Ralstonia solanacearum Type III Effector RipAY Is a Glutathione-Degrading Enzyme That Is Activated by Plant Cytosolic Thioredoxins and Suppresses Plant Immunity.

Takafumi Mukaihara,Tadashi Hatanaka,Kenji Oda,Masahito Nakano,Frederick M Ausubel

doi:10.1128/mbio.00359-16

Abstract

ABSTRACTThe plant pathogen Ralstonia solanacearum uses a large repertoire of type III effector proteins to succeed in infection. To clarify the function of effector proteins in host eukaryote cells, we expressed effectors in yeast cells and identified seven effector proteins that interfere with yeast growth. One of the effector proteins, RipAY, was found to share homology with the ChaC family proteins that function as γ-glutamyl cyclotransferases, which degrade glutathione (GSH), a tripeptide that plays important roles in the plant immune system. RipAY significantly inhibited yeast growth and simultaneously induced rapid GSH depletion when expressed in yeast cells. The in vitro GSH degradation activity of RipAY is specifically activated by eukaryotic factors in the yeast and plant extracts. Biochemical purification of the yeast protein identified that RipAY is activated by thioredoxin TRX2. On the other hand, RipAY was not activated by bacterial thioredoxins. Interestingly, RipAY was activated by plant h-type thioredoxins that exist in large amounts in the plant cytosol, but not by chloroplastic m-, f-, x-, y- and z-type thioredoxins, in a thiol-independent manner. The transient expression of RipAY decreased the GSH level in plant cells and affected the flg22-triggered production of reactive oxygen species (ROS) and expression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes in Nicotiana benthamiana leaves. These results indicate that RipAY is activated by host cytosolic thioredoxins and degrades GSH specifically in plant cells to suppress plant immunity.

Highlights

Ralstonia solanacearum is a Gram-negative soilborne bacterium that causes bacterial wilt in more than 200 plant species [1]
Identification of R. solanacearum type III effector proteins that interfere with yeast growth
We demonstrated that R. solanacearum type III effector RipAY is a GSH-degrading enzyme

Summary

Introduction

Ralstonia solanacearum is a Gram-negative soilborne bacterium that causes bacterial wilt in more than 200 plant species [1]. This pathogen enters the plant roots through natural openings and wound sites and proliferates in the vascular system. The bacterium secretes large amounts of extracellular polysaccharides (EPS) and cell-wall-degrading enzymes inside plants, which block water transport and destroy the vascular system, causing severe wilting and death of infected plants [2, 3]. To counter effector-mediated suppression of PTI, plants have evolved to have another system, called effectortriggered immunity (ETI), that recognizes true pathogens by directly or indirectly detecting pathogen-secreted effectors through the disease-resistance (R) proteins inside plant cells [8]. Pseudomonas syringae pv. tomato DC3000 type III effector HopQ1 suppresses PTI by activating the production of cytokinin, probably functioning as a cytokinin-activating enzyme through its nucleoside hydrolase activity [17]

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Results

Conclusion