Myrosinase: insights on structural, catalytic, regulatory, and environmental interactions

Bhat, Rohini; Vyas, Dhiraj

doi:10.1080/07388551.2019.1576024

Cited by 77 publications

(80 citation statements)

References 135 publications

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“…We excluded from our priority list a number of proteins that were significantly enriched in infected vs. non-infected plants due to the number of candidates to analyze and their apparent lack of significance in viral replication process based on literature. This includes for instance the most enriched protein, a myrosinase present in Brassica crops with anti-microbial activity and involved in defense against herbivores [33].…”

Section: Resultsmentioning

confidence: 99%

Immunocapture of dsRNA-bound proteins provides insight into tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector

Incarbone

Clavel

Monsion

et al. 2019

Preprint

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27 Plant RNA viruses form highly organized membrane-bound virus replication complexes 28 (VRCs) to replicate their genome and multiply. VRCs contain both virus-and host-encoded 29 proteins, and have been investigated through a number of experimental approaches, including 30 genetic screens on surrogate model experimental hosts and pull-down of viral replicase 31 proteins. Double-stranded RNA (dsRNA) is an obligate intermediate of the replication 32 process of all RNA viruses and is the trigger of potent antiviral defenses in all eukaryotes. In 33 this work, we describe the use of a GFP-tagged dsRNA-binding protein (B2:GFP) to pull 34 down viral replicating RNA and associated proteins in planta. Following infection of A.35 thaliana constitutively expressing B2:GFP with tobacco rattle virus (TRV), we were able 36 through GFP immunoprecipitation to pull down dsRNA. Mass spectrometry analysis of the 37 dsRNA-B2:GFP-bound proteins from TRV-infected plants revealed the presence of (i) viral 38 proteins such as the replicase, which attested to the successful isolation of VRCs, and (ii) a 39 number of host proteins, some of which have previously been involved in virus infection. To 40 verify whether the identified host proteins localized at TRV replication complexes, we 41 selected nine candidates, generated genetic fusions with RFP, and transiently expressed them 42 in healthy and TRV-infected N. benthamiana stably expressing B2:GFP. Confocal 43 microscopy analysis revealed that eight out of nine candidates showed dramatic re-44 localization upon infection, and seven of these co-localized with B2-labeled TRV replication 45 complexes, providing ample validation for the immunoprecipitation results. We therefore 46 propose B2:GFP-mediated pull down of dsRNA to be a novel and robust method to explore 47 the proteome of VRCs in planta. 48 49 50 51 AUTHOR SUMMARY 52 Viruses are an important class of pathogens that represent a major problem for human, animal 53 and plant health. They hijack the molecular machinery of host cells to complete their 54 replication cycle, a process frequently associated with the production of double-stranded 55 RNA (dsRNA) that is regarded as a universal hallmark of infection by RNA viruses. Here we 56 exploited the capacity of a GFP-tagged dsRNA-binding protein stably expressed in transgenic 57 Arabidopsis to pull down dsRNA and associated proteins upon virus infection. In this manner 58 we specifically captured short and long dsRNA from tobacco rattle virus (TRV) infected 59 plants, and successfully isolated viral proteins such as the replicase, which attested to the 60 successful isolation of virus replication complexes (VRCs). More excitingly, a number of 61 host proteins, some of which have previously been involved in virus infection, were also 62 captured. Remarkably, among a set of nine host candidates that were analyzed, eight showed 63 dramatic re-localization to viral factories upon infection, and seven of these co-localized 64 dsRNA-labeled VRCs. Being compatible with any plant virus as ...

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Section: Resultsmentioning

confidence: 99%

Immunocapture of dsRNA-bound proteins provides insight into tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector

Incarbone

Clavel

Monsion

et al. 2019

Preprint

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“…Another Gln (Q) residue enables the hydrolysis of this intermediate with assistance from water and ascorbate. Classical myrosinases (with QE catalytic residues) use ascorbate as a cofactor and proton donor to facilitate the release of bound glucose (Burmeister et al., 1997; Wittstock and Burow, 2010; Bhat and Vyas, 2019). In contrast, atypical myrosinases have two catalytic Glu residues (EE), which function as acid/base catalyst in the active site.…”

Section: Distinct Molecular and Biochemical Properties Of Myrosinasesmentioning

confidence: 99%

“…They do not require ascorbate. In addition, atypical myrosinases have two basic amino acid residues at different positions (+6 and +7) for glucosinolate binding compared to +0 position arginine residue of classical myrosinases (Wittstock and Burow, 2010; Nakano et al., 2017; Shirakawa and Hara-Nishimura, 2018; Bhat and Vyas, 2019). Classical myrosinases are glycosylated, activated by low concentrations of ascorbate, and accepted GLSs as the only substrates (Chen and Halkier, 1999; Chen and Andreasson, 2001).…”

Section: Distinct Molecular and Biochemical Properties Of Myrosinasesmentioning

confidence: 99%

Chemodiversity of the Glucosinolate-Myrosinase System at the Single Cell Type Resolution

et al. 2019

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Glucosinolates (GLSs) are a well-defined group of specialized metabolites, and like any other plant specialized metabolites, their presence does not directly affect the plant survival in terms of growth and development. However, specialized metabolites are essential to combat environmental stresses, such as pathogens and herbivores. GLSs naturally occur in many pungent plants in the order of Brassicales. To date, more than 200 different GLS structures have been characterized and their distribution differs from species to species. GLSs co-exist with classical and atypical myrosinases, which can hydrolyze GLS into an unstable aglycone thiohydroximate-O-sulfonate, which rearranges to produce different degradation products. GLSs, myrosinases, myrosinase interacting proteins, and GLS degradation products constitute the GLS-myrosinase (GM) system (“mustard oil bomb”). This review discusses the cellular and subcellular organization of the GM system, its chemodiversity, and functions in different cell types. Although there are many studies on the functions of GLSs and/or myrosinases at the tissue and whole plant levels, very few studies have focused on different single cell types. Single cell type studies will help to reveal specific functions that are missed at the tissue and organismal level. This review aims to highlight (1) recent progress in cellular and subcellular compartmentation of GLSs, myrosinases, and myrosinase interacting proteins; (2) molecular and biochemical diversity of GLSs and myrosinases; and (3) myrosinase interaction with its interacting proteins, and how it regulates the degradation of GLSs and thus the biological functions (e.g., plant defense against pathogens). Future prospects may include targeted approaches for engineering/breeding of plants and crops in the cell type-specific manner toward enhanced plant defense and nutrition.

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“…In addition, the mechanisms underlying the degradation of total GLS amount independent of TGG1 and TGG2 during early developmental stages remain to be clarified (Barth and Jander, 2006). Given that up-regulation of particular BGLUs has yet to be observed under these conditions, we should consider the post-translational regulation of myrosinase activities with regard to myrosinase-associated proteins or small molecule elicitors such as ascorbate (Wittstock et al, 2016a; Bhat and Vyas, 2019; Chen et al, 2019). Under non-disruptive conditions, the physiological functions of myrosinases, including TGGs, are probably controlled more strictly and dynamically than expected till now.…”

Section: Concluding Remarks and Future Perspectivementioning

confidence: 99%

“…Knowledge on the chemical diversity embedded in the GLS metabolism and on the molecular mechanisms underlying the GLS–myrosinase system is frequently updated. For example, energetical investigations have been made of GLS biosynthetic genes (Halkier, 2016; Barco and Clay, 2019), end products directed by specifier proteins (Wittstock et al, 2016a), tissue localization via GLS transporters (Jørgensen et al, 2015; Halkier, 2016), differences in GLS contents among species and accessions (Kliebenstein and Cacho, 2016), and proteins that interact with myrosinases to regulate their activity and stability (Bhat and Vyas, 2019; Chen et al, 2019). However, most of the insights that have been gained regarding the molecular basis and physiological importance of GLS breakdown are based on the intercellular and tissue damage-dependent GLS–myrosinase system.…”

Section: Introductionmentioning

confidence: 99%

Atypical Myrosinase as a Mediator of Glucosinolate Functions in Plants

Sugiyama

Hirai

2019

Front. Plant Sci.

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Glucosinolates (GLSs) are a well-known class of specialized plant metabolites, distributed mostly in the order Brassicales. A vast research field in basic and applied sciences has grown up around GLSs owing to their presence in important agricultural crops and the model plant Arabidopsis thaliana , and their broad range of bioactivities beneficial to human health. The major purpose of GLSs in plants has been considered their function as a chemical defense against predators. GLSs are physically separated from a specialized class of beta-thioglucosidases called myrosinases, at the tissue level or at the single-cell level. They are brought together as a consequence of tissue damage, primarily triggered by herbivores, and their interaction results in the release of toxic volatile chemicals including isothiocyanates. In addition, recent studies have suggested that plants may adopt other strategies independent of tissue disruption for initiating GLS breakdown to cope with certain biotic/abiotic stresses. This hypothesis has been further supported by the discovery of an atypical class of GLS-hydrolyzing enzymes possessing features that are distinct from those of the classical myrosinases. Nevertheless, there is only little information on the physiological importance of atypical myrosinases. In this review, we focus on the broad diversity of the beta-glucosidase subclasses containing known atypical myrosinases in A. thaliana to discuss the hypothesis that numerous members of these subclasses can hydrolyze GLSs to regulate their diverse functions in plants. Also, the increasingly broadening functional repertoires of known atypical/classical myrosinases are described with reference to recent findings. Assessment of independent insights gained from A. thaliana with respect to (1) the phenotype of mutants lacking genes in the GLS metabolic/breakdown pathways, (2) fluctuation in GLS contents/metabolism under specific conditions, and (3) the response of plants to exogenous GLSs or their hydrolytic products, will enable us to reconsider the physiological importance of GLS breakdown in particular situations, which is likely to be regulated by specific beta-glucosidases.

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Myrosinase: insights on structural, catalytic, regulatory, and environmental interactions

Cited by 77 publications

References 135 publications

Immunocapture of dsRNA-bound proteins provides insight into tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector

Immunocapture of dsRNA-bound proteins provides insight into tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector

Chemodiversity of the Glucosinolate-Myrosinase System at the Single Cell Type Resolution

Atypical Myrosinase as a Mediator of Glucosinolate Functions in Plants

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