Elicitor-Induced Cytoskeletal Rearrangement Relates to Vacuolar Dynamics and Execution of Cell Death: In Vivo Imaging of Hypersensitive Cell Death in Tobacco BY-2 Cells

Abstract

Disintegration of the vacuolar membrane (VM) has been proposed to be a crucial event in various types of programmed cell death (PCD) in plants. However, its regulatory mechanisms are mostly unknown. To obtain new insights on the regulation of VM disintegration during hypersensitive cell death, we investigated the structural dynamics and permeability of the VM, as well as cytoskeletal reorganization during PCD in tobacco BY-2 cells induced by a proteinaceous elicitor, cryptogein. From sequential observations, we have identified the following remarkable events during PCD. Stage 1: bulb-like VM structures appear within the vacuolar lumen and the cortical microtubules are disrupted, while the cortical actin microfilaments are bundled. Simultaneously, transvacuolar strands including endoplasmic microtubules and actin microfilaments are gradually disrupted and the nucleus moves from the center to the periphery of the cell. Stage 2: cortical actin microfilament bundles and complex bulb-like VM structures disappear. The structure of the large central vacuole becomes simpler, and small spherical vacuoles appear. Stage 3: the VM is disintegrated and a fluorescent dye, BCECF, leaks out of the vacuoles just prior to PCD. Application of an actin polymerization inhibitor facilitates both the disappearance of bulb-like vacuolar membrane structures and induction of cell death. These results suggest that the elicitor-induced reorganization of actin microfilaments is involved in the regulation of hypersensitive cell death via modification of the vacuolar structure to induce VM disintegration.

Organisation and regulation of the cytoskeleton in plant programmed cell death

“…39,42 In other studies microtubules, like F-actin, concentrate at the infection site and undergo bundling, 42 but some studies 43 found no changes in the microtubule organisation upon pathogen attack. Treatment with cryptogein induces depolymerisation of microtubules 41,45 providing further support of the idea that microtubules are involved in HR.…”

“…Treatment of tobacco BY-2 cells with the elicitor, cryptogein, from Phytophtora cryptogea, also causes reorganisation and bundling of F-actin (Figures 1l and m). 41 Later stages of HR are often accompanied by depolymerisation of F-actin. 42,43 Microtubule organisation is also affected by defence responses, but the response is variable.…”

“…51 Moreover, cryptogein-induced PCD in tobacco tissue culture cells is accelerated in the presence of the actin-depolymerising drug bistheonellide (Figure 2b). 41 This suggests that actin depolymerisation has a key role in the induction of HR-PCD. Cytochalasin D did not affect the induction of the HR in wildtype Arabidopsis when coinoculated with Pseudomonas syringae pv tomato, but in an Atadf4 mutant that normally suppresses HR mediated by P. syringae pv tomato, it allowed a high percentage of leaves to generate the HR.…”

“…The first is characterised by the formation of bundled actin cables, and has been reported in several examples of HR and developmental PCD. 12,13,40,41,[60][61][62] This is likely to involve reduced activity of severing and barbed end capping proteins, and activity of bundling proteins (Figure 3a), though no candidates have been identified in these systems yet. In the second scenario, observed in the SI response, F-actin initially undergoes depolymerisation, followed by the formation of actin foci.…”

Involvement of Adapter Protein Complex 4 in Hypersensitive Cell Death Induced by Avirulent Bacteria

“…On the other hand, vacuolar membrane collapse is observed during infection by avirulent viruses and fungi. This process releases the vacuolar contents into the cytoplasm and leads to hypersensitive cell death as well, thereby preventing pathogen growth (Hatsugai et al, 2004Kuroyanagi et al, 2005;Higaki et al, 2007).…”