Dataset Information

Stable pH Suppresses Defense Signaling and is the Key to Enhance Agrobacterium-Mediated Transient Expression in Arabidopsis Seedlings.

ABSTRACT: Agrobacterium-mediated transient expression is a powerful analysis platform for diverse plant gene functional studies, but the mechanisms regulating the expression or transformation levels are poorly studied. Previously, we developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST, for Arabidopsis seedlings. In this study, we found that AGROBEST could promote the growth of agrobacteria as well as inhibit the host immunity response. When the factor of agrobacterial growth is minimized, maintaining pH at 5.5 with MES buffer was the key to achieving optimal transient expression efficiency. The expression of plant immunity marker genes, FRK1 and NHL10, was suppressed in the pH-buffered medium as compared with non-buffered conditions in Col-0 and an efr-1 mutant lacking the immunity receptor EFR recognizing EF-Tu, a potent pathogen- or microbe-associated molecular pattern (PAMP or MAMP) of A. tumefaciens. Notably, such immune suppression could also occur in Arabidopsis seedlings without Agrobacterium infection. Furthermore, the PAMP-triggered influx of calcium ions was compromised in the pH-buffered medium. We propose that the enhanced transient expression efficiency by stable pH was due to inhibiting calcium ion uptake and subsequently led to suppressing immunity against Agrobacterium.

SUBMITTER: Wang YC

PROVIDER: S-EPMC6244089 | biostudies-other | 2018 Nov

REPOSITORIES: biostudies-other

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Stable pH Suppresses Defense Signaling and is the Key to Enhance Agrobacterium-Mediated Transient Expression in Arabidopsis Seedlings.

Wang Yi-Chieh YC Yu Manda M Shih Po-Yuan PY Wu Hung-Yi HY Lai Erh-Min EM

Scientific reports 20181120 1

Agrobacterium-mediated transient expression is a powerful analysis platform for diverse plant gene functional studies, but the mechanisms regulating the expression or transformation levels are poorly studied. Previously, we developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST, for Arabidopsis seedlings. In this study, we found that AGROBEST could promote the growth of agrobacteria as well as inhibit the host immunity response. When the factor ...[more]

PMID: 30459348

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Project description:BACKGROUND:Transient gene expression via Agrobacterium-mediated DNA transfer offers a simple and fast method to analyze transgene functions. Although Arabidopsis is the most-studied model plant with powerful genetic and genomic resources, achieving highly efficient and consistent transient expression for gene function analysis in Arabidopsis remains challenging. RESULTS:We developed a highly efficient and robust Agrobacterium-mediated transient expression system, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which achieves versatile analysis of diverse gene functions in intact Arabidopsis seedlings. Using ?-glucuronidase (GUS) as a reporter for Agrobacterium-mediated transformation assay, we show that the use of a specific disarmed Agrobacterium strain with vir gene pre-induction resulted in homogenous GUS staining in cotyledons of young Arabidopsis seedlings. Optimization with AB salts in plant culture medium buffered with acidic pH 5.5 during Agrobacterium infection greatly enhanced the transient expression levels, which were significantly higher than with two existing methods. Importantly, the optimized method conferred 100% infected seedlings with highly increased transient expression in shoots and also transformation events in roots of ~70% infected seedlings in both the immune receptor mutant efr-1 and wild-type Col-0 seedlings. Finally, we demonstrated the versatile applicability of the method for examining transcription factor action and circadian reporter-gene regulation as well as protein subcellular localization and protein-protein interactions in physiological contexts. CONCLUSIONS:AGROBEST is a simple, fast, reliable, and robust transient expression system enabling high transient expression and transformation efficiency in Arabidopsis seedlings. Demonstration of the proof-of-concept experiments elevates the transient expression technology to the level of functional studies in Arabidopsis seedlings in addition to previous applications in fluorescent protein localization and protein-protein interaction studies. In addition, AGROBEST offers a new way to dissect the molecular mechanisms involved in Agrobacterium-mediated DNA transfer.

Project description:BACKGROUND:Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. RESULTS:We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. CONCLUSION:The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis.

Dataset Information

Stable pH Suppresses Defense Signaling and is the Key to Enhance Agrobacterium-Mediated Transient Expression in Arabidopsis Seedlings.

Publications

Stable pH Suppresses Defense Signaling and is the Key to Enhance Agrobacterium-Mediated Transient Expression in Arabidopsis Seedlings.

Similar Datasets

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