Ralstonia solanacearum type III effector RipV2 encoding a novel E3 ubiquitin ligase (NEL) is required for full virulence by suppressing plant PAMP-triggered immunity.

Ralstonia solanacearum causes bacterial wilt disease in a broad range of plants, primarily through type Ⅲ secreted effectors. However, the R. solanacearum effectors promoting susceptibility in host plants remain limited. In this study, we determined that the R. solanacearum effector RipV2 functions as a novel E3 ubiquitin ligase (NEL). RipV2 was observed to be locali in the plasma membrane after translocatio into plant cells. Transient expression of RipV2 in Nicotiana benthamiana could induce cell death and suppress the flg22-induced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, mediating such effects as attenuation of the expression of several PTI-related genes and ROS bursts. Furthermore, we demonstrated that the conserved catalytic residue is highly important for RipV2. Transient expression of the E3 ubiquitin ligase catalytic mutant RipV2 C403A alleviated the PTI suppression ability and cell death induction, indicating that RipV2 requires its E3 ubiquitin ligase activity for its role in plant-microbe interactions. More importantly, mutation of RipV2 in R. solanacearum reduces the virulence of R. solanacearum on potato. In conclusion, we identified a NEL effector that is required for full virulence of R. solanacearum by suppressing plant PTI.

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

UNLABELLED: The 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. IMPORTANCE: Ralstonia solanacearum is the causal agent of bacterial wilt disease of plants. This pathogen injects virulence effector proteins into host cells to suppress disease resistance responses of plants. In this article, we report a biochemical activity of R. solanacearum effector protein RipAY. RipAY can degrade GSH, a tripeptide that plays important roles in the plant immune system, with its γ-glutamyl cyclotransferase activity. The high GSH degradation activity of RipAY is considered to be a good weapon for this bacterium to suppress plant immunity. However, GSH also plays important roles in bacterial tolerance to various stresses and growth. Interestingly, RipAY has an excellent safety mechanism to prevent unwanted firing of its enzyme activity in bacterial cells because RipAY is specifically activated by host eukaryotic thioredoxins. This study also reveals a novel host plant protein acting as a molecular switch for effector activation.

Deciphering the route of Ralstonia solanacearum colonization in Arabidopsis thaliana roots during a compatible interaction: focus at the plant cell wall.

The compatible interaction between the model plant, Arabidopsis thaliana, and the GMI1000 strain of the phytopathogenic bacterium, Ralstonia solanacearum, was investigated in an in vitro pathosystem. We describe the progression of the bacteria in the root from penetration at the root surface to the xylem vessels and the cell type-specific, cell wall-associated modifications that accompanies bacterial colonization. Within 6 days post inoculation, R. solanacearum provoked a rapid plasmolysis of the epidermal, cortical, and endodermal cells, including those not directly in contact with the bacteria. Plasmolysis was accompanied by a global degradation of pectic homogalacturonanes as shown by the loss of JIM7 and JIM5 antibody signal in the cell wall of these cell types. As indicated by immunolabeling with Rsol-I antibodies that specifically recognize R. solanacearum, the bacteria progresses through the root in a highly directed, centripetal manner to the xylem poles, without extensive multiplication in the intercellular spaces along its path. Entry into the vascular cylinder was facilitated by cell collapse of the two pericycle cells located at the xylem poles. Once the bacteria reached the xylem vessels, they multiplied abundantly and moved from vessel to vessel by digesting the pit membrane between adjacent vessels. The degradation of the secondary walls of xylem vessels was not a prerequisite for vessel colonization as LM10 antibodies strongly labeled xylem cell walls, even at very late stages in disease development. Finally, the capacity of R. solanacearum to specifically degrade certain cell wall components and not others could be correlated with the arsenal of cell wall hydrolytic enzymes identified in the bacterial genome.

An HrpB-dependent but type III-independent extracellular aspartic protease is a virulence factor of Ralstonia solanacearum.

The host specificity of Ralstonia solanacearum, the causal organism of bacterial wilt on many solanaceous crops, is poorly understood. To identify a gene conferring host specificity of the bacterium, SL341 (virulent to hot pepper but avirulent to potato) and SL2029 (virulent to potato but avirulent to hot pepper) were chosen as representative strains. We identified a gene, rsa1, from SL2029 that confers avirulence to SL341 in hot pepper. The rsa1 gene encoding an 11.8-kDa protein possessed the perfect consensus hrp(II) box motif upstream of the gene. Although the expression of rsa1 was activated by HrpB, a transcriptional activator for hrp gene expression, Rsa1 protein was secreted in an Hrp type III secretion-independent manner. Rsa1 exhibited weak homology with an aspartic protease, cathepsin D, and possessed protease activity. Two specific aspartic protease inhibitors, pepstatin A and diazoacetyl-d,l-norleucine methyl ester, inhibited the protease activity of Rsa1. Substitution of two aspartic acid residues with alanine at positions 54 and 59 abolished protease activity. The SL2029 rsa1 mutant was much less virulent than the wild-type strain, but did not induce disease symptoms in hot pepper. These data indicate that Rsa1 is an extracellular aspartic protease and plays an important role for the virulence of SL2029 in potato.

Endo- and exopolygalacturonases of Ralstonia solanacearum are inhibited by polygalacturonase-inhibiting protein (PGIP) activity in tomato stem extracts.

Polygalacturonases (PGs) of wild-type and non-virulent phenotype conversion mutant (PC) strains of Ralstonia solanacearum were compared by investigating their activities and their inhibition by polygalacturonase-inhibiting proteins (PGIPs) from tomato stems. In cultures of wild-type strain ToUdk2, slimy (s), retarded slimy (rs) and non-slimy (ns) colonies appeared. The conversion of the 's' into the 'rs' colony form coincided with the beginning of PG production. PG activity of the PC strain increased about 5 h earlier (at 6 hpi), and was up to 35 times higher in media supplemented with two different tomato stem extracts or polygalacturonic acid, compared to the wild-type at 6 hpi, and generally 4-8 times higher across test media and time. By hydrophobic interaction chromatography (HIC), fluorophor-assisted carbohydrate-polyacrylamid-gel electrophoresis (FACE-PAGE) and mass spectrometry analyses, endo-PG PehA, exo-PGs PehB and PehC were identified. PGs of the PC mutant consisted mainly of endo-PG. The increased PG production after supplementing the medium with tomato cell wall extract was reflected by a higher activity of exo-PGs for both strains. Total PGs (endo-PG and exo-PGs) activities were inhibited by PGIPs of tomato stem extracts. PGIP activity was concentration dependent, constitutively present, and not related to resistance nor susceptibility of tomato recombinant inbred lines to R. solanacearum. The proteinaceous character of the inhibiting component was inferred from ammonium sulphate precipitation. For the first time a plant PGIP activity against a bacterial pathogen is reported. Observations support that endo- and exo-PG synthesis is governed by a sensitive regulatory network, which, in interaction with PGIP and cell wall degradation products, leads to generation or avoidance of elicitor-active oligomers, and, thus, may contribute to the development of the compatible or incompatible interaction.