Dataset Information

Early Arabidopsis root hair growth stimulation by pathogenic strains of Pseudomonas syringae.

ABSTRACT:

Background and aims

Selected beneficial Pseudomonas spp. strains have the ability to influence root architecture in Arabidopsis thaliana by inhibiting primary root elongation and promoting lateral root and root hair formation. A crucial role for auxin in this long-term (1week), long-distance plant-microbe interaction has been demonstrated.

Methods

Arabidopsis seedlings were cultivated in vitro on vertical plates and inoculated with pathogenic strains Pseudomonas syringae pv. maculicola (Psm) and P. syringae pv. tomato DC3000 (Pst), as well as Agrobacterium tumefaciens (Atu) and Escherichia coli (Eco). Root hair lengths were measured after 24 and 48h of direct exposure to each bacterial strain. Several Arabidopsis mutants with impaired responses to pathogens, impaired ethylene perception and defects in the exocyst vesicle tethering complex that is involved in secretion were also analysed.

Key results

Arabidopsis seedling roots infected with Psm or Pst responded similarly to when infected with plant growth-promoting rhizobacteria; root hair growth was stimulated and primary root growth was inhibited. Other plant- and soil-adapted bacteria induced similar root hair responses. The most compromised root hair growth stimulation response was found for the knockout mutants exo70A1 and ein2. The single immune pathways dependent on salicylic acid, jasmonic acid and PAD4 are not directly involved in root hair growth stimulation; however, in the mutual cross-talk with ethylene, they indirectly modify the extent of the stimulation of root hair growth. The Flg22 peptide does not initiate root hair stimulation as intact bacteria do, but pretreatment with Flg22 prior to Psm inoculation abolished root hair growth stimulation in an FLS2 receptor kinase-dependent manner. These early response phenomena are not associated with changes in auxin levels, as monitored with the pDR5::GUS auxin reporter.

Conclusions

Early stimulation of root hair growth is an effect of an unidentified component of living plant pathogenic bacteria. The root hair growth response is triggered in the range of hours after bacterial contact with roots and can be modulated by FLS2 signalling. Bacterial stimulation of root hair growth requires functional ethylene signalling and an efficient exocyst-dependent secretory machinery.

SUBMITTER: Pecenkova T

PROVIDER: S-EPMC5591418 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Publications

Early Arabidopsis root hair growth stimulation by pathogenic strains of Pseudomonas syringae.

Pecenková Tamara T Janda Martin M Ortmannová Jitka J Hajná Vladimíra V Stehlíková Zuzana Z Žárský Viktor V

Annals of botany. 20170901 3

<h4>Background and aims</h4>Selected beneficial Pseudomonas spp. strains have the ability to influence root architecture in Arabidopsis thaliana by inhibiting primary root elongation and promoting lateral root and root hair formation. A crucial role for auxin in this long-term (1week), long-distance plant-microbe interaction has been demonstrated.<h4>Methods</h4>Arabidopsis seedlings were cultivated in vitro on vertical plates and inoculated with pathogenic strains Pseudomonas syringae pv. macul ...[more]

PMID: 28911019

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Project description:Interactions between epiphytic bacteria and herbivorous insects are ubiquitous on plants, but little is known about their ecological implications. Aphids are devastating crop pests worldwide, so understanding how epiphytic bacteria impact aphid populations is critically important. Recent evidence demonstrates that plant-associated bacteria, such as Pseudomonas syringae, can be highly virulent to one species of aphid, the pea aphid (Acyrthosiphon pisum). Currently, we have no knowledge on how broad this phenomenon is across diverse aphid species that are of high agricultural concern. In controlled experiments using oral exposure in an artificial diet, we challenged five aphid species of agricultural importance with three strains of P. syringae that vary in virulence to the pea aphid. These strains also vary in epiphytic ability and comprise two phytopathogens and one non-plant-pathogenic strain. In general, differences in virulence to aphids remained relatively constant across strains regardless of the aphid species, except for the bird cherry-oat aphid (Rhopalosiphum padi), which is significantly less susceptible to two P. syringae strains. We demonstrate that lower infection incidence likely plays a role in the reduced susceptibility. Importantly, these data support previous results showing that interactions with epiphytic bacteria are important for aphids and may play a large, but underappreciated, role in insect population dynamics. Our study illustrates a potential role of epiphytic bacteria in the biological control of aphid pests broadly but suggests the need for more research encompassing a greater diversity of pest species.IMPORTANCE Sap-sucking aphids are insects of huge agricultural concern, not only because of direct damage caused by feeding but also because of their ability to transmit various plant pathogens. Some bacteria that grow on leaf surfaces, such as Pseudomonas syringae, can infect and kill aphids, making them potentially useful in the biological control of pest aphids. However, only one aphid species, the pea aphid (Acyrthosiphon pisum), has been tested for infection by P. syringae Here, we challenged five aphid species of agricultural importance with three strains of P. syringae that vary in virulence to the pea aphid. We found that four of these aphid species were susceptible to infection and death, suggesting that these bacteria are broadly useful for biological control. However, one aphid species was much more resistant to infection, indicating that more testing on diverse aphid species is needed.

Project description:Using cell sorting technique, we collected Arabidopsis root hair protoplasts (transgenic line harboring root hair specific gene promoter ::GFP, here named GFP) and nonGFP protoplasts( whole root except root hair). Then, we extracted total proteins and RNA for proteome and RNASeq. RNAseq data have already been deposited into NCBI (accession numbers SRR364677 and SRR364678). Total proteins were separated by 1D SDS-PAGE, followed by gel slicing, in gel digested and run ESI-LC-MS/MS on LTQ Orbitrap Velos. We set two biological repeats and each sample was scanned two times (two technique repeats). We then combined all ms/ms raw data from all fractions and technique repeats from one biological sample into one file to indentify proteins by searching Arabidopsis protein database with software Proteome Discoverer (with two searching engines mascot and sequester). Protocol: Protoplasts from EXP7 and non-GFP protoplastswere stored at -80°C, thawed on ice for 5 min, vortexed several times and suspended in 5 x volume of pre-cooled acetone (-20°C) containing 10% (v/v) trichloroacetic acid (TCA) and 0.07% (v/v) 2-mercaptoethanol. Proteins were then precipitated for 2 h at −20°C after thorough mixing. Proteins were collected by centrifuging at 35,000 g (JA-20 108 rotor, Beckman J2-HS) at 4°C for 30 min. The supernatant was removed and the protein pellets were washed three times with cold acetone containing 0.07% (v/v) 2-mercaptoethanol and 1 mMphenylmethanesulfonylfluoride. Protein pellets were dried by lyophilization and stored at -80°C, or immediately extracted using Laemmli buffer (63 mM TrisHCl pH 6.8, 10% glycerol, 2% SDS, 0.0025% bromophenol blue). Protein concentration was determined using Pierce protein assay kit (Pierce, Rockford). Proteins from each sample were separated using a Bis-Tris precast gradient gel (4−16%, Bio-Rad) according to the manufacturer’s instructions. After electrophoresis, the gel was Coomassie-stained using the Colloidal Blue Staining Kit (Invitrogen). The protocol used for in-gel trypsin digestion of proteins in gels was adapted from the method described in Shevchenko et al. (1996). Briefly, protein bands were manually excised and each band was cut into small pieces (~0.5 mm). The gel pieces were washed three times with a solution containing 50% methanol and 5% acetic acid for 2 h, twice with a solution containing 25 mM NH4HCO3 in 50% acetonitrile for 10 min each, and then the gel pieces were dried in a vacuum centrifuge. Reduction of proteins in gel pieces was performed with DTT and alkylation with iodoacetamide, and the gel pieces were washed and dried in a vacuum centrifuge. A trypsin solution in 25 mM NH4HCO3 containing 75 to 100 ng of sequencing grade modified trypsin (Promega) in 25-40 µl was added and incubated with gel pieces for 12-16 h at 37°C. To recover the tryptic peptides, a solution of 30 µl containing 5% formic acid and 50% acetonitril was added to the gel pieces, agitated in a vortex for 30-60 min and withdrawn into a new tube. The recovery was repeated once with 15 µl solution and the resulting two recovers were combined and dried in a vacuum centrifuge. The dried pellet was re-dissolved in 10-20 µl of 0.1% formic acid for LC-MS/MS analysis.

Dataset Information

Early Arabidopsis root hair growth stimulation by pathogenic strains of Pseudomonas syringae.

Background and aims

Methods

Key results

Conclusions

Publications

Early Arabidopsis root hair growth stimulation by pathogenic strains of Pseudomonas syringae.

Similar Datasets

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