English

• 1. Introduction

  Plant diseases present a formidable challenge to agricultural production [1-3]. In recent years, exacerbated by global climate change and alterations in cropping patterns, the frequency and severity of bacterial plant diseases have been steadily rising, leading to escalated damage [4,5]. Approximately 300 out of the known 1600 pathogenic bacteria species are responsible for causing plant bacterial diseases, and there are over 500 known types of plant bacterial diseases. Bacterial diseases such as bacterial wilt, rice bacterial leaf blight, soft rot, and bacterial canker are highly important worldwide [6-10]. Bacterial diseases are ubiquitous and can cause extensive damage, rendering their prevention and control extremely challenging. Once a field is infected, it may become a permanent disease zone, resulting in substantial crop and economic plant losses [11-14]. Currently, agricultural bactericides represent the most efficacious method for disease prevention in agriculture [14-16]. Nevertheless, the available range of bactericides for controlling plant pathogenic bacteria is limited. Furthermore, the rapid reproduction, large population, and mutational capacity of bacteria contribute to the emergence of serious antibiotic resistance issues with frequent application of bactericides [14,17,18].

  Traditional bactericides target key bacterial growth factors to suppress or kill them, which can lead to the emergence and accumulation of antibiotic-resistant mutant pathogenic strains, thereby contributing to the development of antibiotic resistance [19-23]. Therefore, exploring new targets and developing novel antibacterial drugs is a crucial aspect in the development of new pesticides [23-27]. Targeting pathogenic bacterial virulence factors without compromising their survival presents a novel approach to the advancement of new antibacterial medications [28-32].

  Type III secretion system (T3SS), a highly conserved key virulence factor in Gram-negative pathogenic bacteria, is nevertheless non-essentials for bacterial growth, which renders the T3SS an ideal target for the development of new antibacterial drugs [33-35]. Based on previous research, this article introduces the overview of the T3SS and provides a summary of the T3SS inhibitors identified in recent years, including both natural and synthetic T3SS inhibitors, which offers new insights for the development of novel, efficient, and safe chemical substitutes and serves as a reference for the development of new antibacterial drugs.

  2. Research progress

  2.1 The structure and regulation of T3SS

  The T3SS of pathogenic bacteria is composed of more than 20 proteins that form a needle-like complex with highly conserved structural and functional features [36,37]. T3SS is widely present in Gram-negative pathogenic bacteria, including Pseudomonas syringae, Pseudomonas aeruginosa, Erwinia amylovora, Ralstonia solanacearum, Xanthomonas campestris, and Salmonella enterica [38-42]. It consists of several essential components, including the ATPase complex, the C-ring, the inner membrane export apparatus, the basal body, the needle filament or pilus, the tip complex, and the translocation pore. Together, these components form a transmembrane channel that extends across the inner membrane, outer membrane, and host cell membrane of the pathogenic bacteria [43-45].

  The proteins secreted by the T3SS can be classified into four groups: structural component proteins, translocator proteins, effector proteins, and T3SS chaperone proteins [39,46]. The core component of T3SS is the needle-like complex, referred to as the hrp pilus in plant pathogenic bacteria. The hrp pilus was initially discovered in S. typhimurium and subsequently found in other pathogenic bacteria, exhibiting significant conservation in structure and function [47,48]. The hrp pilus is essential for the secretion of T3SS effector proteins and is encoded by the hrpA in P. syringae [49], while it is encoded by the hrpE in Xanthomonas [50]. All Hrp (hypersensitive response and pathogenicity) and Hrc (hypersensitive response and conserved) proteins are required to form the complete hrp pilus [49,51].

  T3SS in Gram-negative pathogens is encoded by the hrp and hrc genes [52,53]. The hrp/hrc gene cluster, which encodes the T3SS, is highly conserved and determines the pathogenicity of the pathogen in host plants as well as the hypersensitive response (HR) in non-host plants [52,54]. The hrp genes were initially reported in P. syringae pv. phaseolicola in 1986 and have subsequently been identified in various other plant pathogens, including X. campestris, Pseudomonas syringae, Erwinia amylovora, and Ralstonia solanacearum [55,56]. The T3SS is closely linked to pathogenicity in bacterial pathogens. Utilizing its needle-like complex, it infiltrates the host cell membrane and releases substantial quantities of effector proteins into the host cell, which suppress the host immunity while simultaneously facilitating the survival and proliferation of the pathogen (Fig. 1) [57-59].

  Figure 1

  

  Figure 1.  Pathogenic bacteria utilize T3SS to deliver virulence proteins into host plant cells. Reproduced with permission [59]. Copyright 2023, Wiley (slightly modified).

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  The hrp gene clusters in plant pathogenic bacteria can be divided into two groups: group I and group II. The hrp group I includes P. syringae and E. amylovora, among which the expression of the T3SS in P. syringae is primarily regulated by the HrpR/S-HrpL pathway [53,60]. In this pathway, the majority of the downstream genes of T3SS are regulated by the HrpL protein, which belongs to the extracytoplasmic function (ECF) family [61]. HrpL activates the expression of hrp genes by binding to the hrp-box (CGAACCNAN₁₄CCACNNA) in the promoter region of the downstream hrp/hrc genes [62-64]. The hrp group II includes R. solanacearum and Xanthomonas spp. [65], Among them, Xanthomonas primarily regulates the expression of hrp genes through the HrpG-HrpX regulatory pathway. HrpG, belonging to the OmpR family of two-component systems, plays a crucial role in positively regulating the expression of hrpX [66]. HrpX, a member of the AraC family, binds to the PIP-box motif (TTCGB-N₁₅-TTCGB; B: C, G, or T) within the hrp/hrc gene promoter and facilitates the transcription of most downstream T3SS genes [67,68].

  2.2 Research progress of T3SS inhibitors

  In recent years, with the increasing threat of bacterial resistance to agriculture and human health [69-71], the screening of novel antibacterial drugs targeting pathogenic bacterial virulence factors has become a significant research focus [72-76]. T3SS is a highly conserved and critical virulence factor in plant pathogenic bacteria that has attracted considerable attention from researchers [77-79]. There have been a significant number of reports on the screening of T3SS inhibitors, which target various pathogenic bacteria as potential novel antibacterial drugs [80], these inhibitors are predominantly sourced from natural products and chemical synthesis. A considerable amount of research has been reported on the utilization of natural products as inhibitors of T3SS in plant pathogens. However, natural products often necessitate artificial structural modifications for widespread application due to factors such as complex structures and limited stability. In contrast, small molecule compounds synthesized artificially exhibit a relatively simple and readily accessible advantage [81-84]. Nevertheless, the broader utilization of synthetic compounds is often hindered by challenges such as insufficient solubility and significant cytotoxicity. To date, researchers worldwide have devised various screening systems and reporter genes to identify T3SS inhibitors. These reporter systems include luciferase (Lux) reporter systems, green fluorescent protein (GFP) reporter systems, β-glucuronidase (GUS) reporter systems, and phospholipase reporter systems [85-87]. The reported T3SS inhibitors primarily consist of cinnamic acid derivatives, heterocyclic derivatives, pyridine carboxamide derivatives, and carboxylic acid compounds, among others.

  2.2.1   Dickeya spp.

  In the process of plant-microbe interaction, plants generate a range of defense responses or substances to combat pathogen invasion, including various secondary metabolites. Phenolic compounds are a significant category of organic intermediates synthesized through the secondary metabolism of plants. In 2008, Yang et al. discovered that plant phenolic compounds, such as o-coumaric acid (OCA, Fig. 2) and trans-cinnamic acid (TCA, Fig. 2), demonstrate noteworthy induction activity on the hrpN gene promoter of D. dadantii 3937 without affecting bacterial growth. The quantitative fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR) results reveal that OCA and TCA induce the expression of T3SS genes, such as dspE (encoding T3SS effector protein), hrpA (encoding T3SS hrp pilus protein), and hrpN (encoding T3SS harpin protein) in vitro. This study has revealed the mechanism by which the induction of T3SS expression by OCA and TCA is regulated through the rsmB-RsmA pathway. This is the first report of plant phenolic compounds stimulating the expression of T3SS genes in plant pathogenic bacteria [88].

  Figure 2

  

  Figure 2.  The structures of compounds OCA and TCA.

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  Subsequently, Li et al. conducted further research on plant phenolic compounds and their analogs in 2009. They discovered that p-coumaric acid (PCA, Fig. 3) exhibited a potent inhibitory effect on the hrpA promoter without compromising bacterial growth. The RT-qPCR results demonstrated that PCA inhibited the transcription levels of dspE, hrpL, hrpN, and hrpS at a concentration of 100 µmol/L. Further investigation revealed that PCA inhibited T3SS expression primarily through the HrpX/Y-HrpS-HrpL pathway, which is the first reported T3SS inhibitor in plant pathogenic bacteria [87].

  Figure 3

  

  Figure 3.  The structure of compound PCA.

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  In 2015, Li et al. screened a range of derivatives of plant phenolic compounds and identified that trans-4-hydroxycinnamohydroxamic acid (TS103, Fig. 4) exhibited an 8-fold greater inhibitory potency on T3SS in comparison to PCA. Through a comprehensive series of studies, it was determined that TS103 exerts its inhibitory effect on T3SS by suppressing hrpY phosphorylation, leading to a decrease in the levels of hrpS and hrpL transcripts, ultimately resulting in the suppression of T3SS expression. Furthermore, TS103 inhibits hrpL at the post-transcriptional level by decreasing the RNA levels of the regulatory small RNA RsmB through the rsmB-RsmA regulatory pathway [89]. This discovery is the first instance of an inhibitor that effectively inhibits T3SS via both transcriptional and post-transcriptional pathways in the phytopathogenic bacteria D. dadantii 3937.

  Figure 4

  

  Figure 4.  The structure of compound TS103.

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  In 2022, Hu et al. utilized a reporter system constructing D. zeae hrpA-GFP to screen for T3SS inhibitors. They identified five compounds that significantly inhibited hrpA promoter activity without affecting bacterial growth, including salicylic acid (SA), p-hydroxybenzoic acid (PHBA), cinnamic alcohol (CA), PCA, and hydrocinnamic acid (HA) (Fig. 5). All the five compounds exhibited a reduction in the HR in non-host tobacco plants and decreased the transcription of the T3SS regulatory gene hrpL and downstream hrp genes. Inoculation experiments demonstrated that these five compounds exhibited significant inhibitory effects on the pathogenicity of D. dadantii 3937 on potato, D. fangzhongdai CL3 on taro, D. oryzae EC1 on rice, and D. zeae MS2 on banana seedlings [90]. These findings provide potential inhibitors for preventing soft rot disease caused by Dickeya spp.

  Figure 5

  

  Figure 5.  The structures of five compounds.

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  2.2.2   Erwinia amylovora

  In 2013, Khokhani et al. established an E. amylovora hrpA-GFP reporter system and employed flow cytometry to screen for phenolic compounds derived from plants. It was discovered that numerous plant phenolic compounds demonstrate inhibitory or inducible activities against E. amylovora T3SS. Among these, compounds TMCA, BA, and EHPES were selected for further investigation (Fig. 6). Preliminary mechanistic studies indicate that two T3SS inhibitors (TMCA and BA) and the T3SS inducer EHPES modulate the expression of T3SS through the HrpS-HrpL pathway. Furthermore, the inhibitor TMCA was found to also suppress the expression of the T3SS system through the rsmB-RsmA pathway. In total, plant phenolic compounds have been confirmed to play a significant role in the regulation of T3SS [91].

  Figure 6

  

  Figure 6.  The structure of TMCA, BA, and EHPES.

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  In 2014, Yang et al. found that salicylidene acylhydrazides compounds displayed inhibition on the gene promoter activity of hrpN, dspE, hrpA, and hrpL in E. amylovora. Specifically, the representative compound 3 (Fig. 7) effectively suppressed the expression of numerous T3SS genes, including the hrpL and avrRpt2 genes, in E. amylovora. Simultaneously, compound 3 also hindered the production of amylovoran, an exopolysaccharide (EPS) essential for biofilm formation. Ultimately, the author opted for crab apple flowers for the inoculation experiment, revealing that compound 3 notably mitigated disease progression in pistils [92].

  Figure 7

  

  Figure 7.  The structure of compound 3.

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  In 2023, Yuan et al. selected TS108 (Fig. 8), a plant phenolic derivative, as the focus of their study to investigate its mechanism of action, based on the research conducted by Khokhani et al. [90]. Subsequent investigations revealed that TS108 exerts a negative regulatory effect on CsrB, a globally regulated small RNA, at the post-transcriptional level, leading to the inhibition of hrpS expression. This finding suggests that TS108 may negatively modulate T3SS gene expression through the CsrB-HrpS-HrpL pathway. Additionally, TS108 had no effect on the expression of T3SS in D. dadantii and P. aeruginosa, implying a potential specificity of TS108 inhibition towards E. amylovora T3SS. In field trials, the incidence of fire blight was reduced by up to 96.7% following treatment with the TS108 compared to untreated trees. These results suggest that targeting pathogen virulence factors could offer a promising strategy for controlling fire blight [93]. This study provides further validation for the development and utilization of T3SS inhibitors targeting pathogens.

  Figure 8

  

  Figure 8.  The structure of compound TS108.

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  2.2.3   Xanthomonas spp.

  In 2017, Fan et al. established a GFP reporter system, hpa1-GFP, in X. oryzae pv. oryzae (Xoo) and investigated the biological activity of 56 plant-derived phenolic compounds and their derivatives using flow cytometry. They discovered that four compounds, namely TS006, TS010, TS015, and TS018 (Fig. 9) effectively suppressed the hpa1 promoter activity without impacting the growth of Xoo. The RT-qPCR experiments demonstrated that the transcription levels of hpa1, hrpE, hrpF, hrcC, hrcT, hrcU, and the regulatory genes hrpG and hrpX were all decreased to varying degrees following treatment with these compounds, which indicates that the compounds effectively suppressed the expression of Xoo T3SS via the HrpG-HrpX pathway. Through Cya-translocation assays, it was observed that four inhibitors had inhibitory effects on the translocation of two non-TAL effectors (PXO_04172 and PXO_03702) within the T3SS. Furthermore, the influence of these four inhibitors on gum gene expression was examined, and it was found that the mRNA levels of the gum genes remained unchanged, indicating that these inhibitors may not affect the production of EPS. Subsequent inoculation experiments confirmed that the four compounds independently inhibited the pathogenicity of X. oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) on rice plants [67]. This study lays a theoretical foundation for the utilization of these four T3SS inhibitors as innovative antibacterial agents combating plant bacterial diseases in agricultural practices.

  Figure 9

  

  Figure 9.  The structures of TS006, TS010, TS015, and TS018.

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  In 2018, Xiang et al. in our group designed and synthesized a set of novel thiazolidin-2-cyanamide derivatives that contain a 5-phenyl-2-furan moiety. These compounds were assayed for activity using the GFP assay system, revealing that XXWII-2, II-3, and II-4 (Fig. 10) inhibited the promoter activities of hpa1, hrpG, and hrpX while not impacting the growth of Xoo. The RT-qPCR experiments unveiled a significant reduction in the mRNA levels of hpa1, hrpE, hrpF, hrcC, hrcT, and hrcU following treatment with these compounds. Furthermore, these compounds were shown to inhibit the HR of Xoo on non-host tobacco plants and the pathogenicity of Xoo on host rice, thereby alleviating the damage caused by rice bacterial leaf blight [94].

  Figure 10

  

  Figure 10.  The structures of compounds XXWII-2, II-3, and II-4.

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  Based on previous research highlighting natural products as small molecules with promising bioactivity, in 2019, Tao et al. in our group performed activity assays on a selection of natural products utilizing the hpa1-GFP reporter system. The three natural product derivatives, CZ-1, CZ-4, and CZ-9 (Fig. 11), were identified as inhibitors of Xoo T3SS. These compounds exhibited effective preventive and therapeutic effects against both rice bacterial leaf blight and rice bacterial leaf streak disease, providing a theoretical basis for the prevention and control of rice diseases [95].

  Figure 11

  

  Figure 11.  The structure of compounds CZ-1, CZ-4, and CZ-9.

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  Research by Xiang et al. demonstrates that 5-phenyl-2-furan is a small molecule scaffold with favorable biological activity. Based on this, in 2019, Tao et al. in our group designed and synthesized a series of 1, 3-thiazolidine-2-thione derivatives containing a 5-phenyl-2-furan moiety. Compound III-7 (Fig. 12) was identified as a potent T3SS inhibitor through a high throughput screening system. The RT-qPCR analysis further confirmed that III-7 effectively suppressed the transcriptional levels of Xoo T3SS. Additionally, compound III-7 showed remarkable therapeutic efficacy against rice bacterial leaf blight [96]. In the same year, Tao et al. designed and synthesized two new series of 1, 3, 4-thiadiazole derivatives containing 5-phenyl-2-furan (Fig. 13) as potential Xoo T3SS inhibitors [97].

  Figure 12

  

  Figure 12.  The structure of compound III-7.

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  Figure 13

  

  Figure 13.  The structures of compounds II and III.

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  In 2019, Jiang et al. in our group designed and synthesized a series of cinnamic acid derivatives. Through screening using the hpa1-GFP reporter system, they identified three compounds, namely I-9, I-12, and I-13 (Fig. 14), which showed potential inhibitory activity against the T3SS of X. campestris pv. campestris (Xcc). It is speculated that these three compounds can potentially decrease the expression of hrp/hrc genes via the HrpG-HrpX pathway, thereby diminishing the pathogenicity of Xcc on radish and Xoo on rice [98].

  Figure 14

  

  Figure 14.  The structures of compounds I-9, I-12, and I-13.

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  Based on the research by Xiang et al. and Tao et al., in 2019, Jiang et al. in our group designed and synthesized a series of S‑thiazol-2-yl-furan-2-carbothioate derivatives. They evaluated the biological activity of the synthesized compounds using a GFP reporter system. Five compounds, specifically JSIII-2, JSIII-4, JSIII-6, JSIII-9, and JSIII-15 (Fig. 15), demonstrated inhibitory effects on the promoter activities of the hpa1, hrpG, and hrpX genes. Following treatment with these five compounds, there was a notable decrease in the transcription levels of hrpE, hrpF, hrcC, and hrcU, suggesting their ability to potentially suppress the expression of the Xoo T3SS via the HrpG-HrpX pathway. Phenotypic assays unveiled that the compounds specifically targeted the T3SS, as they did not impact the secretion of extracellular cellulases, extracellular xylanases, and exopolysaccharides. Additionally, these compounds reduced the pathogenicity of Xoo and Xoc on rice, mitigating both rice bacterial leaf blight and rice bacterial streak disease. Thus, these compounds exhibit significant potential for practical applications [99].

  Figure 15

  

  Figure 15.  The structures of compounds JSIII.

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  In 2019, Ma et al. employed a novel reporter vector that incorporated a fusion of the β-lactamase gene with the signal peptide sequence of a T3SS effector gene to conduct screening for T3SS inhibitors. They observed that the compound BCD03 (3, 4-dichloro-benzyloxy carbonimi-doyl dicyanide, Fig. 16) effectively diminished the HR response of Xoo on tobacco at non-bactericidal concentrations. Moreover, the introduction of compound BCD03 led to a reduction in the secretion of the T3SS effector protein AvrXa27 [100].

  Figure 16

  

  Figure 16.  The structure of compound BCD03.

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  In 2020, Zhou et al. conducted a screening of a library containing 13,129 small molecule compounds, resulting in the identification of ten compounds that exhibited inhibitory effects on the expression of the T3SS in Xcc. Among these compounds, five compounds (A-3, carmofur, HMS3229O07, thioctic acid, and WB 64) showed remarkable therapeutic effects against Xcc virulence in radishes (Fig. 17). By analyzing RT-qPCR, the authors identified that these compounds may exert their inhibitory effects on hrp/hrc genes by specifically targeting the regulatory proteins HpaS and sensor kinase proteins ColS. Meanwhile, Zhou et al. also identified six compounds (Fig. 18) that effectively induced the Xcc T3SS, with pentetic acid being able to induce the HR of Xcc on non-host tobacco and enhance pathogenicity on host radishes [101]. In combination, these small molecules have the potential to serve as valuable tool compounds in the ongoing development of antivirulence candidates to control diseases caused by the plant pathogen Xcc.

  Figure 17

  

  Figure 17.  The structures of ten compounds inhibiting Xcc T3SS.

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  Figure 18

  

  Figure 18.  The structures of six compounds inducing Xcc T3SS.

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  In 2023, Li et al. in our group synthesized a series of pyrimidine derivatives and assessed their activity using a Lux reporter system Xcc-pxopN. The results of the biological activity assays revealed that compound SPF-9 (Fig. 19) displayed significant T3SS inhibitory activity, effectively reducing the HR in tobacco and suppressing the transcription of certain representative hrp/hrc genes. Preliminary mechanistic investigations unveiled that compound SPF-9 exerted its effects by suppressing the HrpG-HrpX pathway of the Xcc T3SS, consequently diminishing the pathogenicity of Xcc. As a promising novel T3SS inhibitor with robust anti-Xcc activity, compound SPF-9 exhibits significant potential for future advancements and development [59].

  Figure 19

  

  Figure 19.  The structure of compound SPF-9.

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  In 2023, Gao et al. in our group synthesized a series of novel ethyl-3-aryl-2-nitropropenoate derivatives, among which compound B9 (Fig. 20) was identified to exhibit enhanced inhibitory activity against Xoo T3SS according to the bioassay results. Compound B9 effectively inhibited the transcription of crucial genes in Xoo T3SS, resulting in a significant reduction in the incidence of rice bacterial leaf blight disease. To enhance the efficacy of the drug against the pathogen, the authors utilized a combination of the T3SS inhibitor and the quorum quenching bacteria Ralstonia pickettii F20, which resulted in a notable improvement in the control of bacterial leaf blight (Fig. 21) [102]. This study not only introduces a novel approach to prevent and control rice bacterial leaf blight, but also provides a theoretical backing for exploring innovative strategies to control pathogenic bacteria in the future.

  Figure 20

  

  Figure 20.  The structure of compound B9.

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  Figure 21

  

  Figure 21.  The working model for controlling Xoo pathogenicity in rice through the combination of the T3SS inhibitor B9 and quorum quenching bacteria F20. Reproduced with permission [102]. Copyright 2023, American Chemical Society.

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  In 2023, a series of 1, 3, 4-thiadiazole thioester derivatives containing 5-phenyl-2-furan were designed and synthesized by Wang et al. in our group. Eight inhibitors (II-2, II-3, II-5, II-6, II-10, II-12, II-13, and II-15, Fig. 13) with excellent protective effects and specific targeting to the Xanthomonas citri subsp. citri jx-6 (Xcci jx-6) T3SS were obtained through inhibitor screening systems, determination of growth curves, tobacco HR, and citrus leaf inoculation. The authors conducted a study on compound II-15 and performed initial investigations into the mechanism of this inhibitor using RT-qPCR and Western blot. The results showed that the inhibitor effectively decreased the expression of Xcci T3SS-related genes and impeded the secretion of effector proteins, thereby reducing the ability of the pathogen to suppress citrus plant immunity, ultimately leading to successful control of citrus canker disease (Fig. 22) [103]. This study is the first to report that 5-phenyl-2-furan derivatives can serve as effective T3SS inhibitors for the control of citrus canker.

  Figure 22

  

  Figure 22.  The mode of action of the T3SS inhibitor II-15. Reproduced with permission [103]. Copyright 2023, American Chemical Society.

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  The extracts from Cryptolepis sanguinolenta (Lindl.) Schlechter, including cryptolepine, neocryptolepine, isocryptolepine, and their derivatives, exhibit excellent antibacterial activity [104,105]. In 2023, Shao et al. in our group synthesized a series of cryptolepine and neocryptolepine derivatives and performed bioassays on these compounds. They observed that compound Z-8 (Fig. 23) selectively targeted the T3SS of Xoo, suppressed the transcription of representative TAL and non-TAL effectors, and effectively alleviated the impact of rice bacterial leaf blight disease to some extent. Ultimately, they co-administered the T3SS inhibitor Z-8 with the quorum quenching bacteria Ralstonia pickettii F20 to control rice bacterial leaf blight caused by Xoo. The results highlighted a synergistic effect when both were used concurrently, which provided experimental evidence and theoretical support for the development of novel antibacterial agents against pathogenic bacteria [106].

  Figure 23

  

  Figure 23.  The structure of compound Z-8.

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  2.2.4   Pseudomonas syringae

  In 2019, Ma et al. observed that the compound BCD03 (Fig. 16) potently reduced the HR response of P. syringae pv. tomato (Pst) on tobacco. In addition, the secretion of the T3SS effector protein AvrPto was markedly reduced after treatment with the compound BCD03 [100].

  In 2020, Kang et al. constructed a Pst hrpA-GFP reporter strain to screen a plant extract library and found that the root extract of Vitis vinifera L. showed remarkable inhibitory activity against the hrpA promoter. After isolating and purifying compounds from the root extract, three resveratrol oligomers hopeaphenol, isohopeaphenol, and ampelopsin A (Fig. 24) were identified that notably decreased the transcription levels of hrpA, hrpL, and hopP1 genes. Moreover, the three compounds showed inhibitory effects on Pst auto-aggregation, indicating that they indeed inhibited the T3SS of Pst. Pathogenicity tests showed that the three compounds reduced the virulence of Pst in tomatoes [107].

  Figure 24

  

  Figure 24.  The structures of hopeaphenol, isohopeaphenol, and ampelopsin A.

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  In 2020, Wang et al. made a significant discovery that crude extracts of Arabidopsis were capable of substantially inhibiting the expression of the T3SS in Pst. Through a series of experiments involving isolation, purification, and structural identification of the crude extracts, the authors successfully identified the active compound responsible for the inhibitory effects as sulforaphane (SFN, Fig. 25), a defense compound naturally found in Arabidopsis. They then demonstrated that SFN effectively suppresses the activity of the T3SS in pathogenic bacteria, leading to a reduction in pathogen virulence and ultimately enhancing the plant's resistance to diseases. Additionally, using a chemical proteomic approach, the authors identified HrpS as the specific target protein of SFN. Their study revealed that SFN directly binds to the cysteine at position 209 of the HrpS protein, thereby inhibiting the formation of the HrpS and HrpR hexamer, which leads to the suppression of the T3SS in pathogenic bacteria. Following this biological mechanism, SFN can selectively diminish the pathogenicity of pathogens while avoiding toxicity to beneficial plant microbiota, thereby assuming a targeted defensive function. This study suggests that plants contain secondary metabolites that are capable of reducing the virulence of pathogens without inducing bacterial death. The study clarified the specific mode of action of SFN, which provides a theoretical and scientific foundation for the study of targets and the subsequent application of T3SS inhibitors [108].

  Figure 25

  

  Figure 25.  The structure of compound SFN.

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  In 2023, He et al. in our group established a high-throughput screening reporter system based on Lux to screen for T3SS inhibitors, revealing that three heterocyclic compounds (II-14, II-15, and II-24, Fig. 26) significantly inhibited the hrpW and hrpL gene promoter activities, demonstrating inhibitory activity superior to the positive control SFN. These three compounds may inhibit the expression of T3SS and subsequently reduce the pathogenicity of Pst through the HrpR/S-HrpL regulatory pathway, while the representative compound II-15 markedly inhibited the secretion of the AvrPto effector protein [109].

  Figure 26

  

  Figure 26.  The structures of compounds II-14, II-15, and II-24.

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  2.2.5   Acidovorax citrulli

  In 2019, Ma et al. developed a reporter vector, pZAC-3502sig-penAC, which fused the β-lactamase gene with the T3SS effector gene penAC signal peptide sequences to screen for T3SS inhibitors of Acidovorax citrulli (A. citrulli). After screening over 12,000 compounds, a series of benzyloxy carbonimidoyl dicyanide (BCDs) derivatives with potent T3SS inhibitory activity were identified, among which compound BCD03 (Fig. 16) showed a notable decrease in the HR response caused by A. citrulli, Pst, and Xoo on non-host tobacco plants. The inhibitory effect of BCD03 on the secretion of T3SS effector proteins from the aforementioned bacteria was validated through western blot experiments. In conclusion, the findings indicate that BCD derivatives hold promise as novel T3SS inhibitors against diverse plant pathogenic bacteria, underscoring their potential value for practical applications [100].

  2.2.6   Ralstonia solanacearum

  In 2019, Puigvert et al. developed a R. solanacearum PhrpY: : luxCDABE reporter system by fusing the hrpY promoter, which encodes the hrp pilus, with the luxCDABE operon. Through investigations, they discovered that salicylidene acylhydrazides derivatives SA1, SA2, and SA3 (Fig. 27) could act as inhibitors of the T3SS in R. solanacearum. Inoculation experiments confirmed that the salicylidene acylhydrazide compounds reduced the severity of disease caused by R. solanacearum, and protected tomato plants from bacterial speck disease induced by Pst [110].

  Figure 27

  

  Figure 27.  The structures of compounds SA1, SA2, and SA3.

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  In 2017, Yang et al. found that the plant natural product umbelliferone (UM, Fig. 28) greatly inhibited the expression of R. solanacearum T3SS by modulating the HrpG-HrpB and PrhG-HrpB pathways, while also suppressing biofilm formation. However, the target and regulatory mechanisms of UM remain unclear [111].

  Figure 28

  

  Figure 28.  The structure of compound UM.

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  During the research process, the research group from Southwest University discovered natural products that induced the expression of the T3SS in R. solanacearum. In 2015, Wu et al. found that oleanolic acid (OA, Fig. 29) significantly induced the expression of the T3SS in R. solanacearum through the HrpG-HrpB pathway [112]. Furthermore, in 2017, Zhang et al. detected that ferulic acid (FA, Fig. 30) induced T3SS expression through the PrhA-prhI/R-PrhJ-HrpG-HrpB signal cascade, highlighting plant-derived compounds as a rich source of regulators for the T3SS in R. solanacearum [113].

  Figure 29

  

  Figure 29.  The structure of compound OA.

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  Figure 30

  

  Figure 30.  The structure of compound FA.

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  In 2023, Guo et al. in our group conducted a bioactivity evaluation of the designed and synthesized amygdalic acid derivatives. During this evaluation, they found that compound F-24 (Fig. 31) significantly inhibited the activity of the hrpY gene promoter, attenuating the HR and pathogenicity of R. solanacearum. Further mechanistic investigations revealed that F-24 inhibits T3SS through the PhcR-PhcA-PrhG-HrpB pathway, suggesting its great potential as a T3SS inhibitor in R. solanacearum [114].

  Figure 31

  

  Figure 31.  The structure of compound F-24.

  DownLoad: Full-Size Img PowerPoint

  3. Conclusion

  In recent years, the increasing threat posed by antibiotic-resistant pathogenic bacteria to agricultural production and human health has propelled the study of novel antibacterial drugs that target the virulence factor T3SS into a prominent research focus. Despite significant progress has been made in identifying various T3SS inhibitors from compound libraries using various screening systems, the targets and mechanisms of most inhibitors remain unknown. In the development of novel pesticides, conducting additional research on the mechanism of action is critical to propel the progress of these innovative inhibitors. The identification of a novel drug target facilitates breakthroughs in the development of a range of new drugs, ultimately leading to significant social and economic benefits. Moreover, the absence of reported practical applications and registered applications of T3SS inhibitors in agriculture emphasizes the need for future research to focus on studying the target and conducting field experiments to facilitate their practical application in agriculture.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Lu-Lu He: Writing – original draft, Resources, Methodology, Investigation, Data curation. Lan-Tu Xiong: Writing – original draft, Resources, Investigation, Data curation. Xin Wang: Writing – original draft, Resources, Formal analysis. Yu-Zhen Li: Writing – original draft, Investigation. Jia-Bao Li: Formal analysis, Data curation. Yu Shi: Writing – original draft, Supervision, Funding acquisition. Xin Deng: Writing – review & editing. Zi-Ning Cui: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

  Acknowledgments

  We acknowledge the financial support from the National Key Research and Development Program of China (No. 2023YFD1701100), the National Natural Science Foundation of China (No. 32072450), the National Science Fund for Distinguished Young Scholars of Guangdong Province (No. 2021B1515020107), the Opening Foundation of Hubei Key Laboratory of Novel Reactor and Green Chemical Technology (No. NRG202306), and the Opening Foundation of Guangdong Province Key Laboratory of Microbial Signals and Disease Control (No. MSDC2023-19).

  
  
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• 

  Figure 1  Pathogenic bacteria utilize T3SS to deliver virulence proteins into host plant cells. Reproduced with permission [59]. Copyright 2023, Wiley (slightly modified).

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  Figure 2  The structures of compounds OCA and TCA.

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  Figure 3  The structure of compound PCA.

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  Figure 4  The structure of compound TS103.

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  Figure 5  The structures of five compounds.

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  Figure 6  The structure of TMCA, BA, and EHPES.

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  Figure 7  The structure of compound 3.

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  Figure 8  The structure of compound TS108.

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  Figure 9  The structures of TS006, TS010, TS015, and TS018.

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  Figure 10  The structures of compounds XXWII-2, II-3, and II-4.

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  Figure 11  The structure of compounds CZ-1, CZ-4, and CZ-9.

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  Figure 12  The structure of compound III-7.

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  Figure 13  The structures of compounds II and III.

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  Figure 14  The structures of compounds I-9, I-12, and I-13.

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  Figure 15  The structures of compounds JSIII.

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  Figure 16  The structure of compound BCD03.

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  Figure 17  The structures of ten compounds inhibiting Xcc T3SS.

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  Figure 18  The structures of six compounds inducing Xcc T3SS.

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  Figure 19  The structure of compound SPF-9.

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  Figure 20  The structure of compound B9.

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  Figure 21  The working model for controlling Xoo pathogenicity in rice through the combination of the T3SS inhibitor B9 and quorum quenching bacteria F20. Reproduced with permission [102]. Copyright 2023, American Chemical Society.

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  Figure 22  The mode of action of the T3SS inhibitor II-15. Reproduced with permission [103]. Copyright 2023, American Chemical Society.

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  Figure 23  The structure of compound Z-8.

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  Figure 24  The structures of hopeaphenol, isohopeaphenol, and ampelopsin A.

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  Figure 25  The structure of compound SFN.

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  Figure 26  The structures of compounds II-14, II-15, and II-24.

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  Figure 27  The structures of compounds SA1, SA2, and SA3.

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  Figure 28  The structure of compound UM.

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  Figure 29  The structure of compound OA.

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  Figure 30  The structure of compound FA.

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  Figure 31  The structure of compound F-24.

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•