A high-throughput method for isolation of salicylic acid metabolic mutants
- George Marek†1,
- Ryan Carver†1,
- Yezhang Ding1,
- Deepak Sathyanarayan1,
- Xudong Zhang1 and
- Zhonglin Mou1Email author
© Marek et al; licensee BioMed Central Ltd. 2010
Received: 24 August 2010
Accepted: 23 September 2010
Published: 23 September 2010
Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor Acinetobacter sp. ADPWH_lux-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.
On the basis of the previously described biosensor-based method, we simplified the tissue collection and the SA extraction procedure. Leaf discs were collected and boiled in Luria-Bertani (LB), and then the released SA was measured with the biosensor. The time-consuming steps of weighing samples, grinding tissues and centrifugation were avoided. The direct boiling protocol detected similar differences in SA levels among pathogen-infected wild-type, npr1 (nonexpressor of pathogenesis-related genes), and sid2 (SA induction-deficient) plants as did the previously described biosensor-based method and an HPLC-based approach, demonstrating the efficacy of the protocol presented here. We adapted this protocol to a high-throughput format and identified six npr1 suppressors that accumulated lower levels of SA than npr1 upon pathogen infection. Two of the suppressors were found to be allelic to the previously identified eds5 mutant. The other four are more susceptible than npr1 to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 and their identity merits further investigation.
The rapid SA extraction method by direct boiling of leaf discs further reduced the cost and time required for the biosensor Acinetobacter sp. ADPWH_lux-based SA estimation, and allowed the screening for npr1 suppressors that accumulated less SA than npr1 after pathogen infection in a high-throughput manner. The highly efficacious SA estimation protocol can be applied in genetic screen for SA metabolic mutants and characterization of enzymes involved in SA metabolism. The mutants isolated in this study may help identify new components in the SA-related signaling pathways.
Salicylic acid (SA) is a key signaling molecule in plant defense against biotrophic pathogens [1, 2]. Upon pathogen attack, SA accumulates in plant cells [3, 4]. Prevention of SA accumulation leads to disease susceptibility , whereas treatment with SA confers resistance to a variety of biotrophic pathogens [6, 7]. Thus, understanding the mechanisms underlying SA accumulation is critical in the study of plant immunity.
Nawrath and Métraux  performed a genetic screen for Arabidopsis mutants that do not accumulate SA after pathogen infection and identified two genetic loci, SID1/EDS5 and SID2/EDS16, which were later shown to encode a chloroplast MATE (multidrug and toxin extrusion) transporter  and an SA biosynthetic enzyme ICS1 (isochorismate synthase) , respectively. In the screen, Nawrath and Métraux used an HPLC-based method to quantify the SA levels in the pathogen-infected leaf tissues from about 4,500 individual M2 plants. Because the HPLC-based method involves extraction of SA in organic solvents, evaporation of organic solvents, chromatographic purification and detection by fluorescence spectroscopy [11, 12], it is extremely costly and time-consuming. To screen for more SA metabolic mutants, a much faster and less expensive method is needed.
Huang et al. recently developed an SA biosensor, named Acinetobacter sp. ADPWH_lux . This strain is derived from Acinetobacter sp. ADP1, and contains a chromosomal integration of a salicylate-inducible luxCDABE operon. The operon encodes a luciferase (LuxA and LuxB) and the enzymes that produce its substrate (LuxC, LuxD and LuxE) so cells that express the cluster emit the 490-nm light spontaneously . The biosensor is highly specific to SA, methyl-SA, and the synthetic SA derivative acetylsalicylic acid , thus suitable for the quantification of SA from crude plant extracts.
We previously described an approach for the simultaneous quantification of free and glucose conjugated SA from Arabidopsis leaf extracts using Acinetobacter sp. ADPWH_lux . Here we present a further shortened protocol for the estimation of SA levels in pathogen-infected leaf tissue. Using the protocol described, we have performed a genetic screen for suppressors of the npr1 (nonexpressor of pathogenesis-related genes) mutant that hyperaccumulates SA during pathogen infection [16, 17].
Rapid Extraction of SA by Direct Boiling of Leaf Discs
High-Throughput Screening for SA Metabolic Mutants
We then attempted to set up a mutant screen aimed at identifying new components involved in regulating SA accumulation. Since the npr1 mutant accumulates significantly higher levels of SA than wild type during pathogen infection, and NPR1 is a key positive regulator of SA-mediated immune responses [19–21], we reasoned that npr1 suppressors, which accumulate less pathogen-induced SA than npr1, would help uncover important regulators of plant immunity. We therefore decided to use the npr1 mutant as starting material for the screen.
Confirmation of the Putative SA Metabolic Mutants Using HPLC
To identify the genetic mutations in 62 and 69, the open reading frames of the two alleles of EDS5 were sequenced. The allele 62 carries a transition mutation converting a TGG to a premature stop condon (TGA) at nucleotide 753 of the coding region, whereas the 69 mutation is caused by a G-to-A transition in the AG from the splice acceptor site in intron 5, which may lead to an abnormal splicing at the border of intron 5/exon 6. These results indicate that the mutant screen identified two new alleles of the previously isolated eds5 mutants .
Pathogen Resistance Test for the SA Metabolic Mutants
Here we present a direct boiling protocol for the rapid estimation of SA from plant tissue using the SA biosensor Acinetobacter sp. ADPWH_lux. This protocol is much faster and less expensive than the previously described biosensor-based approaches [13, 15]. The fast sample preparation procedure, which comprises inoculation of one leaf on each plant, collection of leaf discs, and boiling in LB, significantly reduced the time spent on inoculation, tissue collection, grinding and centrifugation. This method was not designed to accurately determine the SA concentration. Rather, it is intended to estimate SA levels for rapid genetic screens with significant reductions in cost and processing time. We acknowledge that SA levels induced by Psm ES4326 infection can vary quite a lot among individuals of the same genotype, depending on plant growth conditions. Additionally, this method may not be sensitive enough in some applications, such as time-course quantifications of SA levels with pathogen infection. However, the successful genetic screen for suppressors of the npr1 mutant has demonstrated the efficacy of this high-throughput strategy. We hope that the methodology presented in this study can help saturate the genetic screens for SA metabolic mutants, which in turn will facilitate a more thorough understanding of this important plant defense molecule [22, 23].
Plant material and pathogen infection
The wild type used was the Arabidopsis thaliana (L.) Heynh. Columbia (Col-0) ecotype, and the mutant alleles used were npr1-3 , eds5-1  and sid2-1 . EMS mutagenesis was performed as described in . Briefly, one gram of npr1-3 seeds were placed in 25 mL of 0.2% EMS (v/v) in a 50-mL Falcon tube and incubated on a rocking platform for 15 hours. After the seeds were washed eight times with water, they were suspended in 0.1% agarose and sown on soil. M2 seeds were collected in pools when the M1 plants reached to maturity. The M2 plants were germinated, transplanted to 96-pot trays, and then grown at 22~25°C under a 16 hr light/8 hr dark regime for three weeks. Infection of plants with Psm ES4326 was performed as described previously . One leaf on each plant was infiltrated with a suspension of Psm ES4326 (OD600 = 0.001).
Preparation of crude extract
Twenty-four hours after Psm ES4326 infection, a leaf disc was collected from each infected plant using a hole punch and placed in 200 μl of LB in a well of a 96-well PCR plate. The plate was then heated at 95°C for 20 min in a PCR machine and cooled down to room temperature.
Detection of salicylic acid using Acinetobacter sp. ADPWH_lux and HPLC
An overnight culture of Acinetobacter sp. ADPWH_lux was diluted in 37°C LB (1:20) and grown for ~2 hrs at 200 rpm to an OD600 of 0.4. Using a multipipette, 50 μl of biosensor culture was added to each well in a 96-well black cell culture plate, and then 50 μl of the crude extract was added to each well and mixed by pipette action. The plate was incubated at 37°C for 1 hr without shaking before luminescence was read using a Veritas™ Microplate Luminometer (Promega Corporation, Sunnyvale, CA). Measurement of SA with HPLC was done as described by Verberne et al. . Briefly, ~0.1 g tissues were ground in liquid nitrogen and extracted with 1 mL of 90% methanol. After centrifugation at 14,000 g for 10 min, the supernatant was transferred into a microcentrifuge tube. The pellet was extracted with 0.5 mL of 100% methanol and the supernatant was transferred to the same tube and dried in a speed vacuum to final volume of ~50 μL. The residue was resuspended to 500 μL with hydrolysis buffer (0.1 M sodium acetate buffer, pH 5.5). The mixture was equally split into two microcentrifuge tubes [one for free SA, the other for glucose conjugated SA (salicylic acid 2-O-β-D-glucoside or SAG)]. For SAG, 10 units of β-glucosidase were added to the tube. After incubation at 37°C for 1.5 hr, an equal volume of 10% TCA was added to both tubes. After centrifugation at 14,000 g for 10 min, the supernatant was transferred to a fresh tube and partitioned with 1 mL extraction solvent (1 ethylacetate: 1 cyclohexane). The top organic phase was transferred to a new tube, and dried in a speed vacuum to final volume ~25 μL. The residue was resuspended to 0.25 mL with 0.2 M sodium acetate buffer (pH 5.5). After centrifugation at 14,000 g for 10 min, the supernatant was used for HPLC analysis. The sample was eluted with 0.2 M sodium acetate buffer pH 5.5 in 10% methanol at a flow-rate of 0.80 mL/min.
All statistical analyses were performed with the data analysis tools (t-TEST: Two Samples Assuming Unequal Variances) in Microsoft Excel of Microsoft Office 2004 for Macintosh.
We thank Dr. Hui Wang (NERC/Centre for Ecology and Hydrology-Oxford, Oxford, UK) for the SA biosensor strain Acinetobacter sp. ADPWH_lux, and Drs. William Gurley and Sixue Chen (University of Florida, FL) for access to the Veritas™ Microplate Luminometer and the HPLC equipment, respectively. This work was supported by a grant from the Herman Frasch Foundation for Chemical Research and a grant from the National Science Foundation (IOS-0842716) awarded to Z.M., and publication of this article was funded in part by the University of Florida Open-Access Publishing Fund.
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