https://doi.org/10.5061/dryad.qbzkh18t6

Description of the data and file structure

In vitro** split-root system to study Lysobacter capsici effects on root invasion by P. penetrans**

We incubated 20000 surface-sterilized nematodes in 5 ml of bacterial suspension (OD600=0.01) overnight at 22°C on a shaker at 150 rpm. As a control, we incubated surface-sterilized nematodes in sterile tap water under the same conditions. After incubation, we washed nematodes on a 5-µm CellTrics sieve with 10 ml of sterile tap water to remove loosely attached bacterial cells. We inoculated one side of the barley split-root system with 200 nematodes with or without attached bacterial cells. As a control for plant-mediated effects of the bacteria, we inoculated 200 surface-sterilized nematodes to one side of the split-root, and 100000 bacterial cells to the opposite side of the split-root. To sum up, we had three treatments: 1) Sterile (surface-sterilized nematodes), 2) Attachment (nematodes with attached cells of L. capsici), and 3) Systemic (inoculated L. capsici cells physically separated from the nematodes). The inoculated bacterial dilution represented a similar number of bacteria as attached to nematodes in the other treatment. The inoculated split-root systems were kept in randomized complete block design in a climate chamber at 22°C with a 16h/8h photoperiod. At 72h after inoculation, we sampled the roots and stained the invaded nematodes with 1% acid fuchsin (Bybd et al. 1983).

Plant gene expression in response to nematode-attached *L. capsici*

To analyze the expressions of plant defense genes in response to L. capsici attached to the surface of P. penetrans, we used a split-root experimental setup in vitro. In one additional treatment, we inoculated one side of the split-root system only with 100 µL of the dilution 10-6 of the bacterial suspension (OD600=0.01), while the other side of the root was left free to exclude the defense response to L. capsici. In the second additional treatment, the roots were not infected with either nematodes or bacteria. After 24h and 72h, we collected the roots into 2ml microtubes and stored tubes at −80°C until RNA extraction. The tubes with frozen roots were transferred to a pre-cooled block and pulverized using two 4-mm metal beads twice for 40s at the highest speed (30 Hz) in a TissueLyser II (Qiagen). Total RNA was extracted using the FastRNA Pro Green Kit (MP Biomedicals) following the manufacturer's recommendations. DNA traces were removed by DNase I digestion, followed by DNase inactivation using the DNA-free Kit (ThermoFisher Scientific, Waltham, MA, USA). The cDNA synthesis was carried out using Superscript IV and an anchored oligo(dT)20 primer according to the manufacturer's instructions (ThermoFisher Scientific). We performed a quantitative real-time PCR using a CFX Connect Real-Time PCR Detection System (Bio-Rad, Munich, Germany) to quantify the relative expressions of plant defense genes. Of these, 1) the pathogenesis-related proteins (PR1, PR17b) and the Prx7 peroxidase gene are involved in generating reactive oxygen species (ROS), which help limit the nematode invasion; 2) the HSP70 gene enhances the expression of antioxidant enzymes and contributes to the accumulation of PR proteins ; 3) the glucan synthase-like 6 gene (Gsl6) increases callose accumulation, strengthening cell walls against pathogens; 4) the chloroplastic Cu/Zn superoxide dismutase (CSD1) gene plays a key role in the antioxidant defense system, protecting plants against oxidative stress. The relative expression of the housekeeping gene Ubiquitin (UBQ) was quantified as a reference. The reactions were performed using Luna® Universal RT-qPCR Master Mix (New England BioLabs, Frankfurt am Main, Germany), with two technical replicates. The amplifications were carried out using the following conditions: initial denaturation at 95°C for 2 min, 40 cycles of a denaturation step at 95°C for 30 s, annealing step at 60°C for 30s, and extension step at 70°C for 30s, and 80°C for 15s. The fluorescence signal was read at 80°C at each phase of the cycle. The detection cycles (Ct) of the housekeeping gene UBQ were used to correct the Ct of the tested defense genes and to calculate the gene expression. The outcomes were normalized to the non-inoculated root samples using the –ΔΔCt method (Pfaffl 2001).

Files and variables

File: Local_vs_systemic_effect

Description: Root invasion by endoparasitic nematodes as affected by Lysobacter capsici

Variables

Treatments: 
1. Root exposed to nematodes, but not to Lysobacter (control)
2. Root exposed to nematodes that carry Lysobacter on their surface (local effect)
3. Roots exposed to surface-sterilized nematodes, while split-roots in opposite pot were exposed to Lysobacter that induce systemic plant defense 

Parameters: 
1. Number of nematodes inside the root of a single plant
2. Fresh weight (g) of split-root infested by nematodes
3. Fresh weight (g) of opposite split-root without nematodes  
4. Number of nematodes per gram root
5. Shoot fresh weight (g)
6. Shoot dry weight (g)

File: QPCR

Description: Transcript levels of barley defense genes after root invasion of nematodes with/without attached bacteria (Ct values)

Variables

• Treatments:
  1. Control root, not exposured to Lysobacter or nematodes
  2. Split-root invaded by nematodes, while  opposite split-root is exposed to Lysobacter
  3. Split-root exposed to Lysobacter, while opposite split-root is invaded by nematodes
  4. Root exposed to Lysobacter, without nematodes
  5. Root invaded by nematodes with attached Lysobacter
  6. Root invaded by nematodes without attached Lysobacter
  7. No Target Control of PCR
• Parameter: Cycle of threshold (Ct) for barley defence genes PR1, PRX7, HSP70, PR17B, GSL6, CSD1, and for housekeeping reference gene UBQ

Code/software

MS Excel