Dissecting the antibacterial functions of the T6SS-2 cluster in Xanthomonas oryzae for environment and plant protection

Abstract

Plant pathogens pose a serious threat to global food security, and the excessive use of chemical pesticides has raised significant ecological concerns. Harnessing microbial competition presents a promising green technology for crop protection. In this study, we examined the functions of the type VI secretion system (T6SS), a crucial microbial competition tool, in Xanthomonas oryzae pv. oryzae strain PXO99A. PXO99A exhibited significant antibacterial activities dependent on its T6SS-2 cluster. Using genome, secretome, and functional analysis, we systematically predicted T6SS-associated effectors, detected the expression of five putative effectors, and functionally validated the toxicity of a VasX_N family effector PXO_00500 and its immunity protein PXO_RS08595. We further show that PXO99A uses its T6SS-2 to kill a broad range of plant and animal pathogens in vitro and in planta. Inactivation of the T3SS functions abolished virulence but had little effect on the T6SS-2-dependent antibacterial activities. Finally, we demonstrated that the avirulent T3SS-defective mutant is effective in protecting tomatoes from Pseudomonas syringae co-infection. Collectively, these results highlight an effective biocontrol strategy for plant protection.

Importance: The growing concerns over the toxicity, environmental impact, and resistance associated with chemical pesticides underscore the urgent need for alternative pathogen management strategies. In this study, we introduce an innovative approach of "turning waste into treasure" by repurposing plant pathogens as biocontrol-like agents. By elucidating the virulence and antimicrobial functions of Xanthomonas oryzae, we demonstrate that an avirulent mutant can employ its T6SS to effectively combat a broad spectrum of human and plant pathogens. Furthermore, its ability to protect tomato plants underscores its significant potential for sustainable agricultural practices.

Keywords: animal and plant pathogens; biocontrol-like agent; type VI secretion system.

Fig 1

Identification of T6SS effector proteins. (A) Genomic organization of the X. oryzae PXO99A T6SS clusters, including T6SS-1, T6SS-2, and accessory clusters. (B) Volcano plot illustrating differentially secreted proteins in the comparison between WT (PXO99A) and ΔtssB2 strains. (C) MA plot showing secreted proteins identified by mass spectrometry, comparing WT (PXO99A) and ΔtssB2 strains. Secretome data are provided in Data Set S1 for B and C. In panels A–C, “0273” corresponds to PXO_00273, and “RS17560” refers to PXO_RS17560, with all other numbers representing their respective gene identifiers. The “*” symbol highlights proteins identified in both secretome analyses in panels B and C. In panels B and C, each dot represents a single protein, with T6SS proteins highlighted in red and other secreted proteins shown in gray.

Fig 2

Functionality of the X. oryzae T6SS-2. (A) Competition assay of prey cells (E. coli MG1655) after co-incubation with killer strains X. oryzae (parental) and strains containing an inactivating deletion of the T6SS sheath components tssB1 (T6SS-1), tssB2 (T6SS-2), and ΔtssB1-B2 double deletion (T6SS-1 and 2). (B) The secretion of Hcp2 in X. oryzae. The Hcp2 protein was detected by Western blot analysis using an anti-Hcp2 antibody. Detection of the RpoB was used as a control. The position of the molecular size marker (in kDa) is indicated. (C) Schematic diagram of the predicted PXO_00498 and PXO_00500 domain structure. For PXO_00498, TM, transmembrane, TM1 coordinates 560–577, TM2 coordinates 598–614, TM3 coordinates 634–657, and TM4 coordinates 704–726. For PXO_00500, VasX_N, VasX (pore-forming toxin) toxin N-terminal region; TM1 coordinates 724–741, TM2 coordinates 762–780, TM3 coordinates 800–821, and TM4 coordinates 868–890. Secondary structure is predicted by HAMMER. (D) Toxicity assay of PXO_00500 in E. coli. Serial dilutions of E. coli expressing PXO_00500 with either an empty vector (pBAD) or vectors encoding the immunity genes PXO_RS08595 or PXO_RS08605 were analyzed. To direct PXO_00500 to the periplasmic space, a Tat signal peptide was fused to its N-terminus. (E) Interaction of 0500 (PXO_00500) with 8595 (PXO_RS08595) or 8605 (PXO_RS08605). Pull-down analysis was performed using His-GFP (control), His-8595 or 8605, MBP-FLAG (control), and 0500-FLAG. (F) Competition assay of prey cells (ΔPXO_00500-PXO_RS08605) after co-incubation with killer strains PXO99A (parental) and strains containing an inactivating deletion of the T6SS structure components tssB1 (T6SS-1), tssB2 (T6SS-2), and ΔtssB1-B2 double deletion (T6SS-1 and 2). (G) Competition assay of WT and the T6SS-null ΔtssB2 mutant against the effector–immunity deletion mutant ΔPXO_00500-PXO_RS08605 complemented with an empty vector (pBBR) or a vector expressing the immunity protein PXO_RS08595 as indicated. The data point indicates the relative survival of prey cells attacked by WT compared with that by the T6SS-2 mutant. Error bars indicate standard deviation across three biological replicates. Significance was determined by a two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant).

Fig 3

Secretion of PXO_00500 requires chaperone PXO_00501 and VgrG4. (A) Competition analysis of ΔPXO_00501 (chaperone) and ΔvgrG4. Killer strains are indicated, and the prey strain is the ΔPXO_00500-PXO_RS08605 mutant. (B) Interaction of PXO_00500 with PXO_00501 or VgrG4. Pull-down analysis was performed using His-GFP (control), His-0501 or His-VgrG4, MBP-FLAG (control), and 0500-FLAG. Error bars indicate the mean ± s.d. of three biological replicates, and significance was calculated using a two-tailed Student’s t-test (**P < 0.01 and ***P < 0.001).

Fig 4

T6SS-dependent killing of animal and plant pathogens in vitro and in planta. (A) Competition assay with pathogens that could contaminate edible plants and important plant pathogens. Ralstonia solanacearum GMI1000, GMI1000; Pseudomonas syringae pv. tomato PtoT1, PtoT1; P. syringae pv. tomato DC3000, DC3000. WT, X. oryzae PXO99A wild type; ΔtssB2, T6SS-2-null strain; ΔhrcU, T3SS-null strain; ΔtssB2/hrcU, a mutant in which both T3SS and T6SS are inactive. (B) In planta competition assay with X. oryzae PXO99A against the animal pathogens E. coli ETEC H-10407, E. coli DAEC 2787, and the phytopathogen GMI1000. DL, detection limit. Error bars indicate the mean ± s.d. of three biological replicates, and significance was calculated using a two-tailed Student’s t-test (**P < 0.01, ***P < 0.001, and ****P < 0.0001).

Fig 5

X. oryzae T3SS-null (ΔhrcU) protects tomato plants from P. syringae infection. (A) Hypersensitivity response of the X. oryzae in N. benthamiana. WT and its derivatives inoculated with 4-week-old N. benthamiana and observed after 48 hours. WT, X. oryzae PXO99A wild type; ΔtssB1-B2, T6SS-null strain; ΔhrcU, T3SS-null strain; and control, 10 mM MgCl₂. (B) X. oryzae inhibits infection by P. syringae on tomato plants. Four-week-old tomato Ac plants were sprayed with a T3SS-inactivated mutant, P. syringae pv. tomato DC3000 (Pst), and a mixture of the T3SS-inactivated mutant and Pst. Images were captured 11 days post-inoculation, with H₂O used as a control. (C) Disease index was calculated at 11 days post-infection. Error bars indicate the mean ± s.d. of three biological replicates, and significance was calculated using a two-tailed Student’s t-test (***P < 0.001).