ∆ 1-194aa significantly suppressed the expression of all four defense response-associated genes, while XopL Xcc ∆ 290-504aa only negatively affected two marker genes, At2g17740 and At5g57220 (Figure 8).The N-terminal deletion of XopL Xcc altered the subcellular localization and function of this protein in plants, indicating that the N-terminal region of XopL Xcc may play a critical role in suppressing plant immunity.

Discussion
Plant pathogenic bacteria typically secrete T3Es into host cells to modulate host responses, facilitating successful infection and proliferation within the host plant.Xcc 8004 causes economically significant block rot diseases various crops [26], underscoring the importance of elucidating the pathogenesis mechanisms of T3Es.However, the functions and host targets of many T3Es remain largely elusive.Demonstrating the virulence functions of numerous T3Es in Arabidopsis-Xcc 8004 model systems can be challenging due to functional redundancy or similar pathogenicity-related genes [27].Our previous research highlighted the Avr function of XopE2Xcc in the host plant Chinese cabbage cv.Zhongbai-83 [8], whereas XopLXcc was identified as critical for the full virulence of Xcc 8004 on the host plant Chinese radish [18].Initial data from Arabidopsis infections did not reveal significant differences in disease symptoms or bacterial growth among ΔxopE2Xcc, ΔxopLXcc, and the wild-type Xcc 8004 strain, possibly due to masking effects of other T3Es in Xcc 8004.To address potential redundancy issues, we developed a T3E-deficient mutant, the Δ17E strain, with 17 known T3E-coding genes deleted.Using this mutant strain, we demonstrated that XopLXcc contributes to Xcc virulence, whereas XopE2Xcc functions as an Avr protein in Arabidopsis.These results confirm the presence of functional redundancy among certain effector proteins during infection of specific host plants.The Δ17E mutant strain serves as a valuable resource for the functional analysis of one or more T3Es.

XopE2Xcc Triggers Avirulence in Arabidopsis via Upstream Activation of the SA Signaling Pathway
The defense response-associated genes (At1g51890, At2g17740, AtFRK1, and At5g57220) are specifically induced by PTI from bacteria rather than other stress-related signals [28,29].The expression levels of these genes were significantly elevated in Arabidopsis protoplasts transiently expressing XopE2Xcc, suggesting that XopE2Xcc activates their expression, thereby enhancing Arabidopsis PTI.Furthermore, SA-mediated immune responses play a critical role in Arabidopsis defense [4,30,31].Our data revealed that expressing XopE2Xcc in Arabidopsis significantly activated the SA signaling pathway.Genes involved in SA biosynthesis (AtSID2), SA accumulation (AtEDS5, AtPAD4, and AtFMO1),

Discussion
Plant pathogenic bacteria typically secrete T3Es into host cells to modulate host responses, facilitating successful infection and proliferation within the host plant.Xcc 8004 causes economically significant block rot diseases in various crops [26], underscoring the importance of elucidating the pathogenesis mechanisms of T3Es.However, the functions and host targets of many T3Es remain largely elusive.Demonstrating the virulence functions of numerous T3Es in Arabidopsis-Xcc 8004 model systems can be challenging due to functional redundancy or similar pathogenicity-related genes [27].Our previous research highlighted the Avr function of XopE2 Xcc in the host plant Chinese cabbage cv.Zhongbai-83 [8], whereas XopL Xcc was identified as critical for the full virulence of Xcc 8004 on the host plant Chinese radish [18].Initial data from Arabidopsis infections did not reveal significant differences in disease symptoms or bacterial growth among ∆xopE2 Xcc , ∆xopL Xcc , and the wild-type Xcc 8004 strain, possibly due to masking effects of other T3Es in Xcc 8004.To address potential redundancy issues, we developed a T3E-deficient mutant, the ∆17E strain, with 17 known T3E-coding genes deleted.Using this mutant strain, we demonstrated that XopL Xcc contributes to Xcc virulence, whereas XopE2 Xcc functions as an Avr protein in Arabidopsis.These results confirm the presence of functional redundancy among certain effector proteins during infection of specific host plants.The ∆17E mutant strain serves as a valuable resource for the functional analysis of one or more T3Es.

XopE2 Xcc Triggers Avirulence in Arabidopsis via Upstream Activation of the SA Signaling Pathway
The defense response-associated genes (At1g51890, At2g17740, AtFRK1, and At5g57220) are specifically induced by PTI from bacteria rather than other stress-related signals [28,29].The expression levels of these genes were significantly elevated in Arabidopsis protoplasts transiently expressing XopE2 Xcc , suggesting that XopE2 Xcc activates their expression, thereby enhancing Arabidopsis PTI.Furthermore, SA-mediated immune responses play a critical role in Arabidopsis defense [4,30,31].Our data revealed that expressing XopE2 Xcc Arabidopsis significantly activated the SA signaling pathway.Genes involved in SA biosynthesis (AtSID2), SA accumulation (AtEDS5, AtPAD4, and AtFMO1), and downstream signal transduction (AtNPR1 and AtWRKY51) exhibited significantly higher expression levels in XopE2 Xcc -transgenic plants than in control plants.Notably, XopE2 Xcc expression in the SA defective mutant (sid2 mutant) protoplasts failed to enhance the expression of four defense response-associated genes, confirming that XopE2 Xcc elicits the Arabidopsis immune response possible through the SA signaling pathway.Moreover, the expression of genes involved in SA accumulation (AtEDS5, AtPAD4, and AtFMO1) in mutant protoplasts was significantly increased by XopE2 Xcc , indicating that XopE2 Xcc functions upstream of genes related to SA accumulation.These results suggest that XopE2 Xcc triggers Arabidopsis immune responses via an unidentified activator upstream of the SA signaling pathway.Further identification and characterization of the target protein of XopE2 Xcc in plants will enhance our understanding of how XopE2 Xcc modulates plant defense responses.
N-myristoylation involves the covalent attachment of a hydrophobic 14-carbon saturated fatty acid to the glycine at residue two (Gly2), providing primary membrane targeting, signaling for several plant protein kinases and directing proteins to the plant cell plasma membrane [23].Previous research has suggested that XopE2 Xcc , a member of the XopE group, and possibly part of the transglutaminase superfamily, contains a putative Nmyristoylation motif [3].Consistent with findings on Avr Xcc C and XopE2 Xe , our data indicate that the N-myristoylation motif is essential for the subcellular localization and avirulence function of XopE2 Xcc in Arabidopsis [10,23].
Avr proteins are known to play a dual role in triggering ETI in plants with corresponding R proteins, while simultaneously suppressing plant defense in plants without the corresponding R proteins [32][33][34].A previous study has demonstrated that XopE2 Xcc (also known as avrXccE1) induces avirulence of Xcc 8004 in Chinese cabbage cv.Zhongbai-83, but not in Xcc strain 528 T on Early Jersey Wakefield cabbage [35].Our data indicate that the avirulence function of XopE2 Xcc in Arabidopsis is dependent on plasma membrane anchoring for host recognition, suggesting the presence of its corresponding R protein on the plasma membrane of Arabidopsis.Further identification and characterization of the R protein of XopE2 Xcc in Arabidopsis are necessary to enhance our understanding of how XopE2 Xcc modulates plant defense responses.

XopL Xcc Suppresses PTI-Related and SA-Related Genes in Arabidopsis
In this study, we observed significant suppression of four PTI-related genes in XopL Xccexpressing protoplasts, suggesting its ability to inhibit Arabidopsis PTI.These findings are consistent with previous reports indicating that XopL Xcc enhances bacterial infection in Arabidopsis while suppressing the reactive oxygen species (ROS) burst, callose deposition, and other related responses [36].Moreover, we observed significantly lower expression levels of SA-related genes in XopL Xcc -expressing protoplasts than in controls, suggesting that XopL Xcc promotes the synthesis and accumulation of SA, thereby suppressing Arabidopsis immune responses, possibly through the SA signaling pathway.
XopL Xcc belongs to the XopL superfamily, characterized by three LRR domains and a putative XopL E3 ligase box domain (XL box).XopL Xcv can suppress defense gene expression in plants and subvert plant immunity, with the XL box playing an essential role in E3 ubiquitin ligase activity and affecting plastid phenotypes [37,38].However, our in vitro assessments did not reveal any E3 ligase activity for XopL Xcc .Conversely, a truncated form of XopL Xap (lacks the XL box) retained the ability to suppress plant immune responses [39].Our research demonstrates that different N-terminal mutants exhibit varied localizations and effects on plant immunity, highlighting the essential role of the complete set of LRR domains of XopL Xcc in modulating plant immunity.
The LRRs are 20-29 residue sequence motifs present in numerous proteins with diverse functions.All proteins containing these repeats are believed to facilitate proteinprotein interactions [40].Various invasive bacterial proteins were identified to contain multiple LRR motifs [41].The structure of XopL Xcc primarily consists of three LRRs, known ligands and protein-anchoring sites in this region.The crystallographic study revealed that LRRs correspond to β-α structural units.These units are arranged so that the protein forms a curved structure [40].It is hypothesized that the XopL Xcc protein shares a similar three-dimensional structure, and the LRRs are potentially responsible for protein anchoring and membrane localization.Truncation of the C-terminal region of XopL Xcc did not disturb protein conformation, as evidenced by its retained membrane localization ability.Further findings indicated that the membrane localization is likely mediated by the N-terminal region, possibly the LRRs, rather than the C-terminal part.We supposed that the parallel LRRs contribute to stabilizing the structure of β-sheets and α-helices through lateral interactions.Loss of the LRR region may lead to structural alterations that affect protein function.

Conclusions
Our study elucidates the divergent impacts of two T3E proteins on Arabidopsis immune responses.XopE2 Xcc triggers Arabidopsis immune responses via an unidentified activator of the salicylic acid (SA) signaling pathway, whereas XopL Xcc suppresses the expression of PTI-and SA-related genes.The presence of the N-myristoylation motif and N-terminal domain proved crucial for the subcellular localization and virulence function of XopE2 Xcc and XopL Xcc , respectively.These findings significantly enhance our understanding of the mechanisms employed by pathogenic bacteria T3Es and contribute to developing effective strategies for controlling bacterial diseases.

Figure 3 .
Figure 3. Influence of XopE2Xcc and XopLXcc on the expression of defense-related genes in Arab(A-L), The expression levels of defense-related genes in XopE2Xcc or XopLXcc-transfected pro of Arabidopsis Col-0 were measured using real-time qRT-PCR, respectively.The mRNA leve genes were normalized with Atactin2, and the relative expression levels were determined in plasts transfected with the control vector.The a\b\c\d\e labels represent significant differe = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistica ence.

Figure 3 .
Figure 3. Influence of XopE2 Xcc and XopL Xcc on the expression of defense-related genes in Arabidopsis.(A-L),The expression levels of defense-related genes in XopE2 Xcc or XopL Xcc -transfected protoplasts of Arabidopsis Col-0 were measured using real-time qRT-PCR, respectively.The mRNA levels of all genes were normalized with Atactin2, and the relative expression levels were determined in protoplasts transfected with the control vector.The a\b\c\d\e labels represent significant differences (n = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistical difference.

Figure 4 .
Figure 4.Influence of XopE2Xcc on the expression of defense response-associated genes in sid2 mutant protoplasts.(A-L), The expression levels of defense-associated genes in XopE2Xcc or XopLXcctransfected protoplasts of Arabidopsis sid2 mutant were measured using real-time qRT-PCR, respectively.The mRNA levels of all genes were normalized with Atactin2, and the relative expression levels were determined in protoplasts transfected with the control vector.The a\b\c\d labels represent significant differences (n = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistical difference.

Figure 6 .
Figure 6.Influence of mutant XopE2Xcc(G2A) protein on the expression of defense response-associated genes in Arabidopsis.(A-H),The expression of response-associated genes in XopE2Xcc and XopE2Xcc (G2A) transfected protoplasts was measured using real-time qRT-PCR, respectively.The

Figure 6 .
Figure6.Influence of mutant XopE2Xcc(G2A) protein on the expression of defense response-associated genes in Arabidopsis.(A-H),The expression of response-associated genes in XopE2Xcc and XopE2Xcc (G2A) transfected protoplasts was measured using real-time qRT-PCR, respectively.The a\b\c\d\e labels represent significant differences (n = 5, p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistical difference.

Figure 6 .
Figure 6.Influence of mutant XopE2 Xcc (G2A) protein on the expression of defense responseassociated genes in Arabidopsis.(A-H),Theexpression of response-associated genes in XopE2 and XopE2 Xcc (G2A) transfected protoplasts was measured using real-time qRT-PCR, respectively.The a\b\c\d\e labels represent significant differences (n = p < 0.05, two-way ANOVA with Tukey's HSD test).The same letters mean no statistical difference.

Pathogens 2024, 13 , 448 10 of 14 Figure 8 .
Figure 8. Influence of various mutant XopLXcc proteins on the expression of defense response-associated genes in Arabidopsis.

Figure 8 .
Figure 8. Influence of various mutant XopL Xcc proteins on the expression of defense responseassociated genes in Arabidopsis.