The Rapid Methylation of T-DNAs Upon Agrobacterium Inoculation in Plant Leaves

Abstract

Agrobacterium tumefaciens has been foundational in the development of transgenic plants for both agricultural biotechnology and plant molecular research. However, the transformation efficiency and level of transgene expression obtained for any given construct can be highly variable. These inefficiencies often require screening of many lines to find one with consistent and heritable transgene expression. Transcriptional gene silencing is known to affect transgene expression, and is associated with DNA methylation, especially of cytosines in symmetric CG and CHG contexts. While the specificity, heritability and silencing-associated effects of DNA methylation of transgene sequences have been analyzed in many stably transformed plants, the methylation status of transgene sequences in the T-DNA during the transformation process has not been well-studied. Here we used agro-infiltration of the eGFP reporter gene in Nicotiana benthamiana leaves driven by either an AtEF1α-A4 or a CaMV-35S promoter to study early T-DNA methylation patterns of these promoter sequences. The T-DNA was examined by amplicon sequencing following sodium bisulfite treatment using three different sequencing platforms: Sanger sequencing, Ion Torrent PGM, and the Illumina MiSeq. Rapid DNA methylation was detectable in each promoter region just 2–3 days post-infiltration and the levels continued to rapidly accumulate over the first week, then steadily up to 21 days later. Cytosines in an asymmetric context (CHH) were the most heavily and rapidly methylated. This suggests that early T-DNA methylation may be important in determining the epigenetic and transcriptional fate of integrated transgenes. The Illumina MiSeq platform was the most sensitive and robust way of detecting and following the methylation profiles of the T-DNA promoters. The utility of the methods was then used to show a subtle but significant difference in promoter methylation during intron-mediated enhancement. In addition, the method was able to detect an increase in promoter methylation when the eGFP reporter gene was targeted by siRNAs generated by co-infiltration of a hairpin RNAi construct.

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

“…Voucheret already in 1994 showed that transcriptional trans silencing could start quickly in developing seed, but complete inactivation might require few weeks [45]. After massive leaf infiltration with Agrobacterium, rapid methylation of T-DNA was detectable in the promoter region just 2–3 day post-infiltration and the levels continued to rapidly accumulate over the 1st week and then steadily up to 21 days [46]. In mitotically dividing cells, the maintenance methylation of newly synthesised DNA strands must be quick enough to ensure the replication of the epigenetic information between the two subsequent S-phases of the cell cycle.…”

Genome-wide identification and characterization of the GDP-L-galactose phosphorylase gene family in bread wheat

“…Cloning of the correct gene family member and gene orientation was confirmed by Sanger sequencing at the Australian Genome Research Facility Ltd. A. tumefaciens (GV3101 pMP90) containing the constructs of interest (except TaGGP2 -D) were co-infiltrated with A. tumefaciens containing a P19 construct [88] to prevent post-transcriptional gene silencing into the younger leaves of 6-week-old N. benthamiana plants (LAB strain) grown in a plant growth room (16 h daylength, 24 °C) [89]. Infiltration conditions were conducted as previously described [90]. Infiltration with A. tumefaciens containing the P19 construct alone was used as a control.…”

Functional Characterization of the Grapevine γ-Glutamyl Transferase/Transpeptidase (E.C. 2.3.2.2) Gene Family Reveals a Single Functional Gene Whose Encoded Protein Product Is Not Located in Either the Vacuole or Apoplast

“…Relative expression assays were run using the Eco Real-Time PCR System (Illumina) and resulting data analyzed with the supplied Illumina Eco software. Final reaction volumes were 10 µL and are as described in Philips et al (2019), with primer sequences presented in ( Supplementary Table 1 ). All liquid handling steps were performed using an Eppendorf epMotion 5070 liquid handling robot.…”