Insights into the transcriptomic response of the plant engineering bacterium Ensifer adhaerens OV14 during transformation

Evelyn Zuniga-Soto,Ewen Mullins,Fiona M Doohan,David A Fitzpatrick

doi:10.1038/s41598-019-44648-8

Abstract

The ability to engineer plant genomes has been primarily driven by the soil bacterium Agrobacterium tumefaciens but recently the potential of alternative rhizobia such as Rhizobium etli and Ensifer adhaerens OV14, the latter of which supports Ensifer Mediated Transformation (EMT) has been reported. Surprisingly, a knowledge deficit exists in regards to understanding the whole genome processes underway in plant transforming bacteria, irrespective of the species. To begin to address the issue, we undertook a temporal RNAseq-based profiling study of E. adhaerens OV14 in the presence/absence of Arabidopsis thaliana tissues. Following co-cultivation with root tissues, 2333 differentially expressed genes (DEGs) were noted. Meta-analysis of the RNAseq data sets identified a clear shift from plasmid-derived gene expression to chromosomal-based transcription within the early stages of bacterium-plant co-cultivation. During this time, the number of differentially expressed prokaryotic genes increased steadily out to 7 days co-cultivation, a time at which optimum rates of transformation were observed. Gene ontology evaluations indicated a role for both chromosomal and plasmid-based gene families linked specifically with quorum sensing, flagellin production and biofilm formation in the process of EMT. Transcriptional evaluation of vir genes, housed on the pCAMBIA 5105 plasmid in E. adhaerens OV14 confirmed the ability of E. adhaerens OV14 to perceive and activate its transcriptome in response to the presence of 200 µM of acetosyringone. Significantly, this is the first study to characterise the whole transcriptomic response of a plant engineering bacterium in the presence of plant tissues and provides a novel insight into prokaryotic genetic processes that support T-DNA transfer.

Highlights

The soil-borne plant pathogen Agrobacterium tumefaciens underpins Agrobacterium-mediated transformation (AMT) as the primary platform for the generation of engineered crop varieties, which cover ~189.8 million hectares globally[1] (ISAAA, 2018)
Recorded as the percentage of blue foci visible on treated A. thaliana roots, the first signs of transient transformation were noted by day 1 (D1) (3.9%), which increased significantly (P < 0.05) through to day 3 (D3) (33.7%) and day 5 (D5) (87.2%), decreasing slightly to 84.2% at 7 days post-treatment (Fig. 1)
Thirty-six sequencing libraries/samples were created with raw reads ranging from 10 to 12 million per sample and a total of 406.9 million reads were recorded across the experiment

Summary

Introduction

The soil-borne plant pathogen Agrobacterium tumefaciens underpins Agrobacterium-mediated transformation (AMT) as the primary platform for the generation of engineered crop varieties, which cover ~189.8 million hectares globally[1] (ISAAA, 2018). 7-day time course as measured via histochemical GUS staining. Genes that are deemed non-essential but exert a positive influence on the ability to transform a plant genome, while absent from the S. meliloti strain 1021 genome were found present in the genome of E. adhaerens OV1412. When E. adhaerens OV14 carries a Ti equivalent-plasmid (e.g. pCAMBIA 5105) containing a complement of vir genes it acquires the ability to accommodate, transfer and successfully integrate stable copies of T-DNA. The processes supporting this remain unknown, as does the rate of vir gene transcription relative to whole genome activity and the role (if any) of individual E. adhaerens OV14 replicons in supporting EMT

Methods

Results

Conclusion