- Open Access
AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings
© Wu et al.; licensee BioMed Central Ltd. 2014
Received: 7 March 2014
Accepted: 28 May 2014
Published: 18 June 2014
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.
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 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.
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.
Agrobacterium-mediated DNA transfer is currently the most facile and versatile method to deliver gene constructs into the nucleus for gene function analysis in diverse plant species [1–3]. Although stable integration of physiologically active and regulated transgenes is the ultimate goal, transient gene expression via Agrobacterium-mediated DNA transfer in different plant tissues offers a simple and fast method to analyze transgene functions, which is amenable for high-throughput screens. The transient expression assay is also ideal for systematic dissection of the exquisite and complex processes of Agrobacterium–plant interactions and DNA transfer events [4–7].
Agrobacterium tumefaciens is a soil phytopathogen that naturally infects plant wound sites and causes crown gall disease via delivery of transferred (T)-DNA from bacterial cells into host plant cells through a bacterial type IV secretion system (T4SS) . Although Agrobacterium is considered a wound-associated pathogen, it can transfer DNA into diverse host cells or tissues under unwounded conditions [9–13]. Interestingly, most of the Arabidopsis mutants that are resistant to Agrobacterium transformation identified by root explant assays remain highly transformable by floral dip transformation . The mechanisms and plant factors involved in Agrobacterium- mediated transformation may differ between wounded and unwounded cells or different tissues. However, the mechanisms underlying Agrobacterium infection in unwounded cells/tissues have not been explored.
In plant biology research, Arabidopsis mesophyll-protoplast transfection [15, 16] and Agrobacterium- mediated leaf infiltration in Nicotiana benthamiana are the well-established and commonly used platforms for transient gene expression analysis. The Arabidopsis mesophyll-protoplast transient expression system allows for versatile and high-throughput analyses of diverse gene functions and signal transduction pathways; advanced skills with training and practice are essential for successful use of this powerful tool for gene function studies [16, 18, 19]. Agrobacterium- mediated transient expression methods by leaf infiltration have been developed for a wide range of plants including Nicotiana, lettuce, tomato, and Arabidopsis[20–23]. However, the use of 4- to 5-week-old adult plants with manual infiltration has limited application in high-throughput analyses. Furthermore, although Arabidopsis is the most-studied model plant with superbly annotated genome sequences and powerful genetic and genomic resources mostly available for the Columbia (Col) accession, achieving highly efficient and consistent transient expression in Col by adult leaf infiltration is challenging [22, 24].
The use of young seedlings for Agrobacterium-mediated transient expression assays will greatly simplify and amplify the power of the method. Indeed, Agrobacterium-mediated transient expression in Arabidopsis seedlings has been recently developed for fast and robust analysis of protein subcellular localization and protein–protein interactions [25–27]. The system’s requirement for high-density Agrobacterium cells and vacuum infiltration  or chemical treatment (e.g., the addition of surfactant Silwet L-77)  to achieve high cellular transformation efficiency could induce innate immunity and stress responses in plants, which globally alters cellular, physiological, and signaling processes and severely retards growth [28, 29]. Thus, developing a system that circumvents a plant defense barrier may be a key to enhance transient expression efficiency in Arabidopsis seedlings. Furthermore, such a fast, robust, and highly efficient transient expression system could support gain-of-function studies of diverse genes and signaling pathways in planta.
Pattern-triggered immunity (PTI) induced by a microbe- or pathogen-associated molecular pattern (MAMP or PAMP) is the first line of active defense in both plants and animals against pathogens [28–30]. Previous studies have suggested that Agrobacterium- mediated transformation efficiency may be compromised when plants recognize Agrobacterium MAMPs by corresponding pattern-recognition receptors (PRRs) to trigger PTI and block Agrobacterium infection [22, 24]. The elongation factor Tu (EF-Tu) receptor mutant efr-1, which cannot sense EF-Tu MAMP, showed increased Agrobacterium- mediated transient expression efficiency, as did transgenic Arabidopsis expressing a potent bacterial effector AvrPto to suppress PTI signaling with agroinfiltration of 4- to 5-week-old leaves [22, 24]. However, whether these immune-compromised Arabidopsis plants are amenable to increase Agrobacterium- mediated transient expression efficiency in young seedlings has not been tested. Defining the condition for reliable and highly efficient transformation in healthy Col-0 seedlings will be extremely valuable but has never been achieved.
In this study, we systematically investigated various biological factors and growth variances to define a combination of key factors that contribute to the unprecedentedly high transient transformation and reporter gene expression efficiency in Arabidopsis seedlings. As a result of these investigations, we developed an optimized AGROBEST (Agrobacterium-mediated enhanced seedling transformation) method that enabled high transient transformation and expression efficiency in both efr-1 mutant and Col-0 Arabidopsis seedlings. Importantly, we demonstrated the versatile applicability of AGROBEST in gain-of-function studies for the MYB75 transcription factor in specific target-gene activation and for GIGANTEA (GI) reporter gene expression regulated by the Arabidopsis circadian clock. The AGROBEST method is a fast, simple, reliable, and versatile tool for systematic gene function analysis and a new tool for dissecting the Agrobacterium-mediated DNA transfer processes.
Cotyledons of young Arabidopsis EF-TU receptor mutant is highly susceptible to Agrobacterium-mediated transient transformation
Buffered medium at pH 5.5 with AB salts is critical for high transient expression efficiency
Key components in AB-MES are AB salt (17.2 mM K2HPO4, 8.3 mM NaH2PO4, 18.7 mM NH4Cl, 2 mM KCl), minerals (1.25 mM MgSO4, 100 μM CaCl2, 10 μM FeSO4), glucose (2% w/v), and buffering with MES (50 mM) to pH 5.5. We thus tested whether one of these components is responsible for the increased transient expression efficiency. The addition of AB salts with MES buffered at pH 5.5 in MS medium was sufficient to result in comparable levels of GUS expression as with ABM-MS (Figure 2B). Therefore, AB salts alone, pH 5.5 buffered by MES, or both, are critical for the increased transient expression efficiency. Strikingly, all MS media with the addition of AB salts buffered with MES or sodium phosphate at pH 5.5 showed comparable and strong GUS activity as that with ABM-MS (Figure 2C). However, omitting AB salts resulted in ~50% reduction in GUS activity, and no GUS activity was detected with MS medium buffered with sodium phosphate at pH 7.0 in the presence or absence of AB salts. Thus, buffered pH at 5.5 and the presence of AB salts in MS co-cultivation medium are the two key factors for this high transient expression efficiency. We named this optimized infection method AGROBEST (Agrobacterium-mediated enhanced seedling transformation).
Disarmed Agrobacterium strain C58C1(pTiB6S3ΔT)H enables highly efficient AGROBEST-mediated transient expression in Col-0 seedlings
AGROBEST achieves higher transient expression efficiency than existing methods in both efr-1 and Col-0 seedlings
Impact of seedling age and infection time on transient expression efficiency of AGROBEST in efr-1 seedlings
Widespread transient transformation events in different organs and cell types
Studies of protein subcellular localization and protein–protein interactions
Because Arabidopsis plants are less amenable for transient expression analysis, both fluorescent protein localization and bimolecular fluorescence complementation (BiFC) studies are often conducted in protoplasts via transfection or in N. benthamiana leaves via agroinfiltration because of the high transient expression efficiency [16, 17]. Here, we co-infected two A. tumefaciens strains carrying a binary vector for 35S::Venus-intron or 35S::NLS-RFP in efr-1 seedlings and detected both cytoplasmic and nuclear fluorescence signals for Venus and nuclear localization of NLS-RFP in separate or the same cells (Figure 7K). Our assay is also feasible for BiFC studies, which is supported by the interaction of two known interacting proteins, F-box protein TIR1 (transport inhibitor response 1) and ASK1 (Arabidopsis Skp1-like protein) , in the nucleus (Figure 7L). Thus, AGROBEST is an ideal system for subcellular localization and protein-protein interaction studies.
AGROBEST for the expression analysis of a circadian clock reporter gene
AGROBEST for functional assays of transcription factor MYB75
AGROBEST enables high transient transformation and expression efficiency in intact Arabidopsis young seedlings
In this study, we developed a simple, fast, reliable, and robust transient expression system named AGROBEST and uncovered the key factors enabling 100% of infected seedlings with high transgene expression efficiency in Arabidopsis seedlings. Remarkably, AGROBEST appears to achieve the highest transient expression efficiency in the EF-Tu receptor efr-1 mutant as compared to the wild-type Col-0, flagellin receptor mutant fls2, and DEX-inducible AvrPto transgenic line. This result is consistent with a previous finding in agroinfiltrated Arabidopsis adult leaves showing increased transient GUS expression efficiency in efr-1. Because of no detectable phenotype impairing the growth and development in the efr mutant , the use of the efr mutant has an advantage over DEX-inducible AvrPto in seedling stages. Thus, more selected elimination of specific PRRs such as EFR with minimal effects on hormonal signaling, cell death and seedling growth may be a preferred system for Agrobacterium- mediated high transient expression efficiency. Interestingly, N. benthamiana leaves, which are commonly used for Agrobacerium-mediated transient transformation, also lack the EFR receptor .
Unexpectedly, we discovered that AGROBEST also enables high transient expression efficiency in wild-type Col-0 seedlings. The significantly higher transient expression activity by AGROBEST than the FAST and Marion et al. methods likely accounts for the success of our gain-of-function experiments, which have not been shown previously [26, 27]. Of note, efr-1 seedlings remained poorly transformed by FAST method as compared with the significantly increased transient expression in efr-1 by AGROBEST or the Marion et al. method. The reason underlying this discrepancy is unknown, but the yellowish and retarded-growth seedlings after co-cultivation with Agrobacterium in the dark for 2 days by the FAST method may contribute to the observed phenotype. Our AGROBEST method, applying a lower density of Agrobacterium cells (OD600 0.02 as opposed to OD600 0.5 for the FAST method and OD600 2 for the Marion et al. method) for co-cultivation with seedlings without any mechanical treatment (e.g., vacuum infiltration) or chemical treatment (e.g., the addition of surfactant Silwet L-77) offers advantages to maintain infected seedlings with normal growth and a physiological state without injury. The success of transiently expressing the circadian rhythm reporter in Arabidopsis seedlings may open a new platform to rapidly test the circadian behaviors of Arabidopsis mutants, bypassing the process of introducing a circadian reporter gene into the mutants by crossing. Most remarkably, AGROBEST allows for high transient expression of the MYB75 transcription factor and subsequently upregulates the expression of its downstream gene CHS in both efr-1 and Col-0 seedlings. This result suggested the broad application of AGROBEST to study transcription factor action.
Widespread and differential transient transformation events in different organs and cell types
AGROBEST has a breakthrough performance by enabling high and homogeneous transient GUS expression efficiency in shoots of 100% infected Col-0 or efr-1 seedlings. The successful transient expression in roots, although with less efficient transformation events (~70% of seedlings with GUS staining in roots), is also remarkable and not previously detected [26, 27]. Interestingly, preferential transformation events occurring at the initiation sites of lateral roots or the root elongation zone of infected intact seedlings were also previously detected in wounded Arabidopsis roots . High transformation of Arabidopsis roots may require further loosening or opening of cell walls or wounding, which was not included in our infection conditions. Because we observed similar transient expression levels and transformation efficiency in roots of Col-0 and efr-1 seedlings (Additional file 1: Table S1), EFR may play no or little role in seedling root transformation efficiency under our infection conditions. Consistently, EFR is expressed at low levels in Col-0 seedling roots , which were not responsive to the EF-Tu peptide elf26, as evidenced by limited induction of immune marker genes and callose deposition in the roots of Col-0 seedlings . Because the flg22 peptide derived from Agrobacterium flagellin is inactive in Arabidopsis[24, 34, 35] and the flagellin receptor mutant exhibited similar transformation efficiency as Col-0 in our seedling assays, the flagellin receptor FLS2 may not be involved in Agrobacterium-triggered plant innate immune responses and therefore did not compromise Agrobacterium-mediated transient gene expression. Future investigations could examine whether the absence of the peptidoglycan receptor  or yet-to-be identified receptors in recognizing additional MAMPs such as polysaccharides  could increase the transformation efficiency in seedling roots.
Key factors for high transient transformation/expression efficiency
During this course of our method development, we also uncovered new factors critical for the high transient transformation/expression efficiency in Arabidopsis seedlings. One factor is the addition of AB salts in MS medium buffered with acidic pH 5.5 during Agrobacterium infection, which allows for significantly higher transient expression efficiency than in MS medium alone. Another breakthrough is the use of the disarmed A. tumefaciens strain C58C1(pTiB6S3ΔT)H, which offers the highest transient expression efficiency with the least adverse impact on plant growth over other tested strains. Root growth was severely inhibited on infection with other tested A. tumefaciens strains including the transfer-incompetent ΔvirB2. These data indicate that the transport of T-DNA and T4SS effectors into plant cells by a virulent C58 strain may not suppress host immune responses like that observed in T3SS effectors from Pseudomonas syringae. We observed that C58C1(pTiB6S3ΔT)H achieved higher transient expression efficiency in both Col-0 and efr-1 seedlings than other A. tumefaciens strains tested. The agent also had little impact on root growth inhibition of infected seedlings by the ABM50 method (Figure 3). The results suggested that the A. tumefaciens strain C58C1(pTiB6S3ΔT)H is the main factor affecting the root growth difference. EFR may play a minor role in root growth inhibition because we observed slightly stronger root growth inhibition in Col-0 than efr-1 seedlings infected with C58C1(pTiB6S3ΔT)H. This finding is consistent with limited root growth inhibition detected in Col-0 seedlings in response to EF-Tu peptide elf18 as compared with strong root growth inhibition induced by flg22 . The observed inverse association of root growth inhibition and transient expression efficiency suggested that C58C1(pTiB6S3ΔT)H may circumvent a plant defense barrier to enable high transient expression levels in Arabidopsis seedlings. However, interestingly, root length was significantly lower in Col-0 and efr-1 seedlings with C58C1(pTiB6S3ΔT)H infection than in uninfected seedlings (MOCK), despite the significantly higher transient expression efficiency with the AGROBEST than the ABM50 method (Figure 3). Thus, although C58C1(pTiB6S3ΔT)H remains a strain causing the least inhibition in seedling root growth as compared to other A. tumefaciens strains, whether the observed root growth inhibition results from PTI contributing to reduce transient expression efficiency requires future investigation. Other factors in addition to PTI may contribute to the enhanced transient expression efficiency by AGROBEST.
C58C1(pTiB6S3ΔT)H has been known to achieve high transformation efficiency in several plant species including Arabidopsis, but the underlying mechanism is not known. The nomenclature of Agrobacterium strains used in plant transformation experiments is often simplified, which causes confusion and could sometimes be misleading. C58C1(pTiB6S3ΔT)H is often simplified as C58C1 in the plant community. C58C1 is in fact named after curing pTiC58 from the wild-type virulent strain C58, and rifampicin (Rif)-resistant strains are selected from C58C1 for convenient use to acquire various disarmed Ti plasmids transferred from different Agrobacterium strains [52, 53]. Therefore, C58C1(pTiB6S3ΔT)H is a Rif-resistant C58C1 harboring the octopine-type Ti plasmid pTiB6S3 with the removal of the T-DNA region  and containing a pCH32 helper plasmid with increased expression of virulence genes virG and virE2. GV3101(pMP90) is a C58-derived disarmed strain, in which pMP90 is a nopaline-type Ti plasmid, pTiC58, with the removal of T-DNA . Therefore, in theory, C58C1(pTiB6S3ΔT)H should share the same chromosomal background with GV3101(pMP90) and only differ in the use of different Ti plasmids and the presence of the helper plasmid pCH32. Future work to determine which genetic factor(s) contribute to increased transient expression efficiency with less growth inhibition by C58C1(pTiB6S3ΔT)H will shed light on understanding the molecular mechanisms underlying the observed high transient transformation and expression efficiency.
In this study, we developed a valuable and novel method, named AGROBEST, and uncovered the key factors enabling this unprecedented high transient transformation and reporter gene expression efficiency in the immune receptor mutant efr-1 and in wild-type Col-0 Arabidopsis seedlings. The applicability for transient expression of MYB75 in activating downstream gene expression in a Col-0 background further suggested that AGROBEST may be a feasible method to use in examining transcription factor actions or gain-of-function studies in different Arabidopsis ecotypes/genotypes. Because most plants do not harbor EFR, which is only present in Brassicaceae, the established method may be applicable in other plant species. This fast, sensitive, and quantitative assay was routinely used with culture plates, which are easily scaled up for quick and systematic screens. Importantly, this method nicely compliments the commonly used Arabidopsis mesophyll-protoplast transfection [15, 16] and Agrobacterium- mediated leaf infiltration in N. benthamiana for gene functional studies and provides advantages for its high reproducibility without advanced skills. Furthermore, AGROBEST may be an alternative method for evaluating Agrobacterium virulence and discovering and dissecting gene functions involved in various steps of Agrobacterium-mediated DNA transfer. The method may help unravel the mechanisms underlying Agrobacterium infection in unwounded cells/tissues.
Materials and growth condition
Strains, plasmids, and primer sequences used in this study are in Additional file 2: Table S2 and Additional file 3: Table S3. The bacterial growth conditions and procedures for plasmid and mutant constructions are described in Additional file 4: Methods S1. Arabidopsis thaliana plants included ecotype Columbia-0 (Col-0), Wassilewskija (Ws-2), T-DNA insertion mutants efr- 1 (SALK_044334) and fls2 (SALK_093905) and the DEX-inducible AvrPto transgenic line generated in a Col-0 background were obtained from the Arabidopsis Biological Resource Center (Ohio). Seeds were sterilized in 50% bleach (v/v) containing 0.05% Triton X-100 (v/v) for 10 min, rinsed 5 times with sterile water, and incubated at 4°C for 3 days. For germination, 10 seeds were transferred to 1 ml 1/2 MS liquid medium (1/2 MS salt supplemented with 0.5% sucrose (w/v), pH 5.5 [pH adjusted to 5.7 by KOH but pH 5.5 after autoclaving], in each well of a 6-well plate. Germination and growth took place in a growth room at 22°C under a 16-hr/8-hr light–dark cycle (75 μmol m-2 s-1).
Agrobacterium infection in Arabidopsis seedlings
For AGROBEST infection assay, A. tumefaciens was freshly streaked out from -80°C glycerol stock onto a 523 agar plate for 2-day incubation at 28°C. A fresh single colony from the plate was used to inoculate 5 ml of 523 liquid medium containing appropriate antibiotics for shaking (220 rpm) at 28°C for 20–24 hr. For pre-induction of A. tumefaciens vir gene expression, A. tumefaciens cells were pelleted and re-suspended to OD600 0.2 in various liquid media including LB, LB-MES (LB with 10 mM MES, pH 5.7) [53, 54] or AB-MES (17.2 mM K2HPO4, 8.3 mM NaH2PO4, 18.7 mM NH4Cl, 2 mM KCl, 1.25 mM MgSO4, 100 μM CaCl2, 10 μM FeSO4, 50 mM MES, 2% glucose (w/v), pH 5.5)  with different concentrations of acetosyringone (AS; 0, 50 or 200 μM) without antibiotics, then shaken (220 rpm) at 28°C for 12–16 hr. Before infection, A. tumefaciens cells were pelleted and re-suspended in desired co-cultivation liquid media to OD600 0.02. The growth medium of Arabidopsis seedlings was replaced with 1 ml A. tumefaciens cells freshly prepared above and incubated in the same growth room until further analysis. Three-day-old seedlings were treated with 10 μM DEX for 1 day before infection for the following 3 days. When the removal of Agrobacterium cells was required, co-cultivation medium was removed after the chosen infection time and replaced with 1 ml freshly prepared MS medium containing 100 μM Timentin and incubated for additional days before analysis. The procedures for the seedling transient transformation assay using the method optimized by Marion et al. and FAST Method developed by Li et al. were performed [26, 27] and described in Additional file 4: Methods S1. Unless indicated, 10 seedlings grown in each well were infected and 3 biological repeats were performed in each independent experiment.
Plant RNA extraction and quantitative RT-PCR
RNA was extracted from Arabidopsis seedlings as described . An amount of 4 μg total RNA was used to synthesize first-strand cDNA with SuperScript III Reverse Transcriptase (Invitrogen) and oligo dT primer. Quantitative PCR involved the Applied Biosystems QuantStudio 12 K Flex Real Time PCR machine and Power SYBRR Green PCR Master Mix (Invitrogen). Arabidopsis ACTIN 2 (At3g18780) or UBC21 (At5g25760) was an internal control.
GUS staining and activity assays
Seedlings were stained with 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) at 37°C for 6 hr unless indicated or quantified with a fluorescence substrate (4-methylumbelliferyl-β-D-glucuronide [MUG]) as described . For MUG assay, fluorescence was determined using a 96 microtiter-plate reader (Bio-Tek Synergy Mx, 356 nm excitation 455 nm emission with ±20 nm filter) and calculation of specific GUS enzyme activity was based on the standard curve of 0.5–500 pmole (0.5, 5, 50 and 500 pmole) 4-MU standards obtained from the same microtiter plate. For relative GUS activity, the fluorescence signal value was normalized by an equal amount of proteins with subtraction of the background fluorescence signal detected by the mock control.
Fluorescence signals were observed by use of a Zeiss LSM 510 Meta Confocal microscope. Venus signals were observed at 488-nm excitation with an HFT 488/514-nm filter and emission with NTF 515- and BP 505- to 530-nm filters. RFP signals were observed at 488-nm excitation with an HFT 405/488-nm filter and emission with NFT 545 and LP 650 filters.
Transient expression of MYB75 and anthocyanin content assay
Four-day-old seedlings were infected with A. tumefaciens strain C58C1(pTiB6S3ΔT)H carrying the control or MYB75-expressing binary vector in ABM-MS liquid medium for 3 days. The co-cultivation medium was then replaced with 1 ml fresh MS medium (1/2 MS, 2% sucrose (w/v), pH adjusted to 5.7 by KOH but pH 5.5 after autoclaving) containing 100 μM Timemtin and then incubated for 3 days. For anthocyanin content assay, seedlings were blot-dried briefly, weighed, ground into powder with liquid nitrogen and mixed with 1 ml extraction buffer (0.12 M HCl, 18% isopropanol (v/v)). The mixture was boiled for 90 sec and centrifuged at 16000 × g for 15 min. The supernatant was collected and measured at OD535 (A535) and OD650 (A650). Anthocyanin content was calculated as A535 - (2.2 × A650)/fresh weight (g) .
Transient expression of GI::LUC2 and bioluminescence measurement
Four-day-old seedlings were infected with A. tumefaciens strain C58C1(pTiB6S3ΔT)H carrying p1390-GI::LUC2 or empty vector (pCAMBIA1390) in ABM-MS co-cultivation medium. At 3 dpi, each seedling was transferred to MS medium (1/2 MS, pH adjusted to 5.7 by KOH but pH 5.5 after autoclaving) containing 100 μM Timentin and 0.5 mM luciferin in a black 96-well plate. Bioluminescence activity was measured and analyzed as described .
Luciferase activity assay
Arabidopsis seedlings after infection were surface sterilized with 1% bleach (0.05% sodium hypochlorite) for 5–10 min and washed with sterile water 3 times to remove bacteria before assay. The washing step is essential to minimize the background signals expressed in bacteria because of the use of intron-less LUC2 reporter. For photography, 10 seedlings infected by each method were placed in a clean 15-cm square Petri dish and covered with 100 μl 1 mM luciferin. Luciferase intensity was imaged by use of the XENOGEN IVIS lumina system with 5-sec exposure time. Bioluminescence assay involved the luciferase assay system (Promega). Briefly, 10–15 seedlings after a washing were blot-dried with tissue paper before being frozen with liquid nitrogen and stored at -80°C. Seedlings were ground into fine powder by liquid nitrogen, mixed with 300 μl cell-culture lysis reagent (Promega), and centrifuged at 16000 × g for 10 min at 4°C. Supernatant was 100× diluted with cell-culture lysis reagent. In total, 20 μl cell lysate was mixed with 100 μl Luciferase Assay Reagent and the signal was detected by use of lumat LB 9507 (Berthold Technologies). The bioluminescence signal was normalized to the protein amount of each sample quantified by the Bradford protein assay (Bio-Rad).
The authors thank Hau-Hsuan Hwang, Lay-Sun Ma, Jer-Sheng Lin, and Po-Yuan Shih for discussion and critical reading of the manuscript; and Yajie Niu and Hoosun Chung for preliminary transient expression tests on different seedling ages. We thank Dr. Inhwan Hwang for NLS-RFP; Drs. Stanton Gelvin and Lan-Ying Lee for pBISN1, Venus-intron and BiFC vectors; and Ms. Mei-Jane Fang from the Cell Biology Core Laboratory at the Institute of Plant and Microbial Biology, Academia Sinica, for excellent technical support on confocal microscopy. This work was supported by research grants from the National Science Council of Taiwan (NSC 99-2918-I-001-005 and NSC 101-2321-B-001-033 to E. M. Lai, NSC100-2311-B-001-028-MY3 to S. H. Wu) and the US National Institutes of Health (R01GM60493 and R01GM70567 to J. Sheen).
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