Rapid in vivo analysis of synthetic promoters for plant pathogen phytosensing
© Liu et al; licensee BioMed Central Ltd. 2011
Received: 2 September 2011
Accepted: 17 November 2011
Published: 17 November 2011
We aimed to engineer transgenic plants for the purpose of early detection of plant pathogen infection, which was accomplished by employing synthetic pathogen inducible promoters fused to reporter genes for altered phenotypes in response to the pathogen infection. Toward this end, a number of synthetic promoters consisting of inducible regulatory elements fused to a red fluorescent protein (RFP) reporter were constructed for use in phytosensing.
For rapid analysis, an Agrobacterium-mediated transient expression assay was evaluated, then utilized to assess the inducibility of each synthetic promoter construct in vivo. Tobacco (Nicotiana tabacum cv. Xanthi) leaves were infiltrated with Agrobacterium harboring the individual synthetic promoter-reporter constructs. The infiltrated tobacco leaves were re-infiltrated with biotic (bacterial pathogens) or abiotic (plant defense signal molecules salicylic acid, ethylene and methyl jasmonate) agents 24 and 48 hours after initial agroinfiltration, followed by RFP measurements at relevant time points after treatment. These analyses indicated that the synthetic promoter constructs were capable of conferring the inducibility of the RFP reporter in response to appropriate phytohormones and bacterial pathogens, accordingly.
These observations demonstrate that the Agrobacterium-mediated transient expression is an efficient method for in vivo assays of promoter constructs in less than one week. Our results provide the opportunity to gain further insights into the versatility of the expression system as a potential tool for high-throughput in planta expression screening prior to generating stably transgenic plants for pathogen phytosensing. This system could also be utilized for temporary phytosensing; e.g., not requiring stably transgenic plants.
Transgenic techniques have become a powerful tool to address important biological and agricultural challenges, and there is great potential for the production of designer plants using modern biotechnology tools [1, 2]. Many of these applications are addressed by the use of transgenic techniques, including the introduction of homologous or heterologous genes in plants with modified functions and altered expression patterns.
The overall goal of our project is to design plants for the purpose of early detection of plant pathogen infection, which we propose would be attainable employing pathogen inducible promoters fused to reporter genes for altered phenotypes in response to the pathogen infection. The use of "tuned" inducible promoters is a key design feature when constructing transgenic plants as "phytosensors." Inducible plant defense is controlled by signal transduction pathways, inducible promoters and cis-acting regulatory elements (REs) corresponding to key proteins involved in defense, and pathogen-specific responses. These cis-acting regulatory elements are conserved among plant species, enabling them to be used to construct synthetic inducible promoters in heterologous expression systems [3–5]. Upon detection of a pathogen, we expect a gain-of-function response in the form of expression of the visible marker gene.
Our initial study demonstrated the possible utilization of these cis-acting regulatory elements in building phytosensors . This prompted us to construct a number of synthetic promoters consisting of selected cis-acting regulatory elements fused to a red fluorescent protein pporRFP reporter gene (from the hard coral Porites porites; ) for use in phytosensing. However, examining a suite of synthetic promoter constructs for their suitability and potential applications in phytosensing involves the generation of many stably transgenic plants harboring many different constructs. Although there is no substitute for stable plant transformation for complete transgenic construct characterization, current procedures are time-consuming, laborious, and not suited for high-throughput assays. Alternatively, transient expression through agroinfiltration is a simple and useful method and has been demonstrated to be effective in many plant species [8–19]. The use of transient gene expression assays offer an opportunity to study a large number of transgene constructs rapidly, which particularly would be advantageous for evaluating the transcriptional activity of different promoters and the interaction between transcription factors and cis-acting regulatory sequences presented in plant promoters [3, 10, 12, 17, 20, 21].
In the present study, we have evaluated an Agrobacterium-mediated transient expression assay to assess the inducibility of a number of synthetic promoter constructs in vivo. Our results demonstrate that this transient expression is a robust and efficient method for in vivo assays of promoter constructs in response to biotic and abiotic agents. The use of synthetic promoters combined with the agroinfiltration assay provides a robust screening method to rapidly evaluate plant-pathogen interactions prior to stable plant transformation.
Results and Discussion
In order to examine the inducubility of the synthetic promoter constructs in response to pathogen infection, tobacco leaves were inoculated with the bacterial pathogens 24 h after initial agroinfiltation of each synthetic promoter construct and a time-course analysis of pporRFP expression was conducted. As shown in Figure 3, the synthetic promoter constructs, with or without B_A enhancer, exhibited inducibility in response to P. syringae pv. tomato within 24-48 hpi. This result, in fact, reflects the rapid HR reaction caused by the bacterial pathogen during the incompatible interaction with tobacco. Yet, at time point 24 hpi, P. syringae pv. tomato showed a higher inducibility of the pporRFP expression in synthetic promoter constructs containing PR1 and SARE regulatory elements. We believe this result was most likely observed because of the fact that the signal transduction of HR reaction is primarily through the SA-dependent pathway. Accordingly, among the regulatory elements, ERE conferred the least inducibility of pporRFP expression in response to P. syringae pv. tomato, implying less association of ethylene signaling with HR reaction. Time course analysis of pporRFP expression in response to P. syringae pv. tabaci indicated a gradual increase of RFP expression over time following bacterial inoculation (Figure 3, 4b). These results reflect the fact that the normal-sensitive "wildfire" disease symptom, caused by the bacterial pathogen during the compatible interaction with tobacco develops within 72 hpi. In response to P. marginalis infection, the four regulatory elements individually gave rise to a low level of induction of the pporRFP expression at all the time points (Figure 3). The only exception was observed for the ERE regulatory element, which conferred relatively higher induction of the pporRFP expression compared to other regulatory elements. Furthermore, depending on the synthetic promoter constructs used, the increase in pporRFP expression over time caused by different bacterial pathogens was also variable. For example, as shown in Figure 3, while PR1 and SARE regulatory elements conferred rapid induciblity of pporRFP expression at time point of 24 h after P. syringae pv. tomato infection, ERE showed a low level of inducibility after P. syringae pv. tomato infection but relatively higher level of induciblity over time after P. syringae pv. tabaci and P. marginalis infections.
Our experimental system hinges on inducible regulation of cis-acting elements upon bacterial pathogen exposure for early pathogen detection using Agrobacterium-mediated transient assay. Agroinfiltration had been used to study the functional activity of promoters and/or genes during bacterial pathogen, virus, abiotic or environmental stresses [29, 30]. In our experiments, the Agrobacterium-mediated transient expression was used together with gain-of-function experiments of inducible regulatory elements for its suitability, inducibility and potential applications in early bacterial pathogen phytosensing. We have demonstrated that agroinfiltration of tobacco leaves with synthetic promoter constructs has the potential to validate the inducibility of the regulatory elements in response to the corresponding phytohormone treatment. Moreover, it is capable of examining the responsiveness of the pporRFP reporter to bacterial pathogens in both compatible and incompatible interactions. These observations indicate that the Agrobacterium-mediated transient expression can be used as a rapid screening tool for in vivo analysis of promoter constructs for pathogen phytosensing before conducting stable plant transformation experiments, thus narrowing the most appropriate and effective constructs to use for stable phytosensing experiments. We do not believe it can make absolute predictions for the results of stable transformation; stable transformation involves integration into the plant genome and thus every new transformation event could confer a variety of different synthetic promoter response activities. In addition, this system could also be utilized for temporary phytosensing without the need for deploying stable transformants, since levels of transient expression may be much higher than that of stable transgenic plants [16, 31], which is a major advantage for phytosensing. Both transient and stable transgenic expression systems should exhibit congruent expression patterns [16, 19, 32, 33]. Among the four regulatory elements we tested, ERE motif confers the highest expression level while JAR motif confers the lowest (Figures 2, 3 and 4). We therefore, conclude that ERE, PR1 and SARE motifs have more application potential for pathogen phytosensing than the JAR motif.
The use of this expression system for temporary pathogen phytosensing has several benefits when compared to other systems. It avoids the position effects of transgene insertion, requires much less time for measurement of gene expression, and eliminates the possibility of escape of transgenes into the environment. Nevertheless, a few limitations should be considered with regard to our transient expression system for use in pathogen phytosensing. Large variation in expression is a major disadvantage. Leaf size and position, age of plants and growth conditions can affect the transgene expression. The slight discrepancy between inducibility of expression of 4 × JAR RFP and B4 × JARA RFP in response to bacterial pathogens may come from the variation (Figure 3i, j). The OD of infiltration is another major consideration for increasing repeatable results [11, 13, 24, 25, 27]. It is always advantageous to adjust the OD of bacterial pathogens to achieve the expected HR (high concentration) or disease symptoms (low concentration) [24–27]. Post-transcriptional gene silencing should also be considered, since plant endogenous defenses can hinder the level and duration of transient expression of reporter genes .
We have tested the suitability, inducibility and potential of gain-of-function analyses of synthetic promoters containing inducible regulatory elements using Agrobacterium-mediated transient expression for the purpose of engineering transgenic plants for early detection of pathogen infection. We have demonstrated that agroinfiltration of tobacco leaves has potential to validate the sensitivity and inducibility of the regulatory elements in response to the corresponding phytohormone treatment. Moreover, it is capable of examining the responsiveness of the pporRFP reporter to bacterial pathogen infection in both compatible and incompatible interactions. Our results indicate that the Agrobacterium-mediated transient expression can be used as a rapid screening tool for in vivo analysis of inducible regulatory elements for pathogen phytosensing, allowing high-throughput in planta expression screening before conducting stable plant transformation. It could also be utilized for temporary phytosensing which does not require deploying stable transformants.
Details of construction of pSK min35SGUS vector consisting of distinct cis-acting regulatory elements (REs) without or with CaMV 35S enhancer motifs have been given in our previous study . This vector plasmid was originally constructed for β-glucuronidase (GUS) reporter expression with the ability to swap GUS for fluorescent protein reporters for use in a fluorescent phytosensing system, an in vivo system. The pSK min35SGUS vector plasmid containing four copies of distinct REs: pathogenesis-related (PR1), salicylic acid responsive element (SARE), ethylene responsive element (ERE), or jasmonic acid responsive element (JAR) (sequences reported in Ref. 6) were selected for replacing GUS with a red fluorescent protein (RFP) reporter (Figure 1). For enhanced synthetic promoters, version 2 of the CaMV 35S enhancer motif where the RE tetramer was placed between B (-415 to -90) and A1 (-90 to -46) regions of 35S promoter  was used in the present study (Figure 1).
The red fluorescent protein pporRFP from coral Porites porites was used to replace the GUS reporter for potential in vivo reporting. Prior to that, PCR-mediated site-directed mutagenesis was performed to remove the HindIII restriction site at the position of +138 in pporRFP cDNA [GenBank accession number DQ206380] with nucleotide G replaced by A but without changing the encoded amino acid. The GUS reporter of the pSK vector plasmids was then replaced with the RFP reporter (Figure 1). These distinct synthetic promoter-RFP fusion cassettes in pSK vector plasmids were named as: pSK (4 × PR1), pSK (4 × SARE), pSK (4 × ERE), pSK (4 × JAR), pSK (B 4 × PR1 A), pSK (B 4 × SARE A), pSK (B 4 × ERE A), pSK (B 4 × JAR A). Appropriate negative control vectors (empty vectors -4635S RFP and B_A RFP) were produced by digestion of pSK (4 x PR1) and pSK (B 4 × PR1 A) with XbaI and SpeI (to remove the regulatory element tetramer) followed by self-ligation.
For use in agroinfiltration, each synthetic promoter-RFP fusion cassette was excised from the pSK vector constructs and was inserted into the SacI-HindIII site of the binary vector pZP222 .
Tobacco (Nicotiana tabacum cv. Xanthi) plants were grown in a growth chamber at 25°C under fluorescent white light in a 16:8 h light/dark cycle. Six-week-old plants were used for agroinfiltration assays.
Preparation of Agrobacteriumsuspension
Agrobacterium tumefaciens strain GV3085 was transformed with each individual construct by electroporation. A. tumefaciens containing individual constructs was grown on yeast extract peptone [(YEP) 10 g/L yeast extract, 10 g/L peptone, 5 g/L NaCl, 15 g/L agar] solid medium supplemented with rifampicin (50 mg/L), spectinomycin (200 mg/L), and streptinomycin (50 mg/L) at 28°C for 2 days. One single colony was inoculated in 2 ml YEP liquid medium supplemented with the above-mentioned antibiotics and grown for ~2 h at 28°C. One milliliter of this starter culture was then inoculated in 25 ml YEP liquid medium and grown for overnight at 28°C. Agrobacterium cells were collected by centrifugation for 10 min at 3000 g and resuspended in 25 ml infiltration medium (50 mg/ml d-glucose, 50 mM MES, 2 mM NaPO4.12H2O, and 0.1 mM acetosyringone). Centrifugation followed by resuspension in the infiltration medium was repeated for 2 times as above. The bacterial suspension was adjusted to a final OD600 of 0.3 for agroinfiltration.
Agroinfiltration of tobacco leaves
Tobacco plants were removed from the growth chambers and placed under a white fluorescent lamp for 1 h prior to infiltration to open the stomata fully as an aid to infiltration. Infiltration was performed on near fully expanded leaves (~ 5 × 6 cm large, flat, dark green, located in the middle position of the plant) that were still attached to the intact plant. Leaves of the same age on the same branch were used for each experimental test. Each bacterial suspension was infiltrated into leaves of three different plants from the abaxial side of the leaf with a needleless syringe. By infiltration, 100 μl of bacterial suspension was injected into each spot (typically 3-4 cm2 in each infiltrated area). After agroinfiltration, tobacco plants were covered with transparent plastic covers which were sprayed with water and maintained in a growth chamber at 22°C under 16 h light for 24 h. Three biological replicates (i.e., three plants) were used, and the experiments were repeated independently at least three times.
Biotic and abiotic treatments
For chemical treatments, 48 hrs after the initial agroinfiltration, 4 mM salicylic acid (SA), 4 mg/ml ethephon (an ethylene releasing chemical), or 100 μM methyl jasmonate (MeJA) (all from Sigma, St. Louis, MO, USA) was further infiltrated to the same infiltrated spots. For mock control treatments, leaves were infiltrated with water. For bacterial pathogen treatments, Pseudomonas syringae pv. tomato, P. marginalis, and P. syringae pv. tabaci, kindly provided by Dr. Bonnie Ownley, were grown individually at 28°C in tryptic soy broth (TSB) (Becton Dickinson, Sparks, MD, USA) medium overnight. After centrifugation, bacterial cells were resuspended in 10 mM MgCl2, followed by centrifugation and were resuspended again. Twenty-four hours after the initial agroinfiltration, each bacterial suspension was further infiltrated at the same infiltrated spots. For mock control treatments, leaves were infiltrated with 10 mM MgCl2. The numbers of bacteria was estimated in leaf disks (5 mm in diameter) taken from infiltrated areas at different time points post-infection. The discs were ground in 1 ml of 10 mM MgCl2, and serial dilutions were plated out on TSB solid medium. After incubation at 28°C for 24 h, the colonies were counted. All the experiments were repeated independently at least two times.
Determination of pporRFP expression
Expression of pporRFP reporter was measured at time points 0 and 72 h after phytohormone treatments and at time points 0, 24, 48, and 72 h after bacterial pathogen treatments. The treated plants were visualized using an epifluorescence microscope Olympus SZX12 (Olympus, Tokyo, Japan), and the images were captured using imaging software QCapture 2.56 (QImaging, BC, Canada). Fluorescent signal intensity was measured via scanning fluorescence spectrometry using SPEX Fluorolog II spectrophotometer (Horiba Jobin Yvon Inc., NJ, USA). The infiltrated spots were excited at 530 nm, and emission spectra was scanned and recorded from 550 to 640 nm. Intensity was measured at 591 nm in counts per second (cps).
Data processing and statistical analysis
Background subtraction was applied to each measurement of pporRFP expression by using measurements from non-transgenic tobacco as background expression when treated with the biotic and abiotic treatments. Data normalization was then conducted as described . The fold change in the expression of RFP reporter was calculated by using the normalized data at different time points of 24, 48 or 72 h after treatments divided by the normalized data at time point of 0 h. Statistical analyses were conducted by a two-sample t-test (p < 0.05).
We gratefully acknowledge funding by grants from USDA-NIFA. We thank Dr. Bonnie Ownley (University of Tennessee) for providing Pseudomonas cultures and Duncan G. Yeamen for help in maintaining plants and in other assistance.
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