Pythium root rot (PRR) caused by Pythium spp. is an important root rot disease affecting common bean productivity. The objective of this study was to conduct a genome‐wide association analysis of ...resistance to PRR in the common bean of Andean gene pool using single nucleotide polymorphism (SNP) markers. About 260 genotypes of the Andean diversity panel (ADP) were evaluated under screen house conditions using Pythium ultimum isolate MS61 in Uganda. Sixteen significant signals for resistance to PRR were detected on chromosomes Pv01, Pv02, Pv04, Pv05 and Pv09 using 260K GBS‐based and 6K Beadchip SNPs. Common significant signals were detected on Pv02 and Pv09 for PRR. Positional candidate genes associated with significant SNPs on Pv02 were Phvul.002G119700, 16.97 kb near marker S02_25507837 (25.50 Mb), encoding Subtilase family protein, and Phvul.002G278400 near marker ss715645959 (44.79 Mb) encoding Defensin‐like (DEFL) protein involved in plant defence responses. Based on the relatively high heritability estimates observed for PRR in this study, significant SNP markers associated with genomic regions for resistance to PRR could be validated for marker‐assisted breeding in Andean beans.
Abstract
Increasing drought stress poses a severe threat to agricultural productivity. Plants, however, have evolved numerous mechanisms to cope with such environmental stress. Here we report that ...the stress-induced production of a peptide signal contributes to stress tolerance. The expression of phytosulfokine (PSK) peptide precursor genes, and transcripts of three subtilisin-like serine proteases, SBT1.4, SBT3.7, and SBT3.8, were found to be up-regulated in response to osmotic stress. Stress symptoms were more pronounced in sbt3.8 loss-of-function mutants and could be alleviated by PSK treatment. Osmotic stress tolerance was improved in plants overexpressing the PSK1 precursor (proPSK1) or SBT3.8, resulting in higher fresh weight and improved lateral root development in transgenic plants compared with wild-type plants. We further showed that SBT3.8 is involved in the biogenesis of the bioactive PSK peptide. ProPSK1 was cleaved by SBT3.8 at the C-terminus of the PSK pentapeptide. Processing by SBT3.8 depended on the aspartic acid residue directly following the cleavage site. ProPSK1 processing was impaired in the sbt3.8 mutant. The data suggest that increased expression of proPSK1 in response to osmotic stress followed by the post-translational processing of proPSK1 by SBT3.8 leads to the production of PSK as a peptide signal for stress mitigation.
The expression of phytosulfokine precursor genes and processing by the subtilase SBT3.8 are up-regulated in response to osmotic stress, resulting in improved drought tolerance in Arabidopsis.
To relieve endoplasmic reticulum (ER) stress, IRE1 splices XBP1 messenger RNA (mRNA) or engages regulated IRE1-dependent decay (RIDD) of other mRNAs. Upon XBP1 deficiency, IRE1 switches to perform ...RIDD. We examined IRE1 in XBP1-deficient B cells and discovered that IRE1 undergoes phosphorylation at S729. We generated an anti-phospho-S729 antibody to investigate such phosphorylation. Compared with pharmacological ER stress inducers or Toll-like receptor ligands, the bacterial subtilase cytotoxin has an unusual capability in causing rapid and strong phosphorylation at S729 and triggering B cells to express spliced XBP1. To assess the function of S729 in IRE1, we generated S729A knock-in mice and found S729 is critically important for lipopolysaccharide-stimulated plasmablasts to respond to additional ER stress and for antibody production in response to immunization. We further crossed mice carrying an S729A mutation or ΔIRE1 (missing the kinase domain) with B cell-specific XBP1-deficient mice to trigger RIDD and discovered a critical role for S729 in regulating RIDD in B cells.
Post-translationally modified peptides (PMPs) are important regulators of plant growth and development. They are derived from larger inactive precursors by post-translational modification (PTM) and ...proteolytic processing to result in the bioactive peptide signals. We discuss how and why these modifications contribute to the bioactivity of inflorescence deficient in abscission (IDA), phytosulfokine (PSK), and peptides of the Casparian strip integrity factor (CIF) family, as signaling molecules during reproductive development. The emerging picture suggests that PTMs evolved to increase the specificity of interaction of PMPs with cognate receptors and of PMP precursors with processing proteases. Cleavage sites in PMP precursors are recognized by subtilases (SBTs) in a highly specific manner. SBT-mediated processing results in the activation of PMP signals regulating stress-induced flower drop, the formation of the embryonic cuticle, and pollen development.
Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is ...activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva). Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Aspselective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.
The subtilase family (S8), a member of the clan SB of serine proteases are ubiquitous in all kingdoms of life and fulfil different physiological functions. Subtilases are divided in several groups ...and especially subtilisins are of interest as they are used in various industrial sectors. Therefore, we searched for new subtilisin sequences of the family
Bacillaceae
using a data mining approach. The obtained 1,400 sequences were phylogenetically classified in the context of the subtilase family. This required an updated comprehensive overview of the different groups within this family. To fill this gap, we conducted a phylogenetic survey of the S8 family with characterised holotypes derived from the MEROPS database. The analysis revealed the presence of eight previously uncharacterised groups and 13 subgroups within the S8 family. The sequences that emerged from the data mining with the set filter parameters were mainly assigned to the subtilisin subgroups of true subtilisins, high-alkaline subtilisins, and phylogenetically intermediate subtilisins and represent an excellent source for new subtilisin candidates.
Domain of unknown function 26 (DUF26) is a non-catalytic protein domain found only in land plant proteins. These proteins are often associated with defense. Cotton (Gossypium hirsutum L) CRR1 is a ...secreted protein consisting of two DUF26 domains connected by a linker. In this study we report that CRR1 is cleaved by an alkaline subtilase secreted by Verticillium dahliae, a hemi-biotrophic fungal pathogen. Recombinant CRR1 was converted from a 30 kDa glycoprotein into products of ∼15 kDa (CRR1-P) when incubated with secreted protein extracts from V. dahliae cultures. Using this activity as a guide, the protease was purified, and its tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. It was identified as alkaline subtilase G2X826_VERDV, which we named Vd-DUMP for V. dahliaeDUF26 modifying protein. This identification was confirmed by producing active recombinant Vd-DUMP in the yeast Pichia pastoris. Biochemical analysis indicated that Vd-DUMP cleaves CRR1 between the DUF26 domains. Infiltration of Vd-DUMP into Arabidopsis (Arabidopsis thaliana) leaves or cotton cotyledons induced cell death, a response that was absent when Vd-DUMP was chemically inactivated prior to infiltration. This study identifies plant DUF26 proteins, associated with defense, as substrates for alkaline subtilases secreted by fungi that are known to function as elicitors and effectors.
•Fungal alkaline proteases cleave plant duf26 proteins.•Protease activity induces plant cell death.•Fungal effector and plant protein target identified.
Peptide hormones are implicated in many important aspects of plant life and are usually synthesized as precursor proteins. In contrast to animals, data for plant peptide hormone maturation are scarce ...and the specificity of processing enzyme(s) is largely unknown. Here we tested a hypothesis that processing of prosystemin, a precursor of tomato (Solanum lycopersicum) wound hormone systemin, is performed by phytaspases, aspartate-specific proteases of the subtilase family.
Following the purification of phytaspase from tomato leaves, two tomato phytaspase genes were identified, the cDNAs were cloned and the recombinant enzymes were obtained after transient expression in Nicotiana benthamiana.
The newly identified tomato phytaspases hydrolyzed prosystemin at two aspartate residues flanking the systemin sequence. Site-directed mutagenesis of the phytaspase cleavage sites in prosystemin abrogated not only the phytaspase-mediated processing of the prohormone in vitro, but also the ability of prosystemin to trigger the systemic wound response in vivo.
The data show that the prohormoneprosystemin requires processing for signal biogenesis and biological activity. The identification of phytaspases as the proteases involved in prosystemin maturation provides insight into the mechanisms of wound signaling in tomato. Our data also suggest a novel role for cell death-related proteases in mediating defense signaling in plants.
The subtilase cytotoxin (SubAB) belongs to the family of AB
toxins and is produced together with Shiga toxin (Stx) by certain Stx-producing
strains (STEC). For most AB-type toxins, it is assumed that ...cytotoxic effects can only be induced by a complete holotoxin complex consisting of SubA and SubB. However, it has been shown for SubAB that the enzymatically active subunit SubA, without its transport and binding domain SubB, induces cell death in different eukaryotic cell lines. Interestingly, the molecular structure of SubA resembles that of the SubAB complex. SubA alone is capable of binding to cells and then being taken up autonomously. Once inside the host cell, SubA is transported, similar to the SubAB holotoxin, via a retrograde transport into the endoplasmatic reticulum (ER). In the ER, it exhibits its enzymatic activity by cleaving the chaperone BiP/GRP78 and thereby triggering cell death. Therefore, the existence of toxic single SubA subunits that have not found a B-pentamer for holotoxin assembly might improve the pathogenic potential of subtilase-producing strains. Moreover, from a pharmacological aspect, SubA might be an interesting molecule for the targeted transport of therapeutic molecules into the ER, in order to investigate and specifically modulate processes in the context of ER stress-associated diseases. Since recent studies on bacterial AB
toxins contributed mainly to the understanding of the biology of AB-type holotoxins, this mini-review specifically focus on that recently observed single A-effect of the subtilase cytotoxin and addresses whether a fundamental shift of the traditional AB
paradigm might be required.
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