Plant GABA:proline ratio modulates dissemination of the virulence Ti plasmid within the Agrobacterium tumefaciens hosted population

ABSTRACT

Accumulation of amino acids is a common plant response to several biotic and abiotic stresses, even if the roles of these accumulations remain often poorly understood. In a recent study we measured the levels of different amino acids in tumors of Arabidopsis thaliana induced by the phytopathogen Agrobacterium tumefaciens and correlated these data with changes of gene expressions in both organisms. This led to the demonstration that the non-protein amino acid GABA plays an important role for the adaptation of the bacteria to the plant tumor environment, and especially in the control of the virulent Ti plasmid dissemination. Here we present a model that describes how different GABA:proline ratios in the A. thaliana host may have different impacts on the conjugation of A. tumefaciens Ti plasmid, and advance the view that the amino acid metabolism of plant hosts could be critical for the propagation of the virulence genes in A. tumefaciens populations.

The virulence factors of the bacterial phytopathogen A. tumefaciens are encoded by the conjugative Ti plasmid, the dissemination of which is controlled by a quorum-sensing (QS) signaling based on the synthesis and perception of 3-oxo-octanoylhomoserine lactone (OC8HSL) molecules.^1-2 Remarkably the lactonase BlcC encoded by the companion At plasmid of A. tumefaciens C58 is able to cleave the OC8HSL molecules and to negatively affect QS in vitro.³ The blcC gene belongs to the blcABC operon involved in the conversion of gamma-butyrolactone (GBL) to succinate, through gamma-hydroxybutyrate (GHB) and succinic semialdehyde (SSA) intermediates.^4-5 GABA is converted by still unknown GABA-transminase(s) into SSA. Expression of blcABC is tightly controlled by the transcriptional repressor BlcR.³ GHB and SSA can release the repressing activity of BlcR and therefore induce blcABC expression.⁵

In plants GABA and SSA are produced in mitochondria from glutamate in the course of the GABA-shunt metabolic pathway, while GHB is obtained from SSA through glyoxylate reductase activities in the cytosol and the chloroplast. GABA can also be produced by degradation of polyamines. All three SSA, GHB and GABA metabolites were shown to accumulate in different stress conditions.⁶

In a recent study we addressed the question whether the GABA produced by plants can induce expression of the blcC gene in the hosted A. tumefaciens population.⁷ Here we present the general view that dissemination of Ti plasmids is dependent not only on the accumulation in the plant tumors of BlcC-inducers (like GABA), but also on the capacities of these inducers to be taken up by the colonizing bacteria. This view implies that different plant species with different metabolic responses could offer a high or low spread of the A. tumefaciens virulence traits.

GABA transport in A. thaliana and A. tumefaciens

There are 2 characterized GABA ABC-transporters in A. tumefaciens. The first one (Bra) is not specific to GABA and can also import a large variety of amino acids such as proline, alanine, glycine, serine, valine and threonine, which may act as antagonists of the GABA importation.^8-10 The second one (Gts) is GABA specific but is transcriptionally repressed in cell cultures.¹¹ Interestingly an upregulation of the Gts genes was recently observed in A. tumefaciens infection sites and induced tumors, suggesting that interactions with plants can activate this GABA transport system.⁷

In A. thaliana, there are 4 GABA transporters acting at the level of the cell membrane. AtPROT1, 2 and 3 display distinct profiles of expression in tumors, suggesting an active GABA flux throughout the neoplastic organ.⁷ However, these transporters have higher affinities for proline and betaine than for GABA.¹² By contrast, the AtGAT1 transporter is GABA-specific¹³ and its expression seems restricted to specific areas of the tumors.⁷

Both GABA and GABA-transport antagonists accumulate in A. thaliana tumors

GABA accumulates at high level in tumors comparatively to uninfected control tissues (about 200 pmole/mg FW; 10-fold increase). Given the respective biomass of plant and bacterial cells in tumors, this accumulation is most likely due to plant metabolism.¹⁴ However the amino acids known to antagonize GABA transport in A. thaliana (proline) and A. tumefaciens (proline, alanine, glycine, serine, valine and threonine) also abundantly accumulate in tumors, questioning the capacity of GABA to induce blcC expression. In particular GABA:proline ratio is 3:1 in uninfected tissues and goes down to 1:4 in plant tumor.⁷

A high GABA: proline ratio in plant host induces the expression of the quorum-sensing signal-degrading lactonase BlcC in A. tumefaciens

Originally tumors in the A. thaliana her1 background which is deficient for the GABA-Transaminase display a high GABA:proline ratio (5:1) without apparent other modifications of their metabolic profiles; and analysis of gene expression levels revealed an upregulation of the blcC gene in A. tumefaciens populations coming from these tumors comparatively to WT tumors. Moreover conjugation assays showed that efficiency of Ti plasmid transfers decreased in her1 tumors, demonstrating thereby that increasing plant GABA:proline ratios can lead to an inhibition of the Ti plasmid dissemination.⁷

What about other BlcC-inducers?

Unlike GHB for which no accumulation can be detected, SSA is about 10 times more abundant in A. thaliana Col-0 tumors than in non-tumorigenic tissues.⁷ It is therefore possible that SSA also interferes in vivo with A. tumefaciens blcC expression. However levels of SSA accumulation remain modest comparatively to GABA (about 20 pmole/mg FW vs. about 200 pmole/mg FW). Besides no information about transport of SSA in plants or in A. tumefaciens is available so far.

Signaling model

Based on the results obtained in A. thaliana, we propose the general model of Fig. 1. This one depicts how the amino acid metabolism of different plant hosts, and especially the GABA:proline ratios, can exert a negative effect on the dissemination of the virulence genes of A. tumefaciens, through the QS-signal degrading activity of the bacterial BlcC lactonase. Since the maintenance costs of Ti-plasmid are high and might lead to a loss of functional Ti plasmids in A. tumefaciens populations colonizing plant tumors,¹⁵ the impacts of this process appears ecologically essential for the persistence of A. tumefaciens virulence genes. Further investigation about the tumor metabolomes of different plant hosts, and also at different time points of the interaction, should shed additional light on this point. This will emphasize the paradigmatic role of A. tumefaciens-host plant interaction as a model for illustrating quorum-sensing interference in natura.¹⁶

Figure 1.

Glutamate and polyamines are precursors of GABA, SSA and GHB which are synthetized in different plant cell compartments. Glutamate is also a precursor of proline, which acts as an antagonist of the importation of GABA in A. tumefaciens via the Bra ABC-transporter. The A. tumefaciens ABC-transporter Gts is selective for GABA, its activity is not affected in the presence of proline. In A. tumefaciens, GABA is converted into SSA by unknown GABA-T; GBL, GHB and SSA are converted into succinate via enzymes encoded by the blcABC operon. The lactonase BlcC also inactivate the QS-signal OC8HSL. The repressing activity of the transcriptional factor BlcR on the blcABC operon is released in the presence of GHB or SSA. The complex between the transcriptional factor TraR and OC8HSL activates tra and trb operons which operate the horizontal transfer of the Ti plasmid via a dedicated T4SS. The proposed model argued that variation of the GABA:proline ratio in plant tumor controls the expression of the lactonase BlcC in A. tumefaciens, hence the degradation of the QS signal OC8HSL and downregulation of the Ti plasmid transfer between bacterial cells. Legend: GABA, gamma-aminobutyric acid; SSA, succinic semialdehyde; GHB, gamma-hydroxybutyric acid; GBL, gamma-butyrolactone; OC8HSL, 3-oxo-octanoylhomoserine lactone; OC8HS, 3-oxo-octanoylhomoserine; ProT1-3, Proline/GABA transporters 1-3; GAT1, GABA transporter 1; GAD, glutamate decarboxylase; PAO, polyamine oxidase; DAO, diamine oxidase; ALDH, aldehyde dehydrogenase; GABA-T, GABA transaminase; GABP, GABA permease; SSADH, SSA dehydrogenase; GLYR, glyoxylate/succinic semialdehyde reductase; T4SS, Type 4 secretion system. Dashed arrows indicate exchange of metabolites via unknown transporters.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.