Abrogation of disease development in plants expressing animal antiapoptotic genes

Abstract

An emerging topic in plant biology is whether plants display analogous elements of mammalian programmed cell death during development and defense against pathogen attack. In many plant-pathogen interactions, plant cell death occurs in both susceptible and resistant host responses. For example, specific recognition responses in plants trigger formation of the hypersensitive response and activation of host defense mechanisms, resulting in restriction of pathogen growth and disease development. Several studies indicate that cell death during hypersensitive response involves activation of a plant-encoded pathway for cell death. Many susceptible interactions also result in host cell death, although it is not clear how or if the host participates in this response. We have generated transgenic tobacco plants to express animal genes that negatively regulate apoptosis. Plants expressing human Bcl-2 and Bcl-xl, nematode CED-9, or baculovirus Op-IAP transgenes conferred heritable resistance to several necrotrophic fungal pathogens, suggesting that disease development required host-cell death pathways. In addition, the transgenic tobacco plants displayed resistance to a necrogenic virus. Transgenic tobacco harboring Bcl-xl with a loss-of-function mutation did not protect against pathogen challenge. We also show that discrete DNA fragmentation (laddering) occurred in susceptible tobacco during fungal infection, but does not occur in transgenic-resistant plants. Our data indicate that in compatible plant-pathogen interactions apoptosis-like programmed cell death occurs. Further, these animal antiapoptotic genes function in plants and should be useful to delineate resistance pathways. These genes also have the potential to generate effective disease resistance in economically important crops.

Figure 1

Western blot assays of expression of Bcl-2-related proteins in vitro and in transformed tobacco lines. (A) CED-9 expression. Lanes: 1, His-tagged CED-9; 2, wild-type tobacco; 3–10, separate ced-9 transgenic plants. (B) Bcl-2 expression. Lanes: 1, monkey kidney cells expressing His-tagged Bcl-2; 2, wild-type tobacco; 3–10, separate bcl-2-transgenic plants. (C) Bcl-xl expression. Lanes: 1, monkey kidney cells expressing His-tagged Bcl-xl; 2, wild-type tobacco; 3–6, separate bcl-xl-transgenic plants; 7–10, transgenic (susceptible) plants with bcl-xl-G138A.

Figure 2

Antiapoptotic genes confer resistance to necrotrophic fungi in transgenic plants. Detached Glurk tobacco leaves were inoculated with three species of fungi. (A) Leaves inoculated with S. sclerotiorum from plants transformed with bcl-2, ced-9, bcl-xl, or op-iap, as indicated. Leaf V is from a plant transformed with binary vector pZP212 alone. (B) Leaves inoculated with S. sclerotiorum from F₁ progeny of plants transformed with ced-9. (C) Leaves inoculated with B. cinerea from plants transformed with bcl-2 or ced-9, as indicated. (D) Leaves inoculated with B. cinerea from F₁ progeny of plants transformed with bcl-2. (E) Leaves inoculated with C. nicotianae from plants transformed with bcl-2, ced-9, or op-iap, as indicated. In A, C, and E, WT indicates a leaf from an untransformed control plant. In B and D, the leaf shown at Lower Right is from a kanamycin-sensitive F₁ plant.

Figure 3

Transgenic plants expressing antiapoptotic genes have altered response to TSWV. TSWV lesions on detached Glurk tobacco leaves. (A) Three leaves from plants expressing CED-9. (B) Three leaves from Op-IAP-expressing plants. In both A and B, leaf 4 is from an untransformed control plant.

Figure 4

ELISA of TSWV accumulation in detached leaves from transformed tobacco lines. Wells 1 and 2, positive and negative controls, respectively. Wells 3, 5, and 7 are samples from inoculated half-leaves; wells 4, 6, and 8 are from the uninoculated half of the same leaves. Plants are transformed with vector only (wells 3 and 4), with ced-9 (wells 5 and 6), or with op-iap (wells 7 and 8).

Figure 5

Plants expressing wild-type Bcl-xl, but not mutant Bcl-xl, are resistant to S. sclerotiorum. Shown are detached Glurk tobacco leaves inoculated with S. sclerotiorum. Leaves A and B are from plants transformed with bcl-xl, and leaves C and D are from plants transformed with mutant bcl-xl (G138A).

Figure 6

DNA laddering in susceptible tobacco after inoculation with S. sclerotiorum. Ethidium bromide-stained agarose gel of DNA extracted from nontransgenic tobacco (lanes 1 and 3) and the transgenic line expressing Bcl-2 (lanes 2 and 4) after challenge with S. sclerotiorum. DNA was extracted 4 (lanes 1 and 2) and 8 h (lanes 3 and 4) postinoculation.