Programmed Cell Death in stress response

 

 

 

AAL-toxin induced programmed cell death in plants

 

 

 

Our investigations into the interaction of the plant pathogenic fungus Alternaria alternata f.sp. lycopersici with its host, tomato, have led to the identification of a unique role for a plant pathogen resistance gene and the possibility of a conserved pathway for signalling programmed cell death between plants and animals.

 

Plant necrotrophic fungi, such as A.alternata f.sp. lycopersici, must kill their host plant in order to grow on the dead tissue. This particular fungus achieves this by secreting a toxin, AAL-toxin, that is remarkable in that it induces programmed cell death, akin to apoptosis in animals, in the host tissues. AAL-toxin has been shown to inhibit the important enzyme ceramide synthase. Tomato plants that are resistant to A.alternata f.sp. lycopersici are also insensitive to AAL-toxin. The resistance gene Asc is responsible for this. Our results have shown that the Asc-1 gene is able to prevent inhibition of ceramide biosynthesis by AAL-toxin and is itself directly involved in the biosynthesis of ceramide containing lipids (sphingolipids). 

 

This is potentially an exciting finding as ceramide is a potent apoptotic signalling molecule in animals and is clearly important for programmed cell death in plants, suggesting an evolutionary conserved role for ceramide signalling in programmed cell death in animals and plants. In our efforts to understand how ceramide metabolism is important in plant biology and programmed cell death we have used the model plant Arabidopsis thaliana to identify the genes involved in ceramide biosynthesis, including homologues of Asc. A.thaliana plants with a disruption in a particular Asc homologue undergo cell death in response to AAL-toxin. This is associated with particular changes in the biosynthesis of new sphingolipids species and an accumulation of their precursors. Other plants with permanent changes in their sphingolipids show phenotypes reminiscent of hormone mutants providing a tantalising link between ceramide biosynthesis and hormone signalling. We are using the AAL-toxin sensitive Arabidopsis thaliana lines to identify genes that regulate or signal programmed cell-death in response to disruption of ceramide biosynthesis resulting from AAL-toxin treatment.

To obtain more insights into the nature of AAL-toxin induced cell death and to identify genes of potential importance for PCD, we carried out transcription profiling of AAL-toxin-induced cell death with an oligonucleotide array representing 21,500 Arabidopsis genes. Genes responsive to reactive oxygen species (ROS) and ethylene were one of the earliest to be upregulated, suggesting that an oxidative burst and production of ethylene played a role in the activation of the cell death. This notion was corroborated by induction of several genes encoding ROS generating proteins, including a respiratory burst oxidase and germin oxalate oxidase. Cytochemical studies confirmed the oxidative burst and in addition showed synthesis of callose, a feature of the hypersensitive response. A diverse group of transcription factors was also induced. These events were followed by repression of most of the auxin-regulated genes known to be involved in growth and developmental responses. All photosynthesis related genes were repressed. Blocking the synthesis of ethylene or NO significantly compromised cell death. In addition, we identified a heterogeneous group of early-induced genes, some of them never associated with PCD before. The group of early-induced genes included a number of proteases that were previously implicated in developmentally regulated types of PCD, suggesting a more principal role for those proteases in the PCD process.

 

In a parallel approach for identifying PCD-related genes, the AAL-toxin-sensitive Arabidopsis was chemically treated with a mutagen and the M2 seeds were plated on plant growth media supplemented with AAL-toxinin order to select for AAL-toxin-resistant plants. In this so-called Phoenix approach, we were able to identify a number of mutants with increased tolerance to the AAL-toxin compared with the AAL-sensitive knockout. In this way we hope to identify the potentially evolutionary conserved signalling pathway that leads to ceramide induced programmed cell death.

  

 

Tomato mosaic virus resistance  

 

 

 

Programmed cell death is one of the defense mechanisms that are mounted by plants against infection by pathogens. Cell death is initiated when products of the pathogen (Avr-proteins) are detected by the resistance gene products of the plant. One of the pathogens of cultivated tomato is the well-known and thoroughly studied Tomato Mosaic Virus (ToMV). Using classical breeding the Tm-2² resistance against ToMV has been introgressed from the wild tomato species Lycopersicon peruvianum. This resistance has some interesting characteristics: 1) The resistance has shown to be very durable: in its long period of application (approximately 35 years) no virulent virus strains have been isolated that are able to circumvent the resistance. 2) The resistance appears to function very rapidly: visible signs of resistance, such as lesions, are usually not observed and 3) this gene has an allelic counterpart, Tm-2, which shares the Avr-protein, the Movement protein (MP) of ToMV, but has been broken by virulent virus strains and lost, therefore, its horticultural value.

 

The Tm-2² resistance gene has been cloned by our research group. The product of the gene is a typical resistance protein with domains involved in signal transduction and pathogen detection. The allelic gene, Tm-2, has also been isolated. Comparison of these two alleles showed that their gene products differ only in 4 amino acids. This suggests that although the direct or indirect interaction between the MP and the two resistance proteins must be highly comparable, the difference is enough to necessitate mutations at other locations in MP in order to circumvent the resistance. Moreover, while the mutations against the Tm-2 resistance do not affect the virulence of the virus, the ones against the Tm-2² resistance do. Hence, the durability of the Tm-2² resistance is caused by the fact that this resistance apparently targets the Achilles' heel of the virus.

 

The successful introduction of the Tm-2² resistance could be demonstrated in transgenic tomato lines. In addition, we were also able to introduce both the Tm-2² and Tm-2 resistance in a susceptible tobacco line, demonstrating that both the Tm-2² and the Tm-2 resistance could be functionally transferred to Nicotiana tabacum, another species of the Solanaceae family.

Cloning of these genes and the successful introduction of the gene in other plant species allows a detailed study of the structure-function relationships of the protein, the downstream signal transduction network, which leads to the initiation of the various aspects of the resistance, and the reasons for durability. 

  

 

Students projects: 1, 2