Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Lindsey, Benson E.; Rivero, Luz; Calhoun, Chistopher S.; Grotewold, Erich; Brkljačić, Jelena

doi:10.3791/56587-v

Cited by 35 publications

(29 citation statements)

References 14 publications

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“…For the experiments at MPIPZ, Cologne, seeds were stratified for three days in the dark at 4 °C and sown on water-saturated Jiffy pellets (J-7) and allowed to develop at slightly shorter day cycles (10 h light/14 h dark). For experiments in which multiple genotypes of Arabidopsis were used simultaneously, seeds were surface sterilized before sowing by vapor-phase sterilization as described previously 53 .…”

Section: Methodsmentioning

confidence: 99%

Congruent downy mildew-associated microbiomes reduce plant disease and function as transferable resistobiomes

Goossens

Spooren

Baremans

et al. 2023

Preprint

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Root-associated microbiota can protect plants against severe disease outbreaks. In the model-plant Arabidopsis thaliana, leaf infection with the obligate downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) results in a shift in the root exudation profile, therewith promoting the growth of a selective root microbiome that induces a systemic resistance against Hpa in the above-ground plant parts. Here we show that, additionally, a conserved subcommunity of the recruited soil microbiota becomes part of a pathogen-associated microbiome in the phyllosphere that is vertically transmitted with the spores of the pathogen to consecutively infected host plants. This subcommunity of Hpa-associated microbiota (HAM) limits pathogen infection and is therefore coined a resistobiome. The HAM resistobiome consists of a small number of bacterial species and was first found in our routinely maintained laboratory cultures of independent Hpa strains. When co-inoculated with Hpa spores, the HAM rapidly dominates the phyllosphere of infected plants, negatively impacting Hpa spore formation. Remarkably, isogenic bacterial isolates of the abundantly-present HAM species were also found in strictly separated Hpa cultures across Europe, and even in early published genomes of this obligate biotroph. Our results highlight that pathogen-infected plants can recruit protective microbiota via their roots to the shoots where they become part of a pathogen-associated resistobiome that helps the plant to fight pathogen infection. Understanding the mechanisms by which pathogen-associated resistobiomes are formed will enable the development of microbiome-assisted crop varieties that rely less on chemical crop protection.

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Section: Methodsmentioning

confidence: 99%

Congruent downy mildew-associated microbiomes reduce plant disease and function as transferable resistobiomes

Goossens

Spooren

Baremans

et al. 2023

Preprint

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“…A. thaliana seeds (Col-0, bes1-D, BES1-RNAi, and BES1-FLAG) were surface sterilized for 6 h using the vapor generated by hydrochloric acid and sodium hypochlorite (Lindsey et al, 2017). After sterilization, seeds were sown in petri dishes (10 × 1.5 cm) containing half-strength ½ Murashige and Skoog (MS) medium with 0.8% agarose and vernalized at 4°C in the dark for 2 d. Then, these petri dishes were placed in a plant growth chamber at temperature of 23°C day/21°C night, photoperiod of 12 h light/12 h dark, with photosynthetic active radiation (PAR) of 100 mmol•m −2 •s −1 , and relative humidity of 70%.…”

Section: Plant Materials and Treatmentmentioning

confidence: 99%

Integrative transcriptomic and proteomic analyses reveal a positive role of BES1 in salt tolerance in Arabidopsis

Feng

Zhou

et al. 2023

Front. Plant Sci.

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IntroductionSalt stress is a major environmental factor limiting plant growth and development. Previous studies have indicated that the steroidal hormones—brassinosteroids (BRs) are important regulators of plant responses to salt stress. However, the underlying molecular mechanisms have not been fully understood.Methods(1) Phenotypic analysis of bes1-D, BES1-RNAi and their wild-type (Col-0) under salt treatments with different concentrations of NaCl. (2) Transcriptomic and proteomic profiling of BES1-regulated genes and proteins under salt treatment; (3) qRT-PCR validation of selected BES1-regulated genes under salt stress; (4) Transient transcriptional assay of BES1 regulation on its putative target genes in Arabidopsis protoplasts; (5) Electrophoresis Mobility Shift Assay (EMSA) of BES1 binding with its potential target genes.Results and DiscussionPhenotypic analysis indicated that bes1-D, a gain-of-function mutant of the BR-regulated transcription factor BES1 in Arabidopsis showed better salt tolerance than the wild-type plant, while a BES1 RNA interference (BES1-RNAi) line was more sensitive to salt stress. Global gene expression profiling and time series clustering analyses identified a total of 1,170 genes whose expression was boosted in bes1-D under salt stress. Further GO enrichment and gene functional network analyses identified several key modules that are regulated by BES1 and most sensitive to salt stress perturbations, including stress response, response to ABA and ROS, flavonoid biosynthesis and transmembrane transport. A comparative proteomic analysis performed under the same stress conditions supported the results from the transcriptome analysis. In addition, transient gene transcription assays in Arabidopsis protoplasts and in vitro DNA binding assays verified that BES1 regulates the expression of some ion transporter genes directly and indirectly. Taken together, our results support a positive role of BES1 in plant salt tolerance.

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“…Growth conditions and treatments used Seeds were surface sterilized with gas chloride (Lindsey III et al 2017), and stratified for 2 days at 4°C. Seedlings were growth vertically on plates containing 1 % (w/v) agar (Duchefa) with ½ Murashige and Skoog (MS, Duchefa, 0,5g/l MES, 1 % (w/v) sucrose, pH 5.8 adjusted with 1M KOH).…”

Section: Cloning Strategymentioning

confidence: 99%

Auxin coreceptor IAA17/AXR3 controls cell elongation inArabidopsis thalianaroot by modulation of auxin and gibberellin perception

Kubalova

Müller

Dobrev

et al. 2023

Preprint

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The nuclear TIR1/AFB - Aux/IAA auxin pathway plays a crucial role in regulating plant growth and development. Specifically, the IAA17/AXR3 protein participates in root development, and the accumulation of its mutant variant, AXR3-1, which cannot bind auxin, leads to severe root growth phenotype and agravitropism. However, the mechanism by which AXR3 regulates cell elongation is not fully understood. Here we show that the inducible expression of AXR3-1 in the Arabidopsis thaliana root triggers excessive cell elongation that is followed by growth arrest of the root. We exploited this effect to reveal the underlying molecular mechanism of AXR3 action. We show that AXR3-1 acts exclusively in the nucleus where it interferes with the nuclear auxin transcriptional pathway, while the rapid cytoplasmic auxin root growth response is not affected. The analysis of the transcriptome of the induced AXR3-1 roots revealed changes in phytohormone perception and homeostasis. We show that the accumulation of AXR3-1 disturbs auxin homeostasis which leads to excessive auxin accumulation. At the same time, the reaction of the AXR3-1 roots to gibberellin is altered. These results show that the IAA17/AXR3 maintains an optimal cell elongation rate by controlling the auxin response, auxin homeostasis and the interplay with gibberellin signaling.

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Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Cited by 35 publications

References 14 publications

Congruent downy mildew-associated microbiomes reduce plant disease and function as transferable resistobiomes

Congruent downy mildew-associated microbiomes reduce plant disease and function as transferable resistobiomes

Integrative transcriptomic and proteomic analyses reveal a positive role of BES1 in salt tolerance in Arabidopsis

Auxin coreceptor IAA17/AXR3 controls cell elongation inArabidopsis thalianaroot by modulation of auxin and gibberellin perception

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