Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in &lt;em&gt;Arabidopsis thaliana&lt;/em&gt;

Bredow, Melissa; Sementchoukova, Irina; Siegel, Kristen; Monaghan, Jacqueline

doi:10.3791/59437

Cited by 19 publications

(19 citation statements)

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“…This suggests that immune induction by Pto DC3000 EVs does not affect plant growth, unlike treatment with flg22 (Fig. S4B) (Bredow et al , 2019).…”

Section: Resultsmentioning

confidence: 95%

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Biophysical and proteomic analyses suggest functions ofPseudomonas syringaepvtomatoDC3000 extracellular vesicles in bacterial growth during plant infection

Janda

Ludwig²,

Rybak³

et al. 2021

Preprint

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Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. Bacterial EVs contain molecular cargo from the donor bacterium and play important roles in bacterial survival and growth. Here, we describe EV production in plant pathogenic Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), the causal agent of bacterial speck disease. Cultured Pto DC3000 exhibited EV structures both on the cell surface and in the vicinity of bacterial cells, observed as outer membrane vesicle (OMV) release. We used in-solution trypsin digestion coupled to mass spectrometry to identify 369 proteins enriched in EVs recovered from cultured Pto DC3000. The predicted localization profile of EV proteins supports the production of EVs also in the form of outer-inner-membrane vesicles (OIMVs). EV production varied slightly between bacterial lifestyles and also occurred in planta. The potential contribution of EVs to Pto DC3000 plant infection was assessed using plant treatments and bioinformatic analysis of the EV-enriched proteins. While these results identify immunogenic activities of the EVs, they also point at roles for EVs in bacterial defences and nutrient acquisition by Pto DC3000.

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“…This suggests that immune induction by Pto DC3000 EVs does not affect plant growth, unlike treatment with flg22 (Fig. S4B) (Bredow et al , 2019).…”

Section: Resultsmentioning

confidence: 95%

“…EV-induced FRK1 upregulation is in agreement with previous observations (Bahar et al , 2016). We also tested whether treatment with Pto DC3000 EVs could arrest seedling growth, a prototypic PTI response of plants to continual MAMP stimulation (Bredow et al , 2019). Unexpectedly, we observed no significant growth reduction (Fig.…”

Section: Resultsmentioning

confidence: 99%

Biophysical and proteomic analyses suggest functions ofPseudomonas syringaepvtomatoDC3000 extracellular vesicles in bacterial growth during plant infection

Janda

Ludwig²,

Rybak³

et al. 2021

Preprint

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“…Each disk was subsequently transferred to a well of a 96 well microtiter plate containing 100 μl of dH2O. The plates were covered with aluminum foil and incubated at room temperature over night to prevent interference of ROS produced after wounding by allowing de-stressing of the freshly cut leaf disks ( Bredow et al., 2019 ). After overnight incubation, the water was replaced by 200 μl of 0.05% chitosan and 0.5 mM of the luminol derivative L-012 (8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine- 1,4(2H,3H)dione) ( Nishinaka et al., 1993 ) in 10 mM MOPS/KOH buffer (pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

Transcriptome Analysis of Solanum Tuberosum Genotype RH89-039-16 in Response to Chitosan

2020

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Potato ( Solanum tuberosum L.) is the worldwide most important nongrain crop after wheat, rice, and maize. The autotetraploidy of the modern commercial potato makes breeding of new resistant and high-yielding cultivars challenging due to complicated and time-consuming identification and selection processes of desired crop features. On the other hand, plant protection of existing cultivars using conventional synthetic pesticides is increasingly restricted due to safety issues for both consumers and the environment. Chitosan is known to display antimicrobial activity against a broad range of plant pathogens and shows the ability to trigger resistance in plants by elicitation of defense responses. As chitosan is a renewable, biodegradable and nontoxic compound, it is considered as a promising next-generation plant-protecting agent. However, the molecular and cellular modes of action of chitosan treatment are not yet understood. In this study, transcriptional changes in chitosan-treated potato leaves were investigated via RNA sequencing. Leaves treated with a well-defined chitosan polymer at low concentration were harvested 2 and 5 h after treatment and their expression profile was compared against water-treated control plants. We observed 32 differentially expressed genes (fold change ≥ 1; p-value ≤ 0.05) 2 h after treatment and 83 differentially expressed genes 5 h after treatment. Enrichment analysis mainly revealed gene modulation associated with electron transfer chains in chloroplasts and mitochondria, accompanied by the upregulation of only a very limited number of genes directly related to defense. As chitosan positively influences plant growth, yield, and resistance, we conclude that activation of electron transfer might result in the crosstalk of different organelles via redox signals to activate immune responses in preparation for pathogen attack, concomitantly resulting in a generally improved metabolic state, fostering plant growth and development. This conclusion is supported by the rapid and transient production of reactive oxygen species in a typical oxidative burst in the potato leaves upon chitosan treatment. This study furthers our knowledge on the mode of action of chitosan as a plant-protecting agent, as a prerequisite for improving its ability to replace or reduce the use of less environmentally friendly agro-chemicals.

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“…AtPep1(99) and elf18 (100) used for immune assays were synthesized by EZBiolab (USA). AtPep1-induced SGI and ROS burst assays were performed as previously described (101). Infection assays were conducted using virulent Pseudomonas syringae pv.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation-dependent sub-functionalization of the calcium-dependent protein kinase CPK28

Bredow

Bender

Dingee

et al. 2020

Preprint

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Calcium (Ca2+)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca2+-sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1), and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intra- and inter-molecular auto-phosphorylation on Ser318 and can additionally be trans-phosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca2+ activation at physiological concentrations of Ca2+, possibly through stabilization of the Ca2+-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca2+ ] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem-elongation, indicating pathway specific requirements for phosphorylation-based Ca2+-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.

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Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in <em>Arabidopsis thaliana</em>

Cited by 19 publications

References 0 publications

Biophysical and proteomic analyses suggest functions ofPseudomonas syringaepvtomatoDC3000 extracellular vesicles in bacterial growth during plant infection

Biophysical and proteomic analyses suggest functions ofPseudomonas syringaepvtomatoDC3000 extracellular vesicles in bacterial growth during plant infection

Transcriptome Analysis of Solanum Tuberosum Genotype RH89-039-16 in Response to Chitosan

Phosphorylation-dependent sub-functionalization of the calcium-dependent protein kinase CPK28

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