Defense-Related Responses in Fruit of the Nonhost Chili Pepper against Xanthomonas axonopodis pv. glycines Infection

Abstract

Xanthomonas axonopodis pv. glycines (Xag ) is a necrotrophic bacterial pathogen of the soybean that causes bacterial pustules and is a nonhost pathogen of the chili pepper. In the current study, chili pepper fruit wound inoculated in planta with Xag 8ra formed necrotic lesions on the fruit surface and induced several structural and chemical barriers systemically in the fruit tissue. The initial defense response included programmed cell death of necrotizing and necrotized cells, which was characterized by nuclear DNA cleavage, as detected by TUNEL-confocal laser scanning microscopy (CLSM), and phosphatidylserine exposure on cell walls distal to the infection site, as detected by Annexin V FLUOS-CLSM. These two responses may facilitate cell killing and enhance transportation of cell wall materials used for cell wall thickening, respectively. The cells beneath the necrotic tissue were enlarged and divided to form periclinal cell walls, resulting in extensive formation of several parallel boundary layers at the later stages of infection, accompanying the deposition of wall fortification materials for strengthening structural defenses. These results suggest that nonhost resistance of chili pepper fruit against the nonhost necrotrophic pathogen Xag 8ra is activated systematically from the initial infection until termination of the infection cycle, resulting in complete inhibition of bacterial pathogenesis by utilizing organ-specific in situ physiological events governed by the expression of genes in the plant fruit organ.

Keywords: Xanthomonas axonopodis pv. glycines; chili pepper; hypersensitive response; programmed cell death.

Fig. 1

Symptoms on fruits of Capsicum annuum cv. Buguang 4 days after inoculation with Xanthomonas axonopodis pv. glycines 8ra (A) and control wound-inoculated fruits (B).

Fig. 2

Confocal laser scanning microscopy of chili pepper fruit tissues with wounding alone (A, D) and inoculated with Xanthomonas axonopodis pv. glycines 8ra (B, C, E, F) followed by TUNEL (A–C) and Annexin-V FLUOUS staining (D–F). There were no positive signals on the fruit tissues underneath the wound site (WS) in the control inoculation (A, D), but TUNEL-positive signals are evident (arrows) and localized in the nuclei (N) mostly in epidermal and subepidermal cells 24 hours after inoculation (B, C), while Annexin-V FLUOUS-positive signals (arrows) localized on the cell walls distal to the inoculation site (IS) 16 hours (E) and 24 hours (F) after inoculation. (C) Enlarged image of the rectangular area in (B). Scale bars = 50 μm.

Fig. 3

Light microscopy (A) and transmission electron microscopy (B–G) of chili pepper fruit tissues inoculated with Xanthomonas axonopodis pv. glycines 8ra showing structural changes related to hypersensitive and necrotic responses 3 days (A, B), 1 hour (C), 24 hours (D), 48 hours (E), 60 hours (F), and 5 days (G) after inoculation. The inoculated fruit tissue (A) contained necrotic or necrotizing cells (NC) along with highly vacuolated (VC) adjacent cells with necrotized cell walls (arrows). The necrotic or NC (B) were characterized by cytoplasmic shrinkage indicated by separation (S) of the cytoplasm (Cy) from the degenerated cell wall (DW). Initially (C), nuclear (N) features in the inoculated tissues were homogeneously heterochromatic. In later stages (D–G), these features continued to accumulate condensed heterochromatin materials (Ch) that were distributed in the nucleoplasm and finally dispersed in the necrotized cytoplasm (NCy) and in necrotizing (E, F) and necrotized cells (G). Cellular organelles such as plastids (P) and mitochondria (M) in (B), (E) and (F) appeared degenerated, indicating cytoplasmic degeneration. V, vacuole; W, cell wall; No, nucleolus; NW, normal intact cell wall; S, separation of cell wall from cytoplasm. Scale bars = 10.0 μm for (A) and 1.0 μm for (B) through (G).

Fig. 4

Light microscopy (A) and transmission electron microscopy (B–E) of chili pepper fruit tissues inoculated with Xanthomonas axonopodis pv. glycines 8ra showing structural features of cells bounding with necrotic cells with a necrotized cytoplasm (NCy) and walls (NW) and middle lamellae (NM) 3 days (A–D) and 15 days (E) after inoculation. The cells (A) bounding with the necrotic cell contained an intact cytoplasm (ICy) with parallel thin (W) and thick cell walls (TW), around which the incorporation of vesicles (arrows) likely containing cell wall materials into the cell wall to increase cell wall thickening (asterisk) in (B) was observed. The cytoplasms of the cells adjacent to the necrotic cells became dense with cytoplasmic infiltration into the vacuole (V), as indicated by cytoplasmic fragmentation (CF) (C), resulting in a dense cytoplasm with numerous small vacuoles like meristematic cells (D). In (E), structural features at a later stage of infection are evident, showing the freely gathered bacterial cells (Bc) confined in intercellular spaces surrounded by thickened cell walls (TW) that were separated (S) from the NCy, thus protecting the cytoplasm (Cy) from degeneration. In (E), additionally thickened cell walls are noted by arrows. B, inclusion body; M, mitochondria; P, plastids. Scale bars = 5.0 μm for (A) and 2.0 μm for (B) through (E).

Fig. 5

Light microscopy (A–F) and fluorescent microscopy (G, H) of chili pepper fruit tissues one day (A), 4 days (B, E, G), 8 days (C), and 15 days (D, F, H) after inoculation with Xanthomonas axonopodis pv. glycines 8ra. In (A) to (D), the formation of defense structures (boundary layers, BL) is evident, and in (E) and (F), deposition of cell wall fortification materials such as lignin (Lig), and in (G) and (H), suberin (Sub) and cutin (Cut) are present in or under the necrotic tissue (NT) of the inoculation site (IS). The circled area in (B) is to highlight longitudinally enlarged cells in the process of cell division to form boundary layers. Scale bars = 50 μm.