DOI: 10.5061/dryad.qbzkh18t6
title: The soil bacterium Lysobacter capsici attaches to the nematode surface, and triggers induced systemic resistance in barley, impairing the invasion of root-lesion nematodes

description: Root-lesion nematodes pose a global threat to crop yield and stability,
 with their broad host range presenting a formidable challenge for
 effective management. In this study, we investigated the role of the
 nematode-attached bacterium Lysobacter capsici in mitigating the invasion
 of Pratylenchus penetrans into spring barley roots. Using a split-root
 system, we demonstrated that the cuticle-attached bacterium significantly
 reduced nematode invasion by 75% to 77% as compared to the nematodes
 without L. capsici. This reduction was predominantly due to the induction
 of systemic defense in the plants, and partially due to the
 bacterium's local antagonism in contact with the nematode.Compared to
 the nematodes without L. capsici, the reduced root invasion of P.
 penetrans with attached bacteria differed over time and was particularly
 observed at 48- and 72 hours post-inoculation. Notably, the reduction in
 nematode invasion was more pronounced when L. capsici cells were attached
 to the nematode cuticle than when the bacteria were applied systemically
 and were not directly interacting with the nematodes. In addition,
 transcriptional analyses revealed a higher expression of the barley
 pathogenesis-related gene PR1 in response to the nematodes with attached
 bacteria compared to the surface-sterilized nematodes or L. capsici cells.
 This suggests that nematodes carry bacteria inside the roots that activate
 plant defense. Our study showed the potential of L. capsici to effectively
 manage nematode populations and improve plant health by inducing the plant
 immune system upon nematode invasion.
description: Two datasets from invasion of endoparasitic nematodes into roots
 as affected by a bacterial strain of Lysobacter capsici that attaches to
 the surface of the nematodes.
description: # The soil bacterium Lysobacter capsici attaches to the nematode surface,
 and triggers induced systemic resistance in barley, impairing the invasion
 of root-lesion nematodes [https://doi.org/10.5061/dryad.qbzkh18t6
 ](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