Programmed cell death (PCD) is a basic genetically controlled process that functions in the development of multicellular organisms and in their responses to biotic and abiotic stresses.

PCD provides a means to eliminate redundant, damaged or infected cells. In mammals, apoptosis is recognised as a ubiquitous phenomenon with distinct morphological hallmarks, including condensation of the nucleus and the cytoplasm, cleavage of DNA into short 180-bp fragments (DNA laddering) and surface marking of dying cells that are ingested by phagocytes.

At the molecular level, there are two canonical pathways to apoptotic cell death. One involves the interaction of a death receptor with its ligand and the second depends on the participation of mitochondria. The release of a number of factors, most notably cytochrome c, from the mitochondrial intermembrane space is regulated by proapoptotic and antiapoptotic members of the Bcl-2 family. Cross-talk between the two pathways exists.

Caspases

The end result of either pathway is the activation of caspases, a family of Cys proteases conserved evolutionarily from nematodes to humans. Caspases are activated from dormant precursors in the course of apoptosis and introduce breaks after specific Asp (D) residues (hence their name) in a limited set of key cellular protein substrates.

Caspases are among the most specific proteases known and usually no more than one or two breaks per substrate protein molecule are introduced. Caspase-mediated protein fragmentation eventually leads to cell dismantling.The most prevalent executioner caspase in animal cells is caspase-3, which ultimately is responsible for the majority of the effects orchestrating cell death.

In plants, several tissues or whole organs undergo cell death as part of their normal development, such as during senescence or in response to pathogens and environmental stress. Whether or not common mechanisms underlie the implementation of PCD in animals and plants is an important emerging issue.

Although the mechanisms of plant PCD are far less clear, several morphological and biochemical similarities between PCD in animals and plants have been described in different experimental systems, including condensation and shrinkage ofthe nucleus and cytoplasm, DNA laddering, and cytochrome c release from mitochondria.

However, in spite of the striking similarities between the PCD pathways in animals and plants, the case for the existence of any caspases in plants has been controversial. Although some specific inhibitors of animal caspases have been shown to affect development of PCD in plants, no direct homologues of animal caspase genes have been identified in plants.

Missing link

Dr Michael Taliansky and colleagues at SCRI, in collaboration with the Moscow State University team (led by Professor A. Vartapetian), have found this missing link in plant PCD and identified a plant protein with caspase-like protease activity (PCLP), which is a functional analogue of animal caspase (Chichkova et al. 2004).

To test the hypothesis that caspase-like proteases exist and are critically involved in the implementation of PCD in plants, a search was undertaken for plant caspases activated during the N gene-mediated hypersensitive response (HR; a form of pathogen-induced PCD in plants) in tobacco plants infected with Tobacco mosaic virus (TMV). For detection, characterisation and partial purification of a tobacco caspase, the Agrobacterium tumefaciens VirD2 protein, shown here to becleaved specifically at two sites (TATD and GEQD) by human caspase-3, was used as a target.

In tobacco leaves, specific proteolytic processing of the ectopically produced VirD2 derivatives at these sites was found to occur early in the course of the HR triggered by TMV. A proteolytic activity capable of specifically cleaving the model substrate at TATD was partially purified from these leaves. A tetrapeptide aldehyde designed and synthesised on the basis of the elucidated plant caspase cleavage site prevented fragmentation of the substrate protein by plant and human caspases in vitro and counteracted TMV-triggered HR in vivo (Figure 1).

Figure 1
Effect of the caspase-specific inhibitor biotinyl-TATD-CHO on the HR cell death in tobacco Samsun NN plants infected with TMV at 42 and 52 h after inoculation (hpi). Control half-leaves were co-inoculated with diluent.

Therefore, our data provide a characterisation of caspase-specific protein fragmentation in apoptotic plant cells, with implications for the importance of such activity in the implementation of plant PCD. Caspase-like activities have been recently identified in rice, potato, barley, tomato and other crops.

Another important implication of our work is that the Agrobacterium tumefaciens VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity in tobacco. The VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome.

We have demonstrated that that mutagenesis of the VirD2 protein to prevent cleavage by plant caspase increases the efficiency of reporter gene transfer and expression in different plant species including barley and potato (Figure 2). These results indicate that caspase cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection.

Figure 2
Increase in efficiency of green fluorescent protein (GFP) expression in barley plant tissues agroinfiltrated with A. tumefaciens encoding a caspase-resistant VirD2 protein. Plant tissues were agroinfiltrated with A. tumefaciens carrying a GFP gene along with either a wt VirD2 gene (left hand panel) or a mutated, caspase-resistant VirD2 gene (mut VirD2 – right hand panels) and fluorescence was observed by confocal laser scanning microscopy. The type of plant is indicated on the right. Bars, 100 μm.

Cathepsin B

In addition to caspase, Paul Birch, Eleanor Gilroy, Petra Boevink and Ingo Hein have demonstrated that a papain cysteine protease, cathepsin B, is also required for the HR (Figure 3). They showed, using protease inhibitors and virus induced gene silencing in Nicotiana benthamiana, that cathepsin B was required for non-host bacterial HR and for the gene-for-gene interaction between AVR3a from Phytophthora infestans and R3a from potato.

Plant cathepsin B is secreted into the plant apoplast and activated upon secretion. It shows a modest increase in transcript abundance during the HR and could be involved in signalling events leading to PCD (Gilroy et al. 2007).

Figure 3
Virus induced gene silencing of NbCathB in Nicotiana benthamiana abolishes the non-host hypersensitive response to Erwinia amylovora.

References

Chichkova, N.V., Kim, S.H., Titova, E.S., Kalkum, M., Morozov, V.S., Rubtsov, Yu.P., Kalinina, N.O., Taliansky M. and A. Vartapetyan. 2004. A plant caspase-like protease activated uring the hypersensitive response. Plant Cell 16, 157-171.

Reavy, B., Bagirova, S., Chichkova, N.V., Fedoseeva, S.V., Kim, S.H., Vartapetian A.B. and Taliansky M.E. 2007. Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants. Plant Cell Reports 26, 1215-1219.

Method of gene transfer. 2006. British patent Application # 0609420.5

Gilroy, E.M., Hein, I., van der Hoorn, R., Boevink, P.C., Venter, E., McLellan, H., Kaffarnik, F., Pritchard, L., Hrubikova, K., Shaw, J., Holeva, M., Loake, G.J., Lacomme, C., Birch, P.R.J. 2007. Involvement of Cathepsin B in the Plant Disease Resistance Hypersensitive Response. Plant Journal 52, 1-13.