Tomato susceptibility to Alternaria stem canker:Parameters involved in host-specific toxin-induced leaf necrosis

AAL-toxin causes severe necrosis in leaves of susceptible tomato cultivars at nanomolar concentrations. In resistant tomato cultivars harbouring the semi-dominant Alternaria stem canker resistance locus necrosis is also observed, however at much higher toxin concentrations, in both lines the percentage of the leaf area exhibiting necrosis is dependent on toxin concentration and on length of toxin exposure. However, at the same toxin concentration, periods of toxin exposure resulting in similar necrosis are much longer for the resistant than for the susceptible tomato. It was demonstrated that toxin uptake in the leaves does not imply toxin uptake in the cells since a discrepancy was observed between death of protoplasts, isolated from leaves cut for protoplast isolation immediately after incubation on AAL-toxin and necrosis in leaves when further incubated on water. However, when after exposure to AAL-toxin leaves were further incubated on water for 24 h before they were cut for protoplast isolation, a correlation was found between leaf necrosis and death of protoplasts. This suggests that further transport is needed in leaves after toxin uptake, bringing toxin to all the cells, that cannot occur in leaves cut for protoplast isolation. Light plays an important role in AAL-toxin induced necrosis and it was shown that length of light exposure controls necrosis development like toxin concentration and length of toxin exposure. The product of these 3 parameters can provide a good hint to predict the extent of leaf necrosis. The effect of light might be restricted to differentiated leaf tissue, since it was not observed in callus tissue

Cloning, expression and chromosomal localization of a new putative receptor-like protein tyrosine phosphatase

AbstractWe have isolated a mouse cDNA of 5.7 kb, encoding a new member of the family of receptor-like protein tyrosine phosphatases, termed mRPTPμ. The cDNA predicts a protein of 1432 amino acids (not including signal peptide) with a calculated Mr of 161 636. In addition, we have cloned the human homologue, hRPTPμ, which shows 98.7% amino acid identity to mRPTPμ. The predicted mRPTPμ protein consists of a 722 amino acid extracellular region, containing 13 potential N-glycosylation sites, a single transmembrane domain and a 688 amino acid intracellular part containing 2 tandem repeats homologous to the catalytic domains of other tyrosine phosphatases. The N-terminal extracellular part contains a region of about 170 amino acids with no sequence similarities to known proteins, followed by one Ig-like domain and 4 fibronectin type III-like domains. The intracellular part is unique in that the region between the transmembrane domain and the first catalytic domain is about twice as large as in other receptor-like protein tyrosine phosphatases. RNA blot analysis reveals a single transcript, that is most abundant in lung and present in much lower amounts in brain and heart. Transfection of the mRPTPμ cDNA into COS cells results in the synthesis of a protein with an apparent Mr of 195 000, as detected in immunoblots using an antipeptide antibody. The human RPTPμ gene is localized on chromosome 18pter-q11, a region with frequent abnormalities implicated in human cancer

Tomato susceptibility to Alternaria stem canker: Parameters involved in host-specific toxin-induced leaf necrosis

AAL-toxin causes severe necrosis in leaves of susceptible tomato cultivars at nanomolar concentrations. In resistant tomato cultivars harbouring the semi-dominant Alternaria stem canker resistance locus necrosis is also observed, however at much higher toxin concentrations, in both lines the percentage of the leaf area exhibiting necrosis is dependent on toxin concentration and on length of toxin exposure. However, at the same toxin concentration, periods of toxin exposure resulting in similar necrosis are much longer for the resistant than for the susceptible tomato. It was demonstrated that toxin uptake in the leaves does not imply toxin uptake in the cells since a discrepancy was observed between death of protoplasts, isolated from leaves cut for protoplast isolation immediately after incubation on AAL-toxin and necrosis in leaves when further incubated on water. However, when after exposure to AAL-toxin leaves were further incubated on water for 24 h before they were cut for protoplast isolation, a correlation was found between leaf necrosis and death of protoplasts. This suggests that further transport is needed in leaves after toxin uptake, bringing toxin to all the cells, that cannot occur in leaves cut for protoplast isolation. Light plays an important role in AAL-toxin induced necrosis and it was shown that length of light exposure controls necrosis development like toxin concentration and length of toxin exposure. The product of these 3 parameters can provide a good hint to predict the extent of leaf necrosis. The effect of light might be restricted to differentiated leaf tissue, since it was not observed in callus tissue.

CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

Functional inactivation of the pRB pathway is a very frequent event in human cancer, resulting in deregulated activity of the E2F transcription factors. To understand the functional role of the E2Fs in cell proliferation, we have developed cell lines expressing E2F-1, E2F-2, and E2F-3 fused to the estrogen receptor ligand binding domain (ER). In this study, we demonstrated that activation of all three E2Fs could relieve the mitogen requirement for entry into S phase in Rat1 fibroblasts and that E2F activity leads to a shortening of the G(0)-G(1) phase of the cell cycle by 6 to 7 h. In contrast to the current assumption that E2F-1 is the only E2F capable of inducing apoptosis, we showed that deregulated E2F-2 and E2F-3 activities also result in apoptosis. Using the ERE2F-expressing cell lines, we demonstrated that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. Furthermore, CDC25A is defined as a novel E2F target whose expression can be directly regulated by E2F-1. Data showing that CDC25A is an essential target for E2F-1, since its activity is required for efficient induction of S phase by E2F-1, are provided. Finally, our results show that expression of two E2F target genes, namely CDC25A and cyclin E, is sufficient to induce entry into S phase in quiescent fibroblasts. Taken together, our results provide an important step in defining how E2F activity leads to deregulated proliferation

Cell Cycle-Regulated Expression of Mammalian CDC6 Is Dependent on E2F

The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The CDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G(1) controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication