Integrative and deconvolution omics approaches to uncover the Agrobacterium tumefaciens lifestyle in plant tumors

ABSTRACT

Agrobacterium tumefaciens is a plant pathogen which provokes galls on roots and stems (crown-gall disease) and colonizes them. Two approaches combining omics were used to decipher the lifestyle of A. tumefaciens in plant tumors: an integrative approach when omics were used on A. tumefaciens cells collected from plant tumors, a deconvolution approach when omics were used on A. tumefaciens cells exploiting a single tumor metabolite in pure culture assay. This addendum highlights some recent results on the biotroph lifestyle of A. tumefaciens in plant tumors.

Agrobacterium tumefaciens is a soil bacterium of which a subset of its population is virulent because of the carriage of a Ti plasmid coding for the main virulence factors.¹ The virulent A. tumefaciens provokes crown-gall disease in a wide range of plant hosts. Symptoms are root and stem galls (plant tumors) that are caused by transfer of the T-DNA from the Ti plasmid to the plant cells. A. tumefaciens colonizes the surface and intercellular space of the plant tumor tissues. Hence, from an ecological point of view, A. tumefaciens is a soil bacterium that is, conditionally (presence of the Ti plasmid and a compatible host), able to construct and exploit a novel ecological niche: the plant tumors.² The virulence genes (contributing to tumor niche construction) have been – and are still – studied extensively.^3-5 In contrast, with the exception of opine pathways, little is known about the fitness genes contributing to tumor niche exploitation.^6-8 These genetic and functional traits contribute to A. tumefaciens persistence in plant hosts.

Genes coding for opine synthesis and assimilation are carried on the Ti plasmid, along with most of the virulence genes. Opine synthesis genes are located within the T-DNA, while the genes coding for opine exploitation as a nutrient resource or an activator of Ti plasmid conjugation are located outside the T-DNA. Hence, plant tumor tissues, which express the T-DNA, accumulate opines which are exploited by the virulent A. tumefaciens that colonizes the tumors. A mutant defective in the assimilation of the opines nopaline or octopine was less competitive for colonizing plant tumors when this mutant was challenged with a wild-type strain.^6,7 By contrast, such mutant was still able to induce symptoms (tumor weight) similar as the wild type. These phenotypes clearly distinguished virulence genes, which are required for tumor niche construction, and tumor fitness genes, which are required for tumor niche exploitation.

In two recent works,^9,10 we deployed omics in integrative and deconvolution approaches for identifying tumor fitness genes in A. tumefaciens (Figure 1). Integrative approach consisted in omics analyses directly performed on tumor niche samples, while deconvolution approach consisted in omics analyses which were performed in vitro for testing a particular resource or parameter of the tumor niche. In an integrative analysis,⁹ we used transcriptomics for proposing an overview of the A. tumefaciens lifestyle when it colonizes and exploits the tumor niche constructed on Arabidospsis thaliana as compared to free-living condition. Transcriptome analysis revealed a differential expression of some master regulators of cell behavior (Hfq, CtrA, DivK, and PleD), surface and host-interacting components (O-antigen, succinoglucan, curdlan, Ef-Tu, nitric oxide, etc.), metabolic pathways for an exploitation of wide variety of host metabolites (opines, sugars, amino and organic acids, etc.), as well as a different distribution of the upregulated and downregulated genes according to the four replicons (circular and linear chromosomes and At and Ti plasmids). Mutants of some upregulated genes (atu5061, atu5245, atu5344, atu5414, atu5502, and atu5503) in plant tumors, which are encoded by the At plasmid, were constructed and challenged with the wild-type strain in plant tumors. The fitness of these defective mutants was improved, revealing that expression of some At plasmid genes was costly for A. tumefaciens in the tumor niche.⁹ By contrast, mutants of some other loci of the At plasmid such as blc were impaired in their fitness in plant tumors.^10,11 We could conclude that a trade-off between different loci drives the contribution of the At plasmid to A. tumefaciens fitness in plant tumors.

Figure 1.

Integrative and deconvolution approaches for studying tumor niche exploitation by Agrobacterium tumefaciens. See text for a description of the figure.

Using a deconvolution approach,¹⁰ we combined omics for identifying fitness genes. In the first step, the plant tumor metabolites were identified and quantified: this step contributed to describe the tumor niche resources that could be potentially exploited by Agrobacterium. In the second step, transposon-sequencing (Tn-seq) and transcriptomics were used for characterizing the fitness and/or expressed genes when A. tumefaciens grows on a defined metabolite. This combined approach pinpointed the genes pgi, kdgA, pycA, and cisY when A. tumefaciens grew on sucrose, and blcAB, pckA, eno, and gpsA when A. tumefaciens grew on gamma-hydroxybutyrate. Especially, Tn-seq pointed the genes that could not be compensated by functional redundancy when A. tumefaciens exploited a considered resource. In the third step, defective mutants of the genes blc, pckA, and pycA were constructed by reverse genetics. All these genes (blc, pckA, and pycA) were shown to be involved in virulence and/or fitness of A. tumefaciens in plant tumors.

In addition to metabolomics, transcriptomics, and Tn-seq, some other omics such as proteomics could be combined. The assembly of data collected from both the deconvolution and integrative approaches will contribute to decipher the tumor lifestyle of A. tumefaciens.

Funding Statement

This work was supported by the CNRS under Grant I2BC-SE2019; Fundación Ramón Areces under Grant XXIX-2017; and LabEx Saclay Plant Sciences-SPS under Grant ANR-10-LABX-0040-SPS.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.