A phospholipase effector of the type VI secretion system modulates plant reproduction

Abstract

Phytobacteria play diverse roles in plant biology, ranging from promoting health to causing diseases that threaten global food security and the economy. In contrast to the extensive studies of phytopathogens targeting leaves and roots, their impact on plant reproductive processes has been largely overlooked. Here, we demonstrate that a bacterial effector TleB of the type VI secretion system in Xanthomonas oryzae can modulate seed production of Arabidopsis thaliana. Using biochemical, structural, and physiological analyses, we determined TleB as a phospholipase that mediates interspecies microbial competition in X. oryzae. Additionally, TleB plays a key role in the infection of inflorescences by X. oryzae, which leads to significantly reduced seed production. Lipidomic and biochemical assays show that TleB binds to a number of anionic phospholipids that are key signaling molecules. A fluorescence reporter for auxin distribution showed TleB-mediated diminished signals in planta. Additionally, transgenic plants expressing TleB exhibited significantly altered seed counts. These findings introduce a novel paradigm in which phytopathogens can affect plant reproduction in a traditionally non-susceptible host, prompting a reevaluation of diverse phytobacteria-host interactions in reproductive processes and offering new insights into plant health and crop protection.IMPORTANCEPhytobacteria are typically identified as pathogens based on visible effects on leaves and roots; those lacking such phenotypes are often considered nonpathogenic. Similarly, plant hosts that show no phenotypic changes are considered nonhosts and, thus, less studied. Our research challenges this classification by highlighting that bacteria-plant interactions on inflorescences, though less apparent and more delayed, can cause profound impacts on seed production. This discovery not only shifts the focus from the more commonly studied vegetative and root infections to the reproductive aspects of plant-pathogen interactions but also necessitates a reevaluation of host-pathogen dynamics with an emphasis on long-term effects such as seed production.

Keywords: T6SS; Xanthomonas oryzae pv. oryzae; ovule initiation; phosphatidylserine.

Fig 1

Characterization of T6SS phospholipase effector TleB. (A) Organization of the PXO_02029-PXO_02034 operon. The alpha/beta hydrolase domain DUF2235 of TleA and TleB was predicted using HMMER. (B) Toxicity assay of TleA and TleB. Results show the survival of Escherichia coli that express TleA or TleB with an empty vector (pBAD) or a vector carrying the immunity gene tliA or tliB. Sec, periplasmic secretion signal. (C) Interaction of TleB with TliA or TliB. Pull-down analysis was performed using His-GFP (control), His-TliA or TliB, MBP-FLAG (control), and FLAG-TleB. A similar pull-down assay was performed for the interaction of TleA with immunity proteins and shown in Fig. S2H. (D) Competition assay of PXO99A, T6SS-2-null mutant (ΔtssM2), or effector-immunity deletion mutants (ΔtleA and ΔtleB) against E. coli MG1655 (MG1655, co-cultured for 6 hours) or Pseudomonas syringae pv. tomato DC3000 (DC3000, co-cultured for 3 hours) in Nicotiana benthamiana, respectively. (E) Crystal structure of TleB, color-coded from pink (N-terminus) to green (C-terminus). The catalytic triad residues Ser240-Asp280-His347 of TleB are highlighted on the right. (F) UPLC chromatograms of digested lecithin products by purified TleB, TleB^S240A, and TleB^H347A. Peaks were identified using mass spectrometry. (G) Competition assay of PXO99A, T6SS-2-null mutant (ΔtssM2), effector-immunity deletion mutants (ΔtleA and ΔtleB), effector-immunity deletion mutant (ΔtleA-tleB), and ΔtssM2-tleA-tleB against ΔtssM2-tleA-tleB. Error bars indicate the mean ± standard deviation of three biological replicates, and statistical significance was calculated using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. DL, detection limit.

Fig 2

TleB inhibits seed development in A. thaliana. (A) Fat-western immunoblot analysis of TleB. Each dot represents a different phospholipid type: phosphatidylcholine (PC), phosphatidylethanolamine (PE), PS, PA, lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), sphingosine 1-phosphate (S1P), phosphatidylinositol (PI), PI3P, PI4P, PI5P, PI (3,4)P2 (PI 3,4-bisphosphate), PI (3,5)P2, PI (4,5)P2, and phosphatidylinositol 3,4,5-trisphosphate [PI (3,4,5)P3]. (B) UPLC chromatograms of digested PS products by TleB. Products were detected using mass spectrometry. (C) Scanning electron micrographs showing attached bacterial cells on the ovule surface. Mock, 10 mM MgCl₂. (D) Lipidomic analysis of PS content in A. thaliana inflorescences. Phospholipids were profiled using mass spectrometry. Mock, 10 mM MgCl₂. PG, phosphatidylglycerol. (E) and (F) Confocal microscopy of pDR5::eGFP reporter expression in A. thaliana inflorescences treated with mock (10 mM MgCl₂), PXO99A, and ΔtleA-tleB mutant. (G) Images of siliques from A. thaliana treated with the mock (10 mM MgCl₂), PXO99A, ΔT3SS (ΔhrcU), and ΔT6SS-2 (ΔtssM2) mutants. (H) Quantification of seed numbers per silique in A. thaliana treated with the mock (10 mM MgCl₂), PXO99A, ΔT3SS (ΔhrcU), and ΔT6SS-2 (ΔtssM2) mutants. (I) Seed numbers per silique in A. thaliana treated with the mock (10 mM MgCl₂), PXO99A, ΔtssM2, ΔtleB, and tleB^H347A (catalytically inactive chromosomal mutation). (J) Seed numbers in transgenic A. thaliana expressing TleB (Seed numbers indicate normal, fully developed seeds, excluding aborted ones). (K) Phylogenetic analysis of TleB homologs using MEGA7.0 with the Neighbor-Joining method and 1,000 bootstrap values. Using TleB as the query and BlastP, we retrieved the top 24 representative sequences for analysis. The operon structures are indicated, with T6SS-conserved proteins marked in color. Protein sequences are provided in Data S2. Total seeds, including abortive ones, are counted. For panels H and I, the letters displayed above the bar graphs indicate statistically significant differences. Identical letters denote no significant difference between the corresponding groups, while different letters signify a statistically significant difference between the groups. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA for panel H and two-tailed Student’s t-test for panels F, I, and J (*P < 0.05, **P < 0.01, and ****P < 0.0001).

Fig 3

Schematic model of the TleB-mediated host-microbe interactions. The T6SS effector TleB in X. oryzae PXO99A exhibits dual PLA1 and PLA2 phospholipase activities, which aid in microbial competition and host-microbe interaction. By targeting and neutralizing competing microbes, TleB promotes the competitive fitness of PXO99A and successful colonization. By hydrolyzing key anionic phospholipids in the host plant, TleB reduces seed production in A. thaliana. The widespread presence of TleB homologs in various T6SS-encoding phytobacteria suggests a common mechanism affecting plant health. These findings highlight a previously unrecognized impact of bacterial effectors on flowering organs and relatively long-term phenotypic processes in plant-microbe interactions, providing new insights into the complexities of plant-pathogen dynamics and potential strategies for crop protection.