Minisymposium 9: Plant Pathogen/Symbiont Interactions

Abs # 21003: Vacuolar Processing Enzyme Functions in the Early Process of TMV-Induced Hypersensitive Cell Death in Tobacco Leaves

Presenter:	Nishimura, Mikio , mikosome@nibb.ac.jp
Authors	Nishimura, Mikio  (A)   Hatsugai, Noriyuki  (A) (B)  Kuroyanagi, Miwa  (B) (C)  Yamada, Kenji  (A) (C)  Meshi, Tetsuo  (B) (C)  Hara-Nishimura, Ikuko  (B) (C)
Affiliations:	(A): Department of Cell Biology, National Institute for Basic Biology (B): Department of Botany, Graduate School of Science, Kyoto University (C): CREST, Japan Science and Technology Corporation

Programmed cell death (PCD) is a basic physiological process that occurs under various stresses and during the development of animals and plants. The process of controlled cell death in animals is well organized by caspases that are cysteine proteinases. In plants, there is only indirect evidence that caspases are present. Many studies have shown that caspase-like activities regulate plant cell death. However, no plant proteinases exhibiting caspase activities have yet been identified. To identify a functional homolog of caspase in plants, we investigated hypersensitive cell death in tobacco mosaic virus (TMV)-infected tobacco plants. Hypersensitive cell death is a type of well-organized cell death in higher plants. Tobacco leaves were inoculated with TMV and was maintained at 30 ��C for 40 h to proliferate the virus in the inoculated region. The TMV-infected leaves were then subjected to a temperature shift from 30 ��C to 23 ��C to induce necrotic lesions synchronously. Characteristic lesions showing hypersensitive cell death were started to visible in the leaves at 9 h after the temperature shift. A caspase-1-specific inhibitor abolished the lesion formation in the infected leaves, suggesting that a caspase-1-like activity is involved in TMV-induced hypersensitive cell death. A biotinylated caspase-1 inhibitor that was infiltrated into the tobacco leaves specifically bound to the 38- and 40-kDa components of the leaves. We found these components corresponded to two forms of a vacuolar enzyme (vacuolar processing enzyme, VPE), which is a cysteine proteinase responsible for maturation and/or activation of vacuolar proteins. A VPE-specific inhibitor also abolished the lesion formation, as did a caspase-1-specific inhibitor. VPE had caspase-1-like activity toward a caspase-1-specific substrate. Thus, the proteinase responsible for the caspase-1-like activity was identified as VPE. VPE activity rapidly increased to the maximum level at 3 h after the temperature shift and declined before the appearance of lesions in the TMV-infected tobacco leaves. The changes in VPE activity paralleled those in caspase-1-like activity. The hypersensitive cell death was abolished by the VPE/caspase-1 inhibitor when it was infiltrated until 3 h after the temperature shift and not affected when the inhibitor was infiltrated after 6 h. These results suggest that VPE regulates the early process of the TMV-induced hypersensitive cell death in tobacco leaves.