Nicotiana benthamiana is an important model organism of the Solanaceae (Nightshade) family. Several draft assemblies of the N. benthamiana genome have been generated, but many of the gene-models in ...these draft assemblies appear incorrect.
Here we present an improved proteome based on the Niben1.0.1 draft genome assembly guided by gene models from other Nicotiana species. Due to the fragmented nature of the Niben1.0.1 draft genome, many protein-encoding genes are missing or partial. We complement these missing proteins by similarly annotating other draft genome assemblies. This approach overcomes problems caused by mis-annotated exon-intron boundaries and mis-assigned short read transcripts to homeologs in polyploid genomes. With an estimated 98.1% completeness; only 53,411 protein-encoding genes; and improved protein lengths and functional annotations, this new predicted proteome is better in assigning spectra than the preceding proteome annotations. This dataset is more sensitive and accurate in proteomics applications, clarifying the detection by activity-based proteomics of proteins that were previously predicted to be inactive. Phylogenetic analysis of the subtilase family of hydrolases reveal inactivation of likely homeologs, associated with a contraction of the functional genome in this alloploid plant species. Finally, we use this new proteome annotation to characterize the extracellular proteome as compared to a total leaf proteome, which highlights the enrichment of hydrolases in the apoplast.
This proteome annotation provides the community working with Nicotiana benthamiana with an important new resource for functional proteomics.
Abstract
Adapted plant pathogens from various microbial kingdoms produce hundreds of unrelated small secreted proteins (SSPs) with elusive roles. Here, we used AlphaFold-Multimer (AFM) to screen 1879 ...SSPs of seven tomato pathogens for interacting with six defence-related hydrolases of tomato. This screen of 11,274 protein pairs identified 15 non-annotated SSPs that are predicted to obstruct the active site of chitinases and proteases with an intrinsic fold. Four SSPs were experimentally verified to be inhibitors of pathogenesis-related subtilase P69B, including extracellular protein-36 (Ecp36) and secreted-into-xylem-15 (Six15) of the fungal pathogens
Cladosporium fulvum
and
Fusarium oxysporum
, respectively. Together with a P69B inhibitor from the bacterial pathogen
Xanthomonas perforans
and Kazal-like inhibitors of the oomycete pathogen
Phytophthora infestans
, P69B emerges as an effector hub targeted by different microbial kingdoms, consistent with a diversification of P69B orthologs and paralogs. This study demonstrates the power of artificial intelligence to predict cross-kingdom interactions at the plant-pathogen interface.
Subtilases (SBTs), also known as Subtilisin-like serine proteases, are extracellular alkaline protease proteins. SBTs function in all stages of plant growth, development and stress responses. Maize ...(Zea mays L.) is a crop widely used worldwide as food, feed, and industrial materials. However, information about the members and their functions of the SBT proteins in maize is lacking. In this study, we identified 58 ZmSBT genes from the maize genome and conducted a comprehensive investigation of ZmSBTs by phylogenetic, gene duplication event, gene structure, and protein conserved motif analyses. The ZmSBT proteins were phylogenetically classified into seven groups, and collinearity analysis indicated that many ZmSBTs originate from tandem or segmental duplications. Structural and homolog protein comparison revealed ZmSBTs have conserved protein structures with reported subtilase proteins, suggesting the conserved functions. Further analysis showed that ZmSBTs are expressed in different tissues, and many are responses to specific abiotic stress. Analysis of the anther-specific ZmSBT genes showed their expression peaked at different developmental stages of maize anthers. Subcellular localization analysis of selected maize ZmSBTs showed they are located in different cellular compartments. The information provided in this study is valuable for further functional study of ZmSBTs.
•Maize subtilase genes were genome-wide identified for the first time.•Six subtilase genes are specifically expressed in maize anthers.•Most of maize sublilases are localized at extracellular space.•Segmental duplication contributes to the maize SBT gene family expansion.
The plant embryonic cuticle is a hydrophobic barrier deposited de novo by the embryo during seed development. At germination, it protects the seedling from water loss and is, thus, critical for ...survival. Embryonic cuticle formation is controlled by a signaling pathway involving the ABNORMAL LEAF SHAPE1 subtilase and the two GASSHO receptor-like kinases. We show that a sulfated peptide, TWISTED SEED1 (TWS1), acts as a GASSHO ligand. Cuticle surveillance depends on the action of the subtilase, which, unlike the TWS1 precursor and the GASSHO receptors, is not produced in the embryo but in the neighboring endosperm. Subtilase-mediated processing of the embryo-derived TWS1 precursor releases the active peptide, triggering GASSHO-dependent cuticle reinforcement in the embryo. Thus, a bidirectional molecular dialogue between embryo and endosperm safeguards cuticle integrity before germination.
The Pectin methylesterase (PME), Pectin methylesterase inhibitor (PMEI), and Subtilase (SBT) are pectin remodeling enzymes that play an important role in plant growth and development as well as ...response to various biotic and abiotic stress conditions. Withania somnifera is an important medicinal plant that gained attention for its phytochemical properties, but relatively little in-depth genomic exploration has been available, including PME, PMEI, and SBT genes. In the present study, 54 PMEs, 19 PMEIs, and 51 SBTs genes were identified from the W. somnifera transcriptome. A detailed phylogeny was performed, followed by a conserved sequence signature analysis, which revealed the major phylogenetic division with Solanum lycopersicum. Insect infestation can cause a significant increase in the functions of these genes, which were identified using PME activity, methanol content, and emission, often by several orders of magnitude. WsPME28, WsPME32, and WsPME50 are putative genes that are significantly upregulated up to six fold during insect infestation, according to gene expression profile analysis. To study specific relative interactions for a PME-SBT pair, molecular docking analysis was performed and found that the serine residue of WsSBT37 interacts with the processing motif (RKLL) of WsPME28 and releases mature PME. As per our best knowledge, this is the first investigation into the identification and characterization of the PME, PMEI, and SBT gene families in W. somnifera. This study will lay the groundwork for future studies targeting these genes, thus giving a better understanding of the stress tolerance mechanism in W. somnifera, and will serve as a reference for determining the most specific SBT-PME pairs in development and stress conditions.
Display omitted
•A total of 54 PME, 19 PMEI, and 51 SBT genes were identified from Withania somnifera.•Phylogeny, motif analysis & conserved region showed the evolutionary significance of the genes.•PME activity and methanol emission significantly increased during insect infestation.•During biotic stress, the interaction of WsSBT37-WsPME28 causes PME gene activation.
Certain foodborne Shiga toxin-producing Escherichia coli (STEC) strains carry genes encoding the subtilase cytotoxin (SubAB). Although the mode of action of SubAB is under intensive investigation, ...information about the regulation of subAB gene expression is currently not available. In this study, we investigated the regulation of the chromosomal subAB1 gene in laboratory E. coli strain DH5α and STEC O113:H21 strain TS18/08 using a luciferase reporter gene assay. Special emphasis was given to the role of the global regulatory protein genes hfq and hns in subAB1 promoter activity. Subsequently, quantitative real-time PCR was performed to analyze the expression of Shiga toxin 2a (stx2a), SubAB1, and cytolethal distending toxin V (Cdt-V) genes in STEC strain TS18/08 and its isogenic hfq and hns deletion mutants. The deletion of hfq led to a significant increase of up to 2-fold in subAB1 expression, especially in the late growth phase, in both strains. However, deletion of hns showed different effects on the promoter activity during the early and late exponential growth phases in both strains. Furthermore, upregulation of stx2a and cdt-V was demonstrated in hfq and hns deletion mutants in TS18/08. These data showed that the expression of subAB1, stx2a, and cdt-V is integrated in the regulatory network of global regulators Hfq and H-NS in Escherichia coli.
The subtilase cytotoxin (SubAB) is secreted by certain Shiga toxin-producing
Escherichia coli
(STEC) strains and is composed of the enzymatically active subunit SubA and the pentameric ...binding/transport subunit SubB. We previously demonstrated that SubA (10 µg/ml), in the absence of SubB, binds and intoxicates the human cervix cancer-derived epithelial cell line HeLa. However, the cellular and molecular mechanisms underlying the cytotoxic activity of SubA in the absence of SubB remained unclear. In the present study, the cytotoxic effects mediated by SubA alone were investigated in more detail in HeLa cells and the human colon cancer cell line HCT116. We found that in the absence of SubB, SubA (10 µg/ml) is internalized into the endoplasmic reticulum (ER), where it cleaves the chaperone GRP78, an already known substrate for SubA after its canonical uptake into cells via SubB. The autonomous cellular uptake of SubA and subsequent cleavage of GRP78 in cells is prevented by treatment of cells with 10 µM brefeldin A, which inhibits the transport of protein toxins into the ER. In addition, by analyzing the SubA mutant SubA
ΔC344
, we identified the C-terminal SEEL motif as an ER-targeting signal. Conclusively, our results strongly suggest that SubA alone shares the same intracellular transport route and cytotoxic activity as the SubAB holotoxin.
Subtilisin-like proteases (subtilases) are serine proteases that fulfill highly specific functions in plant development and signaling cascades. Over the last decades, it has been shown that several ...subtilases are specifically induced following pathogen infection and very recently an Arabidopsis subtilase (SBT3.3) was hypothesized to function as a receptor located in the plasma membrane activating downstream immune signaling processes. Despite their prevalence and potential relevance in the regulation of plant defense mechanisms and crop improvement, our current understanding of subtilase function is still very limited. In this perspective article, we overview the current status and highlight the involvement of subtilases in pathogen recognition and immune priming.
PDF
