Defining the cytosolic pathway of glutathione degradation in <i>Arabidopsis thaliana</i>: role of the ChaC/GCG family of γ-glutamyl cyclotransferases as glutathione-degrading enzymes and AtLAP1 as the Cys-Gly peptidase

Kumar, Sudhir; Kaur, Amandeep; Chattopadhyay, Banani; Bachhawat, Anand K.

doi:10.1042/bj20141154

Cited by 57 publications

(77 citation statements)

References 45 publications

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“…However, a search in the National Center for Biotechnology Information (NCBI) conserved domain database (Marchler‐Bauer et al ., ) indicated that the RipAY central region (amino acids 126–252; E value = 1.65e‐05; Fig. S6) is similar to proteins from the GGCT‐like superfamily, associated with the degradation of glutathione in a multitude of organisms, including plants (Kumar et al ., ; Oakley et al ., ; Paulose et al ., ). Therefore, we decided to test whether RipAY has an impact on the glutathione content of plant cells.…”

Section: Resultsmentioning

confidence: 98%

The Ralstonia solanacearum type III effector RipAY targets plant redox regulators to suppress immune responses

Sang

Wang

Hóng

et al. 2016

Molecular Plant Pathology

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The subversion of plant cellular functions is essential for bacterial pathogens to proliferate in host plants and cause disease. Most bacterial plant pathogens employ a type III secretion system to inject type III effector (T3E) proteins inside plant cells, where they contribute to the pathogen-induced alteration of plant physiology. In this work, we found that the Ralstonia solanacearum T3E RipAY suppresses plant immune responses triggered by bacterial elicitors and by the phytohormone salicylic acid. Further biochemical analysis indicated that RipAY associates in planta with thioredoxins from Nicotiana benthamiana and Arabidopsis. Interestingly, RipAY displays γ-glutamyl cyclotransferase (GGCT) activity to degrade glutathione in plant cells, which is required for the reported suppression of immune responses. Given the importance of thioredoxins and glutathione as major redox regulators in eukaryotic cells, RipAY activity may constitute a novel and powerful virulence strategy employed by R. solanacearum to suppress immune responses and potentially alter general redox signalling in host cells.

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Section: Resultsmentioning

confidence: 98%

The Ralstonia solanacearum type III effector RipAY targets plant redox regulators to suppress immune responses

Sang

Wang

Hóng

et al. 2016

Molecular Plant Pathology

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“…In fact, our enzyme kinetic analysis revealed that the k cat value of Trx-h5-activated RipAY was much higher than that of a S. cerevisiae class I ChaC protein, Gcg1 (160-fold), although both activated RipAY and Gcg1 show a similar K m value (Table 4). Recently, Kumar et al (29) reported a detailed enzyme kinetic analysis of Arabidopsis ChaC orthologues, AtGGCT2;2 and AtGGCT2;3.…”

Section: Discussionmentioning

confidence: 99%

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins

Fujiwara

Kawazoe

Ohnishi

et al. 2016

Journal of Biological Chemistry

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The plant pathogenic bacterium Ralstonia solanacearum injects more than 70 effector proteins (virulence factors) into the host plant cells via the needle-like structure of a type III secretion system. The type III secretion system effector proteins manipulate host regulatory networks to suppress defense responses with diverse molecular activities. Uncovering the molecular function of these effectors is essential for a mechanistic understanding of R. solanacearum pathogenicity. However, few of the effectors from R. solanacearum have been functionally characterized, and their plant targets remain largely unknown. Here, we show that the ChaC domain-containing effector RipAY/RSp1022 from R. solanacearum exhibits ␥-glutamyl cyclotransferase (GGCT) activity to degrade the major intracellular redox buffer, glutathione. Heterologous expression of RipAY, but not other ChaC family proteins conserved in various organisms, caused growth inhibition of yeast Saccharomyces cerevisiae, and the intracellular glutathione level was decreased to ϳ30% of the normal level following expression of RipAY in yeast. Although active site mutants of GGCT activity were non-toxic, the addition of glutathione did not reverse the toxicity, suggesting that the toxicity might be a consequence of activity against other ␥-glutamyl compounds. Intriguingly, RipAY protein purified from a bacterial expression system did not exhibit any GGCT activity, whereas it exhibited robust GGCT activity upon its interaction with eukaryotic thioredoxins, which are important for intracellular redox homeostasis during bacterial infection in plants. Our results suggest that RipAY has evolved to sense the host intracellular redox environment, which triggers its enzymatic activity to create a favorable environment for R. solanacearum infection.

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“…Although several different GSH degradation enzymes are known in plants (Grzam et al ., ; Martin et al ., ; Ohkama‐Ohtsu et al ., ,b) recent work has shown that the major route in Arabidopsis thaliana is via γ‐glutamylcyclotransferase (GGCT) (Ohkama‐Ohtsu et al ., ). GGCT hydrolyzes GSH (Kumar et al ., ) releasing Cys–Gly and 5‐oxoproline (5‐OP), a cyclized form of Glu. 5‐OP is converted to Glu by 5‐oxoprolinase, an ATP‐dependent enzyme (Ohkama‐Ohtsu et al ., ).…”

Section: Introductionmentioning

confidence: 99%

“…5‐OP is converted to Glu by 5‐oxoprolinase, an ATP‐dependent enzyme (Ohkama‐Ohtsu et al ., ). An aminopeptidase cleaves Cys–Gly (Kumar et al ., ). Arabidopsis contains three GGCT genes (Paulose et al ., ) and the encoded enzymes specifically cleave GSH (Kumar et al ., ).…”

Section: Introductionmentioning

confidence: 99%

Arabidopsis γ‐glutamylcyclotransferase affects glutathione content and root system architecture during sulfur starvation

et al. 2018

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c-Glutamylcyclotransferase initiates glutathione degradation to component amino acids L-glutamate, L-cysteine and L-glycine. The enzyme is encoded by three genes in Arabidopsis thaliana, one of which (GGCT2;1) is transcriptionally upregulated by starvation for the essential macronutrient sulfur (S). Regulation by S-starvation suggests that GGCT2;1 mobilizes L-cysteine from glutathione when there is insufficient sulfate for de novo L-cysteine synthesis.The response of wild-type seedlings to S-starvation was compared to ggct2;1 null mutants. S-starvation causes glutathione depletion in S-starved wild-type seedlings, but higher glutathione is maintained in the primary root tip than in other seedling tissues. Although GGCT2;1 is induced throughout seedlings, its expression is concentrated in the primary root tip where it activates the c-glutamyl cycle. S-starved wild-type plants also produce longer primary roots, and lateral root growth is suppressed. While glutathione is also rapidly depleted in ggct2;1 null seedlings, much higher glutathione is maintained in the primary root tip compared to the wild-type. S-starved ggct2;1 primary roots grow longer than the wild-type, and lateral root growth is not suppressed.These results point to a role for GGCT2;1 in S-starvation-response changes to root system architecture through activity of the c-glutamyl cycle in the primary root tip. L-Cysteine mobilization from glutathione is not solely a function of GGCT2;1.

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Defining the cytosolic pathway of glutathione degradation in Arabidopsis thaliana: role of the ChaC/GCG family of γ-glutamyl cyclotransferases as glutathione-degrading enzymes and AtLAP1 as the Cys-Gly peptidase

Cited by 57 publications

References 45 publications

The Ralstonia solanacearum type III effector RipAY targets plant redox regulators to suppress immune responses

The Ralstonia solanacearum type III effector RipAY targets plant redox regulators to suppress immune responses

RipAY, a Plant Pathogen Effector Protein, Exhibits Robust γ-Glutamyl Cyclotransferase Activity When Stimulated by Eukaryotic Thioredoxins

Arabidopsis γ‐glutamylcyclotransferase affects glutathione content and root system architecture during sulfur starvation

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