Biology subjects

Sanguankiattichai, N.

Publications and source records attributed to Sanguankiattichai, N..

9 recordsLinked to original sources

bioRxiv2026

Polychrome activity profiling distinguishes Cys proteases and their isoforms in plants

Activity-based profiling with fluorescent probes is a powerful tool for the functional characterization of whole enzyme classes in crude proteomes. Here, we discovered that probe cocktails consisting of the E-64 warhead carrying different fluorophores via short linkers distinguishes the labeling of papain-like cysteine proteases and their isoforms because of their differential affinity to these probes. This causes polychrome labeling with specific apparent colors for different protease families and isoforms. Polychrome labeling revealed differential labeling of isoforms of RD21-like proteases carrying C-terminal granulin domains. Molecular modeling of the RD21 isoforms revealed that the C-terminal granulin may create a fluorophore-binding pocket with the substrate-binding groove, implying that the granulin domain influences substrate selectivity. The concept of polychrome labeling may be widely applicable to other chemical probes for the characterization of protein families in all kingdoms of life.

Plant Biology↗

bioRxiv2025

Bacterial pathogen deploys iminosugar galactosyrin to manipulate plant glycobiology

The extracellular space (apoplast) of plants is an important molecular battleground during infection by many pathogens. We previously found that a plant-secreted {beta}-galactosidase BGAL1 acts in immunity by facilitating the release of immunogenic peptides from bacterial flagellin and that Pseudomonas syringae suppresses this enzyme by producing a small molecule inhibitor called galactosyrin. Here, we elucidated the structure and biosynthesis of galactosyrin and uncovered its multifunctional roles during infection. Structural elucidation by cryo-EM and chemical synthesis revealed that galactosyrin is an iminosugar featuring a unique geminal diol attached to the pyrrolidine moiety that mimics galactose binding to the {beta}-galactosidase active site. Galactosyrin biosynthesis branches off from purine biosynthesis and involves three enzymes of which the first is a reductase that is unique in iminosugar biosynthesis. Besides inhibiting BGAL1 to avoid detection, galactosyrin also changes the glycoproteome and metabolome of the apoplast. The manipulation of host glycobiology may be common to plant-associated bacteria that carry putative iminosugar biosynthesis clusters.

plant biology↗

bioRxiv2024

Extracellular plant subtilases dampen cold shock peptide elicitor levels

Recognizing pathogen-associated molecular patterns (PAMPs) on the cell surface is crucial for plant immunity. The proteinaceous nature of many of these patterns suggests that secreted proteases play important roles in their formation and stability. Here, we demonstrate that the apoplastic subtilase SBT5.2a inactivates the immunogenicity of cold-shock proteins of the bacterial plant pathogen Pseudomonas syringae by cleaving within the immunogenic csp22 epitope. Consequently, mutant plants lacking SBT5.2a activity retain higher levels of csp22, leading to enhanced immune responses and reduced pathogen growth. SBT5.2 sensitivity is influenced by sequence variation surrounding the cleavage site and likely extends to CSPs from other bacterial species. These findings suggest that variations in csp22 stability among bacterial pathogens are a crucial factor in plant-bacteria interactions and that pathogens might exploit plant proteases to avoid pattern recognition.

plant biology↗

bioRxiv2024

Subtilase SBT5.2 counterbalances flagellin immunogenicity in the plant apoplast

Most angiosperm plants recognise the flg22 epitope in bacterial flagellin via homologs of cell surface receptor FLS2 and mount pattern-triggered immune responses. However, flg22 is buried within the flagellin protein indicating that proteases might be required for flg22 release. Here, we demonstrate the extracellular subtilase SBT5.2 not only releases flg22, but also inactivates the immunogenicity of flagellin and flg22 by cleaving within the flg22 epitope, consistent with previous reports that flg22 is unstable in the apoplast. The prolonged lifetime of flg22 in sbt5.2 mutant plants results in increased bacterial immunity in priming assays, indicating that SBT5.2 counterbalances flagellin immunogenicity to provide spatial-temporal control and restrict costly immune responses and that bacteria take advantage of the host proteolytic machinery to avoid detection by flagellin having a protease-sensitive flg22 epitope.

plant biology↗

bioRxiv2023

Protein migration and topography mapping reveals endogenous protein processing and homomers in Nicotiana benthamiana.

Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant protein in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as a favorite transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5,040 plant proteins of agroinfiltrated N. benthamiana within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. For instance, this analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed various processing events in vivo. This extensive proteomic dataset can be investigated further and demonstrates that most plant proteins are proteolytically processed and implicates an extensive proteolytic machinery shaping the proteome of agroinfiltrated N. benthamiana.

plant biology↗

bioRxiv2023

Cautionary note on ribonuclease activity of recombinant PR-10 proteins

We studied the biochemical properties of three splicing isoforms of PR-10 from rubber tree (Hevea brasiliensis) and found that purified recombinant HbPR10 can cause RNA degradation in vitro, a well-known activity described for many PR-10 proteins. This ribonuclease activity was observed for all three HbPR10 splicing isoforms and is abolished by boiling. However, inclusion of a negative control proteins revealed that ribonuclease activity rather originates from RNases that are copurified from E. coli, which are overlooked by traditionally used controls such as heat inactivation, RNase inhibitors and negative control proteins obtained with different procedures. The crucial control proteins are missing for at least nine reports on ribonuclease activity in PR-10 proteins published by different laboratories worldwide, indicating that proper controls are frequently overlooked in ribonuclease assays. The raised cautionary note applies to several PR-10 proteins with proclaimed ribonuclease activities and call for the use of different assays and mutant PR-10 proteins as control.

plant biology↗

bioRxiv2020

Proteome-wide Profiling of RNA-Binding Protein Responses to flg22 Reveals Novel Components of Plant Immunity

RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation and are known to contribute to plant immunity. To understand the responses of cellular RBPs to an immune elicitor, we applied RNA interactome capture to Arabidopsis leaves treated with flg22. Strikingly, flg22 induced a pervasive remodelling of the cellular RBPome affecting 186 proteins. Flg22-responsive RBPs included classical RBPs involved in RNA metabolism as well as non-canonical RBPs. RBP responders detected after 2h of treatment are enriched in putative sites for post-translational modifications, which may play a regulatory role. By contrast, changes in RBP abundance becomes increasingly important for the RBPome responses to flg22 after 12h. Plant resistance to Pseudomonas syringae is strongly altered in mutant lines lacking individual flg22-responsive RBPs, supporting the importance of RBP dynamics in plant immunity. This study provides a comprehensive and systematic census of flg22 responsive plant RBPs, discovering novel components of plant immunity.

molecular biology↗

bioRxiv2020

Agromonas: a rapid disease assay for Pseudomonas syringae growth in agroinfiltrated leaves

The lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth and that transient depletion of a host immune gene and transient expression of a T3 effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure-function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers reliable opportunity for further comprehensive analysis of plant immunity. One sentence summaryAgromonas is a rapid and robust disease assay to monitor Pseudomonas syringae growth in agroinfiltrated leaves expressing immune components and their suppressors.

plant biology↗

bioRxiv2017

Host autophagosomes are diverted to a plant-pathogen interface

Filamentous plant pathogens and symbionts invade their host cells but remain enveloped by host-derived membranes. The mechanisms underlying the biogenesis and functions of these host-microbe interfaces are poorly understood. Recently, we showed that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host protein ATG8CL to stimulate autophagosome formation and deplete the selective autophagy receptor Joka2 from ATG8CL complexes. Here, we show that during P. infestans infection, ATG8CL autophagosomes are diverted to the pathogen interface. Our findings are consistent with the view that the pathogen coopts host selective autophagy for its own benefit.

plant biology↗