Resistance to citrus canker induced by a variant of Xanthomonas citri ssp. citri is associated with a hypersensitive cell death response involving autophagy-associated vacuolar processes

Abstract

Xanthomonas citri ssp. citri (X. citri) is the causal agent of Asiatic citrus canker, a disease that seriously affects most commercially important Citrus species worldwide. We have identified previously a natural variant, X. citri A^T , that triggers a host-specific defence response in Citrus limon. However, the mechanisms involved in this canker disease resistance are unknown. In this work, the defence response induced by X. citri A^T was assessed by transcriptomic, physiological and ultrastructural analyses, and the effects on bacterial biofilm formation were monitored in parallel. We show that X. citri A^T triggers a hypersensitive response associated with the interference of biofilm development and arrest of bacterial growth in C. limon. This plant response involves an extensive transcriptional reprogramming, setting in motion cell wall reinforcement, the oxidative burst and the accumulation of salicylic acid (SA) and phenolic compounds. Ultrastructural analyses revealed subcellular changes involving the activation of autophagy-associated vacuolar processes. Our findings show the activation of SA-dependent defence in response to X. citri A^T and suggest a coordinated regulation between the SA and flavonoid pathways, which is associated with autophagy mechanisms that control pathogen invasion in C. limon. Furthermore, this defence response protects C. limon plants from disease on subsequent challenges by pathogenic X. citri. This knowledge will allow the rational exploitation of the plant immune system as a biotechnological approach for the management of the disease.

Keywords: autophagy; biofilm formation; biological control; citrus canker resistance; hypersensitive response; salicylic acid; secondary metabolites.

Figure 1

Host‐specific response triggered by Xanthomonas citri ssp. citri (X. citri) strain A^T. (a) Biofilm formation on Citrus limon and C. clementina leaves at 7 days post‐inoculation (dpi). Red chlorophyll fluorescence and green signals from green fluorescent protein (GFP)‐tagged X. citri strains are shown. XY and ZX are the XY‐ and ZX‐axis‐projected images, respectively. Scale bar, 50 µm. (b) Macroscopic symptoms in C. limon leaves at 20 dpi. Leaves were photographed under white and UV light. Scale bar, 10 mm. (c) Microscopic cell death phenotype (arrows) observed at 48 h post‐inoculation. Scale bar, 150 µm.

Figure 2

Phenolic compounds are involved in the Citrus limon response to Xanthomonas citri ssp. citri (X. citri) strain A^T. (a) Quantitative reverse transcription‐polymerase chain reaction analysis of phenylalanine ammonia lyase (PAL1) and chalcone synthase (CHS1) mRNAs measured at 48 h post‐inoculation (hpi). The relative gene expression (ΔΔCt) fold change of mRNA levels was performed considering non‐treated plants as reference samples and a histone H4 transcript as an endogenous control. Values are expressed as means ± standard deviation (SD) from three independent biological replicates. The dataset marked with an asterisk is significantly different as assessed by Tukey's test (P < 0.05). (b) Light microscopic images of lemon leaves inoculated with X. citri strains. Leaves were photographed at 48 hpi and 7 days post‐inoculation (dpi) under white and UV light. Green fluorescent polyphenolic compounds (arrows) and red chlorophyll fluorescence are observed. The presence of discrete black spots in the C. limon–X. citri A^T interaction is shown enlarged in the top inset. Scale bar, 10 µm. (c) Spectrophotometric determination of flavonoids and anthocyanins at 48 hpi. Values are expressed as means ± SD. Each sample consists of 10 leaf discs (0.5 cm in diameter) obtained from two shoots of three different plants, and 10 biological replicates were performed. The dataset marked with an asterisk is significantly different as assessed by Tukey's test (P < 0.05). A, absorbance.

Figure 3

Xanthomonas citri ssp. citri (X. citri) strain A^T triggers the accumulation of salicylic acid (SA) in Citrus limon. (a) Quantitative reverse transcription‐polymerase chain reaction analysis of NPR1 (nonexpressor of pathogenesis‐related genes 1), WRKY70 transcription factor and pathogenesis‐related (PR1) mRNAs was measured at 48 h post‐inoculation (hpi). The relative gene expression (ΔΔCt) fold change of mRNA levels was performed considering non‐treated plants (NT) as reference samples and the histone H4 transcript as an endogenous control. Values are expressed as means ± standard deviation (SD) from three independent biological replicates. The dataset marked with an asterisk is significantly different as assessed by Tukey's test (P < 0.05). (b) Analysis of SA through liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) performed at 48 hpi and 7 days post‐inoculation (dpi). Values are expressed as means ± SD from three independent biological replicates. The dataset marked with an asterisk is significantly different as assessed by Tukey's test (P < 0.05). DW, dry weight.

Figure 4

Ultrastructural features of Citrus limon leaves inoculated with Xanthomonas citri ssp. citri (X. citri) strains. (a, g) At 0 h post‐inoculation (hpi), the nucleus, vacuole and chloroplast are intact. (b) Bacteria are localized on the leaf surface and (c) within the mesophyll cells. (d) Bacteria colonizing mesophyll tissue. (e) Bacteria in the intercellular space. Arrows, electron‐dense multitextured materials. (f) Breakdown of epidermal tissue and canker formation. (h) Bacteria colonizing the leaf surface and (i) the intercellular spaces. Arrows, epidermal tissue collapse. Top panel shows the magnification of degenerated bacteria. (j) Arrows, vacuole membrane invaginations. (k) Arrow, perforations of nuclear envelope. (l) Arrows, cellular collapse. (m) Arrows, autophagosome‐like vesicles. (n) Cell death and accumulation of electron‐dense multitextured materials. (o–q) Arrows, autophagosome‐like vesicles. Scale bar, 2 µm. b, bacteria; C, canker; CD, cell death; ch, chloroplast; cw, cell wall; db, degenerated bacteria; ep, epidermis; is, intercellular space; ls, leaf surface; m, mitochondria; n, nucleus; sg, starch granules; t, tubular extensions; v, vacuole.

Figure 5

Immunoblot assay to detect free ATG8 and ATG8‐phosphatidylethanolamine (PE) adduct in Citrus limon inoculated with Xanthomonas citri ssp. citri (X. citri) strains. (a) Total protein was extracted from the inoculated tissues at 7 and 20 days post‐inoculation (dpi) and subjected to sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in the presence of urea, followed by immunoblot analyses with ATG8 antibody. The full lines locate the groups of free ATG8 isoforms and ATG8‐PE adducts. Protein profiles in the lower panels were detected by Ponceau S staining of a polyvinylidene difluoride (PVDF) membrane. The experiment was repeated twice using three independent biological replicates, and a representative image is shown. (b) The ATG8‐PE/ATG8 ratios were determined by the inmunodetection experiment shown in (a). NT, non‐treated leaves.

Figure 6

Pre‐inoculation with Xanthomonas citri ssp. citri (X. citri) strain A^T protects Citrus limon from canker disease. (a) Phenotypic response of lemon leaves pre‐inoculated with X. citri A^T tagged with green fluorescent protein (GFP) or mock‐inoculated by cotton swab. At 48 h post‐inoculation, the leaves were subsequently challenged, via spraying, with the pathogenic X. citri T‐GFP strain. Sections from the left panels are shown magnified in the right panels. Leaves were photographed under white and UV light. Scale bar, 10 mm. (b) Number of canker lesions per square centimetre in pre‐inoculated leaves at 20 days post‐inoculation (dpi). Values are expressed as means ± standard deviation (SD) from three independent biological replicates, each involving three different plants and five different leaves per plant. The dataset marked with an asterisk is significantly different as assessed by Student's t‐test (P < 0.05). (c) Canker symptoms, developed at 20 dpi, of lemon leaves co‐inoculated with equal amounts of both bacterial strains by cotton swab. Scale bar, 10 mm.