Overexpression of a defensin enhances resistance to a fruit-specific anthracnose fungus in pepper

Abstract

Functional characterization of a defensin, J1-1, was conducted to evaluate its biotechnological potentiality in transgenic pepper plants against the causal agent of anthracnose disease, Colletotrichum gloeosporioides. To determine antifungal activity, J1-1 recombinant protein was generated and tested for the activity against C. gloeosporioides, resulting in 50% inhibition of fungal growth at a protein concentration of 0.1 mg·mL-1. To develop transgenic pepper plants resistant to anthracnose disease, J1-1 cDNA under the control of 35S promoter was introduced into pepper via Agrobacterium-mediated genetic transformation method. Southern and Northern blot analyses confirmed that a single copy of the transgene in selected transgenic plants was normally expressed and also stably transmitted to subsequent generations. The insertion of T-DNA was further analyzed in three independent homozygous lines using inverse PCR, and confirmed the integration of transgene in non-coding region of genomic DNA. Immunoblot results showed that the level of J1-1 proteins, which was not normally accumulated in unripe fruits, accumulated high in transgenic plants but appeared to differ among transgenic lines. Moreover, the expression of jasmonic acid-biosynthetic genes and pathogenesis-related genes were up-regulated in the transgenic lines, which is co-related with the resistance of J1-1 transgenic plants to anthracnose disease. Consequently, the constitutive expression of J1-1 in transgenic pepper plants provided strong resistance to the anthracnose fungus that was associated with highly reduced lesion formation and fungal colonization. These results implied the significance of the antifungal protein, J1-1, as a useful agronomic trait to control fungal disease.

Figure 1. Expression of J1-1 is related to fruit ripening and induced by fungal infection in pepper fruits.

A, Organ-specific expression of J1-1 protein in leaves, stems, roots, flowers, unripe (UR) and ripe (R) fruits of pepper. β-tubulin was shown as a loading control. An arrowhead indicates the protein band of J1-1. B, Fungal-induced J1-1 accumulation in unripe and ripe pepper fruits infected with C. gloeosporioides. Numbers on the top represent hours after infection (HAI). Immunoblot analysis was performed with total soluble proteins from pepper tissues using polyclonal J1-1 antibody. C, Immunolocalization of J1-1 in unripe (a–c) and ripe (d–f) fruits at 0, 24, and 48 h after inoculation. To localize the protein, transverse sections of pepper fruits were incubated with polyclonal J1-1 antibody that was detected with AEC (3-amino-9-ethylcarbazole) chromogen, shown as red. The arrows indicate fungal spores on the surface of the pepper fruits. Bar  = 50 µm.

Figure 2. J1-1 recombinant protein shows antifungal activity against C. gloeosporioides.

A, Spore germination. B, Appressorium formation. Spore suspensions were amended with 10 µL of the GST/J1-1 recombinant protein or its heated protein to final concentrations of 0.1 mg·mL⁻¹. The protein was heated by incubating at 90°C for 10 min. A minimum of 100 spores were counted per replicate. Each value represents the mean ± SD of three replicates. Means with different letters in each column are significantly different at P<0.05. C, Representative photos of fungi that were treated with 0.1 mg·mL⁻¹ of GST/J1-1 recombinant protein for 48 hours (right). Control was treated with distilled water (left). Arrows indicate appressorium.

Figure 3. Southern blot analysis of transgenic pepper plants carrying J1-1 gene.

A, Schematic diagram of the T-DNA representing restriction enzyme sites and primer sites for i-PCR. LB, T-DNA left border repeat; RB, T-DNA right border repeat; HPT1, hygromycin phosphotransferase I; CaMV35S, CaMV 35S promoter; T_NOS, transcriptional terminator of nopaline synthase (NOS); T_35S, CaMV 35S transcriptional terminator. B, Southern blot analysis. gDNA was digested with HindIII, and hybridized with ³²P-labeled HPT1 probe (left) or rehybridized with the J1-1gene (right). WT, non-transformed wild-type pepper plant. Arrowheads indicate endogenous J1-1 bands.

Figure 4. Northern blot analysis of unripe fruits from transgenic pepper lines.

Lane 1, green fruit (G) from wild-type (WT) plant as a negative control; lanes 2–4, three T₁ transgenic plants representing homozygous progenies; lanes 5–7, three T₁ transgenic plants representing hemizygous progenies; lane 8, ripe fruit (R) from WT plant as a positive control. ³²P-labeled J1-1 was used as a probe, and total RNAs were shown as loading controls in lower panels.

Figure 5. Expression of the J1-1 in the unripe pepper fruits of transgenic plants.

A, Northern blot analysis of the J1-1 transcript. Total RNA from each T₂ progeny was hybridized to a radiolabeled J1-1 probe. Lane 1, unripe green fruits (G) of non-transformed wild-type (WT) pepper plant as a negative control; lanes 2–5, four T₂ transgenic lines representing homozygous progenies; lane 6, ripe fruits (R) of non-transformed pepper as a positive control. B, Immunoblot analysis of the J1-1 protein. Total soluble proteins from T₂ progenies were subjected to immunoblot analysis with polyclonal anti-J1-1 antibody. Total RNA and β-tubulin were shown as loading controls.

Figure 6. Expression of JA-biosynthesis related genes (A) and pathogenesis-related genes (B) in transgenic pepper fruits.

Total RNAs were extracted from the unripe fruits of T₂ transgenic pepper lines (J15, J32, and J51). 10 µg of total RNA was separated in a formaldehyde/agarose gel, transferred onto nylon membrane, and hybridized to radiolabeled respective probes. WT (G), non-transgenic unripe fruits as a negative control; WT (R) non-transgenic ripe fruits as a positive control.

Figure 7. Inhibition of fungal growth in transgenic pepper fruits.

A & C, Microscopic observation of fungal penetration at the infected area in the non-transformed (A) or J15 transgenic (C) unripe pepper fruit at 24 hr after inoculation with C. gloeosporioides. Fungus was stained with 0.1% toluidine blue. B & D, Cross sections of infection sites in the non-transformed (B) and J15 transgenic (D) fruits at 5 day after inoculation. Lactophenol-trypan blue was used for staining. a, appressorium; ih, infection hypha; c, conidium; Ac, acervuli. Arrowheads indicate spores and arrows indicate mycelia. Bar  = 25 µm.

Figure 8. Fungal resistance of transgenic pepper fruits challenged with C. gloeosporioides.

A, Representative photographs of unripe pepper fruits 9 days after infection with the anthracnose fungus. Green mature fruits from transgenic lines and wild-type control plants were inoculated with spores. J15, J19, J32 and J51, homozygous T₂ transgenic pepper lines; WT, non-transformed unripe fruits as a negative control. B, The rate of lesion development from inoculated spots on infected fruits. C, Number of spores in a lesion of the infected fruits. Fifty unripe mature fruits were infected at two spots. The number of spores was counted in the infected area at 9 days after infection. The data are presented as means ± SD from three independent estimations. Means with different letters in each column are significantly different at P<0.05.