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Bacterial pathogen deploys the iminosugar glycosyrin to manipulate plant glycobiology
(AAAS, 2025-04) Chandrasekar, Balakumaran
The extracellular space (apoplast) in plants is a key battleground during microbial infections. To avoid recognition, the bacterial model phytopathogen Pseudomonas syringae pv. tomato DC3000 produces glycosyrin. Glycosyrin inhibits the plant-secreted β-galactosidase BGAL1, which would otherwise initiate the release of immunogenic peptides from bacterial flagellin. Here, we report the structure, biosynthesis, and multifunctional roles of glycosyrin. High-resolution cryo–electron microscopy and chemical synthesis revealed that glycosyrin is an iminosugar with a five-membered pyrrolidine ring and a hydrated aldehyde that mimics monosaccharides. Glycosyrin biosynthesis was controlled by virulence regulators, and its production is common in bacteria and prevents flagellin recognition and alters the extracellular glycoproteome and metabolome of infected plants. These findings highlight a potentially wider role for glycobiology manipulation by plant pathogens across the plant kingdom.
Proteome analysis of soybean root apoplast combined with alphafold prediction reveal macrophomina phaseolina infection strategies and potential targets for engineering resistance
(2024-12) Chandrasekar, Balakumaran
Macrophomina phaseolina (Tassi) Goid. is a hemibiotrophic pathogen that causes charcoal rot (CR) disease in various legumes, including soybean. To date, no reliable resistance gene sources have been identified in soybean or other legumes to combat M. phaseolina. Therefore, the identification of mechanistic targets is crucial for improving resistance against the pathogen. The apoplast is a critical region where intense molecular cross-talk occurs between plants and pathogens, and the outcome of their interactions is determined in this compartment. Here, we employed label-free quantitative (LFQ) proteomics to investigate the dynamics of soybean root apoplast during M. phaseolina infection. We have detected several secreted proteins of M. phaseolina and differential regulation of soybean-secreted proteins in root apoplast during infections. Glycome analysis and callose deposition assays have revealed changes in soybean root cell wall compositions and potential polysaccharide targets of M. phaseolina. AlphaFold 2 (AF2) analysis was instrumental in revealing several interesting sequence-unrelated structurally similar (SUSS) effectors and effectors with novel structural folds secreted by M. phaseolina. Structured-guided engineering of protease-inhibitor complexes is emerging as an important strategy to engineer resistance in plants against pathogens. AlphaFold Multimer (AFM) analysis of candidate-secreted proteins from soybean and M. phaseolina has predicted cysteine and serine protease-inhibitor complexes with high confidence. We have validated these interactions using molecular dynamics (MD) and competitive activity-based protein profiling (ABPP) approaches. Therefore, our work provides insights into Soybean-M. phaseolina interactions in the root apoplast and unveil potential candidates for engineering resistance
Bacterial pathogen deploys iminosugar galactosyrin to manipulate plant glycobiology
(2025) Chandrasekar, Balakumaran
The extracellular space (apoplast) of plants is an important molecular battleground during infection by many pathogens. We previously found that a plant-secreted β-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 β-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.
807-P2 - Pseudomonas syringae produces an inhibitor of a plant defence-related Beta-galactosidase
(IS-MPMI XVIII Congress, 2019-07) Chandrasekar, Balakumaran
During infection, plants and bacteria participate in a dynamic interaction in the apoplast. Secreted hydrolases are among the prominent players in plant defence and are therefore targets of virulence factors. Using Nicotiana benthamiana and Pseudomonas syringae pv. tomato (Pst) DC3000 as a model system, we recently discovered a plant β-galactosidase (BGAL1) that functions in immunity against bacteria by facilitating the hydrolytic release of elicitor peptides from glycosylated flagellin, which activates plant defences. Interestingly, PstDC3000 produce an inhibitor of BGAL1. Here, we identify the biosynthetic gene cluster that is responsible for BGAL1 inhibitor production, which is under the control of type III secretion system regulators hrpR/S/L. Mutant bacteria lacking this gene cluster show reduced virulence in N. benthamiana. Comparative genomics suggests acquisition of this gene cluster via horizontal gene transfer and indicates a correlation between inhibitor production and the identity of flagellin glycans. Partial purification and characterization of the inhibitor has revealed that it is a hydrophilic, heat stable and basic small molecule, similar to a sugar derivative. Our work has uncovered a novel small molecule virulence factor that is secreted into the apoplast to inhibit a plant defence-related enzyme and highlights the role of glycans and apoplastic enzymes in plant-pathogen interactions.
Chemiluminescence-Based Assay to Monitor Early Oxidative Bursts in Soybean (Glycine max) Lateral Roots
(Wiley, 2023-08) Chandrasekar, Balakumaran
The reactive oxygen species (ROS) burst assay is a valuable tool for studying pattern-triggered immunity (PTI) in plants. During PTI, the interaction between pathogen recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) leads to the rapid production of ROS in the apoplastic space. The resultant ROS can be measured using a chemiluminescent approach that involves the usage of horseradish peroxidase and luminol. Although several methods and protocols are available to detect early ROS bursts in leaf tissues, no dedicated method is available for root tissues. Here, we have established a reliable method to measure the PAMP-triggered ROS burst response in soybean lateral roots. In plants, lateral roots are the potential entry and colonization sites for pathogens in the rhizosphere. We have used important PAMPs such as chitohexaose, flagellin 22 peptide fragment, and laminarin to validate our method. In addition, we provide a detailed methodology for the isolation and application of fungal cell wall components to monitor the oxidative burst in soybean lateral roots. Furthermore, we provide methodology for performing ROS burst assays in soybean leaf discs with laminarin and fungal cell walls. This approach could also be applied to leaf and root tissues of other plant species to study the PTI response upon elicitor treatment
Multifaceted roles of plant glycosyl hydrolases during pathogen infections: more to discover
(Springer, 2024-04) Chandrasekar, Balakumaran
Glycosidases or glycosyl hydrolases (GHs) are a family of carbohydrate-active enzymes (CAZymes) that hydrolyze glycosidic bonds in carbohydrates and glycoconjugates. Compared to those of all other sequenced organisms, plant genomes contain a remarkable diversity of glycosidases. Plant glycosidases exhibit activities on various substrates and have been shown to play important roles during pathogen infections. Plant glycosidases from different GH families have been shown to act upon pathogen components, host cell walls, host apoplastic sugars, host secondary metabolites, and host N-glycans to mediate immunity against invading pathogens. We could classify the activities of these plant defense GHs under eleven different mechanisms through which they operate during pathogen infections. Here, we have provided comprehensive information on the catalytic activities, GH family classification, subcellular localization, domain structure, functional roles, and microbial strategies to regulate the activities of defense-related plant GHs. We have also emphasized the research gaps and potential investigations needed to advance this topic of research.
A GH81-type β-glucan-binding protein facilitates colonization by mutualistic fungi in barley
(2023-04) Chandrasekar, Balakumaran
Cell walls are important interfaces of plant-fungal interactions. Host cell walls act as robust physical and chemical barriers against fungal invaders, making them an essential line of defense. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to reinforce their walls and prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each other’s cell walls. These enzymes release a wide range of sugar oligomers into the apoplast, some of which trigger the activation of host immunity via host surface receptors. Recent characterization of cell walls from plant-colonizing fungi have emphasized the abundance of β-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated β-glucan laminarin. Thereby, we identified a glycoside hydrolase family 81-type glucan-binding protein (GBP) as the major β-glucan interactor. Mutation of GBP1 and its only paralogue GBP2 in barley led to decreased colonization by the beneficial root endophytes Serendipita indica and S. vermifera, as well as the arbuscular mycorrhizal fungus Rhizophagus irregularis. The reduction of symbiotic colonization was accompanied by enhanced responses at the host cell wall. Moreover, GBP mutation in barley also increased resistance to fungal infections in roots and leaves by the hemibiotrophic pathogen Bipolaris sorokiniana and the obligate biotrophic pathogen Blumeria graminis f. sp. hordei, respectively. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi, a role that has potentially been appropriated by barley-adapted pathogens.
Reevaluation of the Reliability and Usefulness of the Somatic Homologous Recombination Reporter Lines
(OUP, 2012-11) Chandrasekar, Balakumaran
A widely used approach for assessing genome instability in plants makes use of somatic homologous recombination (SHR) reporter lines. Here, we review the published characteristics and uses of SHR lines. We found a lack of detailed information on these lines and a lack of sufficient evidence that they report only homologous recombination. We postulate that instead of SHR, these lines might be reporting a number of alternative stress-induced stochastic events known to occur at transcriptional, posttranscriptional, and posttranslational levels. We conclude that the reliability and usefulness of the somatic homologous recombination reporter lines requires revision. Thus, more detailed information about these reporter lines is needed before they can be used with confidence to measure genome instability, including the complete sequences of SHR constructs, the genomic location of reporter genes and, importantly, molecular evidence that reconstituted gene expression in these lines is indeed a result of somatic recombination.
Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases
(ACS, 2016-03) Chandrasekar, Balakumaran
Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD+, and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.
Inhibitor Discovery by Convolution ABPP
(Springer, 2016-10) Chandrasekar, Balakumaran
Activity-based protein profiling (ABPP) has emerged as a powerful proteomic approach to study the active proteins in their native environment by using chemical probes that label active site residues in proteins. Traditionally, ABPP is classified as either comparative or competitive ABPP. In this protocol, we describe a simple method called convolution ABPP, which takes benefit from both the competitive and comparative ABPP. Convolution ABPP allows one to detect if a reduced signal observed during comparative ABPP could be due to the presence of inhibitors. In convolution ABPP, the proteomes are analyzed by comparing labeling intensities in two mixed proteomes that were labeled either before or after mixing. A reduction of labeling in the mix-and-label sample when compared to the label-and-mix sample indicates the presence of an inhibitor excess in one of the proteomes. This method is broadly applicable to detect inhibitors in proteomes against any proteome containing protein activities of interest. As a proof of concept, we applied convolution ABPP to analyze secreted proteomes from Pseudomonas syringae-infected Nicotiana benthamiana leaves to display the presence of a beta-galactosidase inhibitor.

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