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

Abstract

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.

Regulation of plant peptide hormones and growth factors by post‐translational modification

“…Substitution of phytaspase cleavage sites precluded PS processing in vitro and inhibited systemin signalling in vivo, indicating that Asp-specific cleavage is required for signal biogenesis (Beloshistov et al 2017). However, which of the 12 tomato phytaspases (Reichardt et al 2018) is responsible for the production of L-systemin in vivo is still unclear. Also unclear is how prosystemin that lacks a secretion signal may meet with extracellular phytaspases.…”

Homology-guided re-annotation improves the gene models of the alloploidNicotiana benthamiana

“…Remarkably, no SBT3 clade family members were identified in N. benthamiana (Table 1, Supplemental Figures S2-S3). Three N. benthamiana subtilases may possess phytaspase activity based on the presence of an aspartic acid residue at the pro-domain junction as well as a histidine residue in the S1 pocket which is thought to bind to P1 aspartic acid (Supplemental Figures S2-S3, Reichardt et al, 2018).…”

Subtilase activity in the intrusive cells mediates haustorium maturation in parasitic plants

“…3). A phylogenetic analysis revealed that the five SBTs upregulated in intrusive cells all belong to Group 1 (Taylor and Qiu, 2017; Reichardt et al ., 2018) (Fig. 3), which contains many SBTs involved in biotic interactions.…”

“…In addition, Group 1 SBTs also expanded in plants that undergo symbiosis with nitrogen-fixing bacteria. Group 1 SBTs also include many that are involved in plant defense (Taylor and Qiu, 2017; Reichardt et al ., 2018). These findings indicate that Group 1 SBTs may have evolved for biotic interactions, including parasitism.…”