Transcriptomics reveal core activities of the plant growth-promoting bacterium Delftia acidovorans RAY209 during interaction with canola and soybean roots

The plant growth-promoting rhizobacterium Delftia acidovorans RAY209 is capable of establishing strong root attachment during early plant development at 7 days post-inoculation. The transcriptional response of RAY209 was measured using RNA-seq during early (day 2) and sustained (day 7) root colonization of canola plants, capturing RAY209 differentiation from a medium-suspended cell state to a strongly root-attached cell state. Transcriptomic data was collected in an identical manner during RAY209 interaction with soybean roots to explore the putative root colonization response to this globally relevant crop. Analysis indicated there is an increased number of significantly differentially expressed genes between medium-suspended and root-attached cells during early soybean root colonization relative to sustained colonization, while the opposite temporal pattern was observed for canola root colonization. Regardless of the plant host, root-attached RAY209 cells exhibited the least amount of differential gene expression between early and sustained root colonization. Root-attached cells of either canola or soybean roots expressed high levels of a fasciclin gene homolog encoding an adhesion protein, as well as genes encoding hydrolases, multiple biosynthetic processes, and membrane transport. Notably, while RAY209 ABC transporter genes of similar function were transcribed during attachment to either canola or soybean roots, several transporter genes were uniquely differentially expressed during colonization of the respective plant hosts. In turn, both canola and soybean plants expressed genes encoding pectin lyase and hydrolases – enzymes with purported function in remodelling extracellular matrices in response to RAY209 colonization. RAY209 exhibited both a core regulatory response and a planthost-specific regulatory response to root colonization, indicating that RAY209 specifically adjusts its cellular activities to adapt to the canola and soybean root environments. This transcriptomic data defines the basic RAY209 response as both a canola and soybean commercial crop and seed inoculant.

. Canola root colonization by Delftia acidovorans RAY209. (A) and (C) = control (uninoculated); (B) and (D) = RAY209 inoculation. Comparison of A and B demonstrates an increase in turbidity surrounding the developing root after 24 days with RAY209 inoculation (red box). Comparison of C and D demonstrates the strength of RAY209 colonization on primary and secondary roots after 24 days.

Plant growth-promoting behaviour of RAY209
To monitor the effect of Delftia acidovorans RAY209 on root development, canola seeds were surface sterilized by submerging in 2% NaClO for 5 min and rinsed with sterile water several times. Three seeds were placed in CYG germination pouches (Mega International, Newport, MN) with 0.5´ Hoagland's Solution (Sigma-Aldrich) and inoculated with 1.0 ´ 10 5 CFU of RAY209 per seed by pipetting (day 0). For effective germination and to maintain sterility, all pouches were covered with tinfoil and placed in a phytotron (Conviron PGR15) with a day/night cycle of 16/8 h and 22/18° C. Tinfoil was removed on day 4 and plants were thinned to two plants per pouch. Images of plant development were taken on day 8 (Figure S2A-B). Plants were harvested on day 15 and the number of lateral roots counted, and root/shoot lengths measured and weighed (Table  S1). Prior to weighing, root/shoot material was dried at 37 °C for 4 days. The number of root hairs on developing primary roots with and without RAY209 inoculation was examined after 36 days by submerging roots in water and observing at 100´ magnification ( Figure S2C-D).  Results are expressed as mean ± standard deviation (n=12 for each treatment). Different letters indicate a significant difference among treatments (p < 0.05).

Validating strongly root-attached RAY209 isolation strategies from canola roots
Canola roots were harvested and plated as previously described in Materials and Methods. Homogenization and low-intensity sonication were tested for the ability to release root-attached RAY209 while preserving cell viability (Figure S5). A subset of processed roots (as previously described in main text) were weighed and submerged in 500 µL PBS, then bath-sonicated using the Low power setting of a Bioruptor Standard UCD-200 (Diagenode) for 5 min, alternating 30 sec on/off [1]. Cell counts were obtained as previously described.

Figure S5. Validation of method for releasing root-attached RAY209 cells from canola roots.
Low-intensity sonication (grey) and homogenization (black) methods were tested for the ability to release root-attached RAY209 cells from the root surface. Sonication was ineffective, as mean cell counts did not differ significantly between days 2 and 7 (p two tail = 0.70). Homogenization was sufficiently effective, as a significant log 10 -fold increase in mean root-attached RAY209 cells was observed (p one tail = 0.03); n = 3 replicates.

Dissolved oxygen concentration measurements
To ensure adequate oxygenation within the liquid plant growth medium, dissolved oxygen concentrations were measured at canola roots with a YSI meter prior to root harvesting on day 2 and day 7 (Figure S8). Before each measurement, the probe was sterilized in a 2% NaClO solution for 3 min and rinsed with sterile water. Care was taken to not disturb the liquid growth medium during data collection. Figure S8. Average dissolved oxygen concentration around canola roots. Dissolved oxygen (dO 2 ) at the roots remained consistent (p two tail = 0.12) throughout the week-long growth period, averaging 4.8 ppm at day 2 and 4.3 ppm by day 7 (n = 3 replicates).