The tomato subtilase family includes several cell death-related proteinases with caspase specificity

Phytaspases are Asp-specific subtilisin-like plant proteases that have been likened to animal caspases with respect to their regulatory function in programmed cell death (PCD). We identified twelve putative phytaspase genes in tomato that differed widely in expression level and tissue-specific expression patterns. Most phytaspase genes are tandemly arranged on tomato chromosomes one, four, and eight, and many belong to taxon-specific clades, e.g. the P69 clade in the nightshade family, suggesting that these genes evolved by gene duplication after speciation. Five tomato phytaspases (SlPhyts) were expressed in N. benthamiana and purified to homogeneity. Substrate specificity was analyzed in a proteomics assay and with a panel of fluorogenic peptide substrates. Similar to animal caspases, SlPhyts recognized an extended sequence motif including Asp at the cleavage site. Clear differences in cleavage site preference were observed implying different substrates in vivo and, consequently, different physiological functions. A caspase-like function in PCD was confirmed for five of the seven tested phytaspases. Cell death was triggered by ectopic expression of SlPhyts 2, 3, 4, 5, 6 in tomato leaves by agro-infiltration, as well as in stably transformed transgenic tomato plants. SlPhyts 3, 4, and 5 were found to contribute to cell death under oxidative stress conditions

The tomato subtilase family includes several cell death-related proteinases with caspase specificity

Phytaspases are Asp-specific subtilisin-like plant proteases that have been likened to animal caspases with respect to their regulatory function in programmed cell death (PCD). We identified twelve putative phytaspase genes in tomato that differed widely in expression level and tissue-specific expression patterns. Most phytaspase genes are tandemly arranged on tomato chromosomes one, four, and eight, and many belong to taxon-specific clades, e.g. the P69 clade in the nightshade family, suggesting that these genes evolved by gene duplication after speciation. Five tomato phytaspases (SlPhyts) were expressed in N. benthamiana and purified to homogeneity. Substrate specificity was analyzed in a proteomics assay and with a panel of fluorogenic peptide substrates. Similar to animal caspases, SlPhyts recognized an extended sequence motif including Asp at the cleavage site. Clear differences in cleavage site preference were observed implying different substrates in vivo and, consequently, different physiological functions. A caspase-like function in PCD was confirmed for five of the seven tested phytaspases. Cell death was triggered by ectopic expression of SlPhyts 2, 3, 4, 5, 6 in tomato leaves by agro-infiltration, as well as in stably transformed transgenic tomato plants. SlPhyts 3, 4, and 5 were found to contribute to cell death under oxidative stress conditions

TUIT, a BLAST-based tool for taxonomic classification of nucleotide sequences

Pyrosequencing of 16S ribosomal RNA (rRNA) genes has become the gold standard in human microbiome studies. The routine task of taxonomic classification using 16S rRNA reads is commonly performed by the Ribosomal Database Project (RDP) II Classifier, a robust tool that relies on a set of well-characterized reference sequences. However, the RDP II Classifier may be unable to classify a significant part of the dataset due to the absence of proper reference sequences. The taxonomic classification for some of the unclassified sequences might still be performed using BLAST searches against large and frequently updated nucleotide databases. Here we introduce TUIT (Taxonomic Unit Identification Tool) – an efficient open source and platform-independent application that can perform taxonomic classification on its own or can be used in combination with RDP II Classifier to maximize the taxonomic identification rate. Using a set of simulated DNA sequences we demonstrate that the algorithm performs taxonomic classification with high specificity for sequences as short as 125 base pairs. TUIT is applicable for 16S rRNA gene sequence classification; however, it is not restricted to 16S rRNA sequences. In addition, TUIT may be used as a complementary tool for effective taxonomic classification of nucleotide sequences generated by many current platforms, such as Roche 454 and Illumina. Standalone TUIT is available online at http://sourceforge.net/projects/tuit/

Author Correction : The tomato subtilase family includes several cell death-related proteinases with caspase specificity

Aquesta és una correcció a l'article 10.1038/s41598-018-28769-

Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity

Caspases are cysteine-dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase-specific proteolytic activity. Nevertheless, plants do display caspase-like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase-like proteases. Here, we report the identification and characterisation of a novel PCD-related subtilisin-like protease from tobacco and rice named phytaspase (plant aspartate-specific protease) that possesses caspase specificity distinct from that of other known caspase-like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD-related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re-imported into the cell during PCD providing insights into how phytaspase operates