Abstract
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 γ-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 ∼30% 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 γ-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.
Highlights
Numerous bacterial pathogens of plants and animals inject virulence proteins, so-called effectors, directly into the host cell cytoplasm through specialized secretion apparatuses, such as the type III secretion system (T3SS).2 The translocated T3SS effector proteins manipulate diverse host cellular processes, including cytoskeletal reorganization, signal transduction, gene expression, vesicular trafficking, autophagy, and DNA replication, to promote infection and cause disease [1, 2]
The T3SS effector proteins exhibit various molecular activities that generally allow bacteria to escape from host immune systems and break down barriers for pathogen growth and dissemination
We revealed that a ChaC domain-containing effector protein, RipAY, from the plant pathogenic bacterium R. solanacearum modulates the abundance of host intracellular glutathione by acting as a eukaryotic thioredoxin-dependent GGCT
Summary
T3SS, type III secretion system; GGCT, ␥-glutamyl cyclotransferase; SD, synthetic dextrose; SGal, synthetic galactose; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Trx, thioredoxin. RipAY, which is one of the R. solanacearum core effectors, has previously been shown experimentally to be an effector injected into host plant cells via T3SS [17], but the molecular function of this effector has yet to be characterized. It has been reported that yeast and mammalian ChaC domain-containing proteins exhibit ␥-glutamyl cyclotransferase (GGCT) activity to degrade glutathione [18]. We could detect robust GGCT activity when RipAY was activated by yeast or plant thioredoxins Both glutathione and thioredoxins are important for maintaining cellular redox homeostasis and indispensable for proper activation of plant innate immunity during pathogen infection [19, 20]. Our data provide new evidence that R. solanacearum perturbs the host redox environment to allow bacterial infection
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