AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings

Abstract

BackgroundTransient 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.ResultsWe 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 β-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.ConclusionsAGROBEST 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.

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…The AGROBEST (Agrobacterium-mediated enhanced seedling transformation) method achieves high transient transformation efficiency in whole seedlings, allowing gain-of-function studies, by using a pH-buffered medium supplemented with AB salts and glucose. It has been shown that transient expression of GUS or LUC of Col-0 or efr-1 seedlings was significantly enhanced when AGROBEST was used during A. tumefaciens inoculation (Wu et al, 2014b). Similar to what was observed for agroinfiltration in adult leaves (Wroblewski et al, 2005), C58C1(pTiB6S3DT-DNA, pCH32) achieves higher transient transformation efficiency than the wild type C58 virulent strain or the C58-derived disarmed strain GV3101(pMP90) in A. thaliana seedlings (Wu et al, 2014b).…”

“…For transient transformation, T-DNA integration into the host genome is not required, and T-DNA expression usually lasts for a few days (Wroblewski et al, 2005;Marion et al, 2008;Jones et al, 2009;Kim et al, 2009;Li et al, 2009;Tsuda et al, 2012;Wu et al, 2014b). It presents useful advantages, in that it allows results of experimental treatments to be observed in a short period of time, as discussed below.…”

“…It has been shown that transient expression of GUS or LUC of Col-0 or efr-1 seedlings was significantly enhanced when AGROBEST was used during A. tumefaciens inoculation (Wu et al, 2014b). Similar to what was observed for agroinfiltration in adult leaves (Wroblewski et al, 2005), C58C1(pTiB6S3DT-DNA, pCH32) achieves higher transient transformation efficiency than the wild type C58 virulent strain or the C58-derived disarmed strain GV3101(pMP90) in A. thaliana seedlings (Wu et al, 2014b). This study also showed an inverse association of root growth inhibition and transient expression efficiency, suggesting that C58C1(pTiB6S3DT-DNA, pCH32) may circumvent a plant defense barrier to enable high transient expression levels in A. thaliana seedlings.…”

MYB75 Phosphorylation by MPK4 Is Required for Light-Induced Anthocyanin Accumulation in Arabidopsis

“…Anthocyanin was measured as previously described with some modifications (Lange et al, 1971;Wu et al, 2014). Arabidopsis seedlings were quickly weighed, boiled for 3 min in extraction solution (propanol:HCl: H 2 O = 18:1:81), and incubated at room temperature in the dark for at least 2 h. Samples were centrifuged, and the absorbance of the supernatants was measured at 535 and 650 nm.…”

The Transcriptional Regulator BBX19 Promotes Hypocotyl Growth by Facilitating COP1-Mediated EARLY FLOWERING3 Degradation in Arabidopsis

“…Arabidopsis transient assays were performed using 5-d-old seedlings infiltrated with Agrobacterium GV3101 carrying pELF3:ELF3:LUC according to a previously described procedure (Wu et al, 2014). Luciferase activity measurements were performed for 36 h with a CCD camera.…”