Identification and Functional Characterization of the Soybean GmaPPO12 Promoter Conferring Phytophthora sojae Induced Expression

Identification of pathogen-inducible promoters largely lags behind cloning of the genes for disease resistance. Here, we cloned the soybean GmaPPO12 gene and found that it was rapidly and strongly induced by Phytophthora sojae infection. Computational analysis revealed that its promoter contained many known cis-elements, including several defense related transcriptional factor-binding boxes. We showed that the promoter could mediate induction of GUS expression upon infection in both transient expression assays in Nicotiana benthamiana and stable transgenic soybean hairy roots. Importantly, we demonstrated that pathogen-induced expression of the GmaPPO12 promoter was higher than that of the soybean GmaPR1a promoter. A progressive 5’ and 3’ deletion analysis revealed two fragments that were essential for promoter activity. Thus, the cloned promoter could be used in transgenic plants to enhance resistance to phytophthora pathogens, and the identified fragment could serve as a candidate to produce synthetic pathogen-induced promoters.


Introduction
Soybean (Glycine max [L.] Merr.) is a legume crop of great economic and agricultural importance across the world. Soybean yields are significantly reduced due to Phytophthora root and stem rot caused by Phytophthora sojae. The disease leads to 1-2 billon dollars in damage globally every year [1]. P. sojae is an oomycete, which is most closely related to algae such as kelp and diatoms, distinct from fungi and a member of the stramenopiles [1][2][3]. Oomycetes contain many other devastating plant pathogens, such as Phytophthora infestans responsible for potato and tomato late blight [4], and Plasmopara viticola and Pseudoperonospora cubensis causing downy mildew on grape and cucumber, respectively [5]. Breeding for disease resistance is the main strategy for combating oomycete diseases. In soybean, 15 Rps (resistance to P. sojae) genes that have been mapped to nine genomic loci are widely used for breeding [6]. Unfortunately, their effectiveness is often transient because pathogens can overcome disease resistance genes after they are widely and/or continually implicated [7]. Genetic engineering has the potential to provide a complement to some of the weaknesses by expressing antimicrobial compounds or components of known defense signaling pathways that confer durable and broad-spectrum resistance [8].
Several distinct strategies have been used in transgenic plants to enhance oomycete disease resistance, such as modifying the resistance signaling pathway by altering the expression of endogenous components, expressing antimicrobial peptides or pathogenesis-related proteins and even pyramiding the cloned R genes [9,10]. A bean gene encoding a polygalacturonase-inhibiting protein (PGIP), which is antagonistic to pathogen polygalacturonase, can protect transgenic tobacco against oomycetes (P. parasitica and Peronospora hyoscyami) [11]. Transgenic tobacco overexpressing the polyamine oxidase (PAO) gene, catalyzing the oxidative catabolism of spermidine and spermine to generate hydrogen peroxide, shows pre-induced disease tolerance against Phytophthora parasitica through activation of the plant systemic acquired resistance (SAR) pathway [12]. Functional stacking of three broad-spectrum potato R genes, Rpi-sto1, Rpi-vnt1. 1, and Rpi-blb3, in susceptible potato leads to a sum of the resistant spectra from the three individual Rpi genes [13]. Despite all efforts, however, the use of transgenic crops that are resistant to oomycete diseases is generally not as successful as conventional plant breeding [8]. This unexpected failure is often due to overexpression of the genes used for disease resistance, leading to a fitness penalty in normal plant growth and development. One possible way to solve the problem is to restrict expression of the transgenic target gene to only when it is needed at the infection sites using pathogen-induced promoters. However, discovery of these promoters largely lags behind identification of the genes for disease resistance [14].
Recently, several pathogen-induced promoters were identified in Arabidopsis, tobacco, and rice [14,15]. One of the best studied pathogen-induced promoters is from the tobacco hsr203J gene whose activation is rapidly induced by incompatible interaction and selectively expressed in response to the hypersensitive response (HR)-inducible bacteria [16]. Transgenic tobacco plants harboring a fusion between the hsr203J promoter and an elicitor (cryptogein) encoding gene from P. cryptogea exhibit HR resembling cell death when infected with several fungal and oomycete pathogens, thus displaying enhanced broad-spectrum disease resistance [17]. Similar results were also observed when the hsr203J promoter fused with popA was expressed in tobacco, although some transgenic lines showed runaway cell death [18]. In contrast, this promoter appears to be very weakly activated following pathogen inoculation in transgenic pears [19]. Thus, the suitability of the hsr203J promoter in different plant-microbe interaction systems remains unclear and the useful pathogeninducible promoters are still insufficient.
Pathogen-inducible promoters usually possess many conserved cis-regulatory elements that are potential binding sites for pathogen-responsive transcription factors [14,20]. Since each element may respond to one or more specific defense signaling pathways, the expression profile of the targeted gene is usually complex and obtaining a promoter that is not only highly specific to but also rapidly and strongly induced by pathogen infections is challenging. One strategy to overcome this complexity is to produce synthetic promoters by stacking or combining only well-characterized individual elements [15,21,22]. Two synthetic promoters, for example, were shown to combine good inducibility with low background in transgenic Arabidopsis [22]. Although testing these synthetic promoters in crop plants for disease resistance is still necessary, use of the synthetic promoters for pathogen phytosensing could potentially monitor plant disease outbreaks in agricultural fields [23]. Considering that rapid in vivo analysis of synthetic pathogen-inducible promoters is available [15,24], identifying functional cis-elements in crop plants is necessary.
Here, a P. sojae-induced promoter from the soybean GmaPPO12 gene was identified and characterized using stable transgenic soybean hairy roots and transient expression assays in N. benthamiana. Our results demonstrated that the gene was an immediate-early P. sojae-induced gene. Deletion mutant analysis revealed that a 113-bp fragment in the GmaPPO12 promoter is important for the activities. To our understanding, this is the first report of a pathogen-induced promoter in soybeans.

Soybean GmaPPO12 is highly induced by P. sojae
Recently, a large-scale microarray experiment involving three soybean cultivars and 72 biological replicates was carried out to analyze the response of soybean plants to infection by the pathogen P. sojae. The overall results showed that almost the entire plant genome undergoes significant transcriptional modulation in response to infection and genetic variation [25]. Here, we took advantage of the available microarray data to screen genes that were highly induced by infection with P. sojae. A gene annotated as Gma.7559.1.S1_s_at was one of the most strongly induced genes. This gene was upregulated 65-fold at 3 dpi (days post inoculation) and 80-fold at 5 dpi in the resistant cultivar V71-370, upregulated 103-fold at 3 dpi and 135-fold at 5 dpi in the moderately resistant cultivar Williams, and upregulated 47-fold at 3 dpi and 179-fold at 5 dpi in the susceptible cultivar Sloan. The expression levels of Gma. 7559.1.S1_s_at at 3 dpi were higher than that of the PR1a gene in the resistant and moderately resistant cultivars and weaker than the PR1a gene in the susceptible cultivar ( Figure  1).
Phylogenetic and functional domain analysis showed that this gene belongs to the soybean polyphenol oxidase (PPO) gene family ( Figure S1). In soybean, 11 PPO genes have been identified and designated as GmaPPO1-11 [26]. Gma. 7559.1.S1_s_at was not identified as one of these genes, and we found that it was an annotated soybean gene Glyma04g14361 [27], in this report designated as GmaPPO12. GmaPPO12 is predicted to encode a protein with 601 amino acids. Plant polyphenol oxidases (PPOs) are enzymes that oxidize ortho-diphenols to ortho-diquinones, or convert monophenols to ortho-diphenols. They are often considered to be defense proteins due to their pathogen and wound-induced expression [26]. GmaPPO12 is an immediate-early P. sojae infection response gene To further validate that the GmaPPO12 gene is an immediate-early P. sojae infection response gene, we used quantitative reverse transcription-polymerase chain reaction (RT-PCR) to characterize its expression profile in soybean roots at 0.5 and 2 h after infection with P. sojae zoospores. Since the GmaPPO12 gene has three other highly conserved homologs, we designed a pair of specific primers to distinguish it from the others ( Figure S1). Figure 2A shows that the transcript levels of GmaPPO12 increased at 0.5 hpi and 2 hpi and showed 53-fold and 33-fold higher mRNA levels, respectively, compared to 0 hpi. The transcript levels of soybean PR1a, a known pathogen-inducible gene, also increased at 0.5 hpi and 2 hpi and showed 23-fold and 28-fold induction, respectively compared to 0 hpi ( Figure 2A). Our results show that the induction levels of GmaPPO12 are much higher than those of PR1a at 0.5 hpi (p < 0.01). Thus, we selected this gene for further analysis and designated its promoter as a P. sojae immediately induced promoter.

GmaPPO12 promoter contains many known ciselements and is not up-regulated by the tested hormones except for ABA
A 1,500-bp fragment harboring the -1500 to +1 region (transcription start site of GmaPPO12 as +1) was isolated from soybean as the candidate pathogen-induced promoter. This fragment (GmaPPO12 promoter) was submitted to PlantCARE [28] to detect the cis-regulatory elements. Several elements that could be classified into three groups were identified within this promoter ( Figure S2). The first group is the basal regulatory elements, including nine CAAT-box elements [29]. The second group includes the well studied pathogen-inducible cis-acting elements, including six GT-1 elements [30], two Wbox elements [31], three WRKY-binding site WRKY elements [30], eight R response MYC binding sites [32], one regulatory defense-related gene CACGTG motif [33]. The third group is involved in abiotic stress or hormone response, such as the two GA-responsive GARE elements [34], one central element of gibberellin response MYB [35], one auxin response factor (ARF) binding sequence SURE [36], three water stress responsive elements MYB2 elements [37], one transcriptional activator ARR1AT [38], three abscisic acid (ABA) -responsive complex RYR elements [39], four ABA or stress responsive MYB1AT elements [40], one light-activated transcription binding site T-box [41], and one plant bZIP protein binding site SUREP [42]. In conclusion, based on the predictive identification of putative cis-elements, we hypothesize that the transcriptional activity of GmaPPO12 is induced by different biotic and abiotic stresses.
Since several predicted hormone-responsive elements exist in the GmaPPO12 promoter region, we used quantitative RT-PCR to examine the effects of five plant hormones on the GmaPPO12 gene ( Figure 2B). We harvested the 7-day-old soybean roots and washed them with water, then immersed them in jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), ethephon (ETH), gibberellic acid (GA), or doubledistilled water (ddH 2 O) (control) for 3 h. The results show that the transcript levels of GmaPPO12 was slightly reduced when treated with JA, SA, ethephon, or GA3, and have a 2.4-fold increase occurred when treated with ABA (p < 0.05). Thus, GmaPPO12 is not up-regulated by the tested hormones except for ABA.

The GmaPPO12 promoter mediates rapid and strong GUS expression upon Phytophthora capsici infection in N benthamiana leaves
To determine the inducibility of the promoter, we generated a construct containing the whole promoter region (1,500-bp) fused with the GUS reporter gene along with a 35S minimal (-46-+8) region ( Figure 3A). The 35S minimal region, 35S fulllength promoter and soybean PR1a promoter were used as controls. We examined GUS expression levels using Agrobacterium-mediated transient expression assay in N.

Figure 2.
Induced expression of GmaPPO12 and GmaPR1a in soybean roots. A. Expression patterns of soybean GmaPPO12 and GmaPR1a. Quantitative RT-PCR analysis was carried out to detect GmaPPO12 and GmaPR1a transcript abundance in 7-day-old soybean roots submerged in suspensions containing 10 5 zoospores of P. sojae per milliliter at 0 hpi, 0.5 hpi and 2 hpi (hours post inoculation). The soybean ACT20 gene was used as a reference gene with designed primers shown in Table S1. Transcript levels represent GmaPPO12 or GmaPR1a mRNA levels compared with reference mRNA levels and then normalized to the treated roots at 0 hpi. Biological triplicates were averaged. A significant difference compared using Student's t-test (** for p < 0.01). Bars indicate the standard error. B. Effects of the tested hormones on accumulation of soybean GmaPPO12 gene transcripts. Quantitative RT-PCR analysis was carried out to detect GmaPPO12 transcript abundance in 7-day-old soybean roots. The roots were submerged in ddH 2 O (control) or JA (100 µM), SA (100 µM), MeJA (100 µM), GA 3 (100 µM), ethephon (100 µM) for 3 h. The soybean ACT20 gene was used as a reference gene. Each column represents the relative transcript expression to the soybean reference gene then normalized to the roots treated with ddH 2 O. Biological triplicates were averaged. Means with different letters at the top are significantly different (p < 0.05 Duncan's multiple range tests), lines designated with the same letter exhibit no significant difference in response to the tested hormones. Bars indicate the standard error. benthamiana leaves. The method is simple and efficient for the quantitative analysis of plant promoters in vivo [24,43].
We examined induction activity upon inoculation with P. capsici zoospores after the Agrobacterium strains were infiltrated into N. benthamiana leaves. Quantitative GUS enzyme activity assays demonstrated that GmaPPO12 showed almost 8.2-and 10.8-fold induction, while PR1a showed 2.1and 4.6-fold induction at 0.5 and 2 hpi ( Figure 3B), respectively. Histochemical staining revealed that GmaPPO12 showed stronger induction than PR1a by P. capsici, although untreated tobacco leaves containing GmaPPO12 or PR1a showed weak background expression ( Figure 3C). To further confirm the results, we used quantitative RT-PCR to measure GUS mRNA levels and found that the transcript expression of GmaPPO12 was 6.1-and 9.5-fold at 0.5 and 2 hpi, and PR1a was 2.2-and 4.1-fold higher compared to uninfected tobacco leaves ( Figure 3D), respectively. As expected, the leaves infiltrated with negative control (the construct only containing 35S minimal promoter) showed very low levels of GUS activity in all the three independent assays, while a full length 35S promoter fused to GUS exhibited high levels regardless of the treatment ( Figure 3B-C). Importantly, the induction levels of GUS by GmaPPO12 promoters were similar to the 35S promoter ( Figure 3B-C), suggesting that the promoter is strongly and rapidly induced upon pathogen infection.

The GmaPPO12 promoter functions in soybean hairy roots
To independently validate that GmaPPO12 is an immediateearly Phytophthora infection gene, the vectors were introduced into soybean hairy roots by Agrobacterium-mediated transformation. The multiple soybean hairy roots were inoculated with P. sojae zoospores, and analyzed by histochemical staining, enzymatic assay, and quantitative RT-PCR. Enzymatic assay showed that GmaPPO12 was induced 6.3-and 10.8-fold and PR1a was induced 1.6-and 3.0-fold at 0.5 and 2 hpi ( Figure 4A), respectively. The histochemical staining shows that the GUS reporter activity driven by GmaPPO12 was significantly higher than that of PR1a and non-treatment with P. sojae (0 hpi) in transgenic hairy roots ( Figure 4B). we also used quantitative RT-PCR to measure GUS mRNA levels and found that the transcript expression of GmaPPO12 was 4.8-and 5.2-fold increases at 0.5 and 2 hpi, respectively, compared to that of PR1a with 1.4-and 2.8-fold increases at 0.5 and 2 hpi ( Figure 4C), respectively. These results demonstrate that the GmaPPO12 promoter, compared to the PR1a promoter, was more strongly induced upon infection in soybean.

Deletion analysis identifies an important responsive region in the promoter
To locate the pathogen-responsive cis-regulatory regions, a series of progressive 5' or 3' truncated GmaPPO12 promoters were fused to a minimal 35S promoter and the GUS reporter gene ( Figure 5A-S3). In Agrobacterium-mediated transient expression of N. benthamiana leaves, the potential expression patterns and pathogen-inducible activities of those promoter mutations were evaluated by histochemical staining and  Figure  6A). These results were also confirmed by a histochemical staining assay ( Figure 6B). Thus, 5' and 3' deletions together suggest that the fragment from -1012 to -413 is essential for promoter activity, although the fragment from -1500 to -1012 is partially required in response to pathogen inoculation.

A 113-bp fragment is a vital pathogen-induced region in GmaPPO12 promoter
To further confirm the activity of the fragment from -1012 to -413 (M1), we generated a plant expression vector containing the fragment ( Figure S3). In N. benthamiana leaves, GUS activity increased 6.0-fold upon induction by P. capsici zoospore infection for 2 h ( Figure 7A). Histochemical staining showed concordant results (Figures 5C-6B). The results support that the fragment from -1012 to -413 (M1) is essential to confer promoter activity in response to induction. On the basis of our above results, we found that a 113-bp fragment from -525 to -413 was shared by three analyzed mutants, they are M1 (-1012 to -413), NT1 (-1012 to +1), and CT1 (-1500 to -413), which showed high pathogen-induced activity (Figures 5-6). We then measured activity of the fragment. In N. benthamiana leaves, the enzymatic assay showed 4.1-fold induction upon infection ( Figure 7A). An obvious induction was also observed in histochemical staining assays ( Figure 7B). This 113-bp fragment contains two basal regulatory CAAT-box elements [29], two ABA or stress responsive MYB1AT elements [40], three R response MYC elements [32], and one bZIP transcription factor binding core DPBF sequence [44] ( Figure S3). Thus, the results suggest that the 113-bp sequence from -525 to -413 can mediate induction activity at around 40% compared to activity of the full length promoter.

Discussion
We are unaware of pathogen-inducible promoters have been identified and characterized in soybeans. Here, we screened the published microarray data [25] and found that the GmaPPO12 gene is strongly induced by P. sojae. We elucidated its expression pattern by quantitative RT-PCR analysis in soybeans, transient expression in N. benthamiana, and stable expression in soybean hairy roots. We found that the gene had three expressional characterizations. First, it is an immediate-early pathogen response gene because it can be induced by P. sojae infection at 0.5 h in soybean, and its promoter also can mediate fast induction activity in N. benthamiana by P. capsici. Second, its induction is strong. The expression levels upon infection are higher than that of the GmaPR1a gene in soybean. Third, the expression is specific to infection. It cannot be induced by the tested hormones in soybean, and its background expression is almost undetectable in soybean and N. benthamiana. This promoter could be used in transgenic plants to enhance their resistance to Phytophthora and perhaps other oomycete diseases. Finally, we identified a small responsive region by progressive 5' and 3' deletions. The small functional segment could serve as a candidate to further identify novel pathogen responsive ciselements or to produce synthetic pathogen-inducible promoters.
The gene is predicted to encode a member of the polyphenol oxidase (PPO) family that catalyzes the oxidation of phenolics to quinones [26,45]. In green plants, the PPO gene family is highly variable in both gene number and structure, except in the genus Arabidopsis [26]. The enzymes play many divergent roles in a variety of processes, including defense processes [46,47]. In tomatoes, for example, PPO overexpression increased resistance to Pseudomonas syringae and several insects [45]. PPOs are also implicated in latex coagulation [48], and the biosynthesis of aurones and lignans [49,50]. Soybean contains 11 PPO isoforms [26], which correlate with its abundant flavonoids. Here, we showed that GmaPPO12 was rapidly and strongly induced by P. sojae infection. However, the role of GmaPPO12 in disease resistance still needs to be addressed.
Patterns of gene expression in soybean upon P. sojae infection have been investigated in several independent studies [25,51,52]. Only 22 soybean genes were identified as upregulated early in infection (3 hrs). The genes included GmaPR1a and a gene encoding flavin adenine dinucleotide (FAD)-linked oxidoreductase [51]. Here, we identified GmaPPO12 as being strongly inducing at 0.5 h postinfection. Its fold change was higher than that of GmaPR1a. Characterization of the GmaPR1a promoter in N. benthamiana leaves and soybean hairy roots also supported the idea that the gene was rapidly and highly induced by Phytophthora pathogens. Moreover, the fold change in GmaPPO12 promoter driven GUS expression was lower than that of the endogenous gene in soybean. Quantitative RT-PCR analysis showed that the gene was upregulated 53-fold at 0.5 hpi in soybean, but the GUS gene was only induced 6.1-fold in N. benthamiana and 4.8-fold in soybean hairy roots, respectively. This difference may be due to that the stress-induced expression of the endogenous gene driven by the native promoter not being similar to the heterologous GUS gene fused with the same promoter, as observed in previous studies [53]. We have noted that the promoter driving GUS expression exhibited unwanted background expression, although the levels were low. One possible reason is that the agroinfiltration associated wounding induced gene expression. Further studies will be required to test the utilization of the cloned promoter to drive functional disease resistance genes.
Promoter deletion analysis revealed that two fragments appeared to be partially sufficient for the induction activity of the GmaPPO12 promoter. The 599-bp and 113-bp fragments accounted for over 60% and 40%, respectively compared to activity of the full length promoter. The identified smaller fragment contained several putative cis-elements that are involved in defense responses, including two MYB binding sites, three MYC binding sites, and one DPBF-box. The MYB binding sites have been identified in the parsley PAL1 promoter as the sites of fungal elicitor-inducible DNA-protein interactions [54], and the tobacco PR1a promoter [55]. The MYC recognition sites play a critical role in MYC regulation of innate immune responses against mycobacterial infection [56]. Testing these elements from soybeans is now a priority so that we can design the synthetic promoters that are specific and highly induced by pathogens.

Plant materials and inoculation
Nicotiana benthamiana was used for transient expression and soybean (cv. Williams) was used to provide stable transgenic soybean hairy roots. The plant seeds were grown in a growth chamber at 25°C under fluorescent white light in a 16: 8-h light/dark cycle. Seven-day-old soybean roots or soybean hairy roots (see below for the methods to obtain transgenic hairy roots) were inoculated with suspensions containing 10 5 zoospores of P. sojae (Strain, P6497) per milliliter. Six-weekold N. benthamiana leaves were immersed in suspensions containing 10 5 zoospores of P. capsici [57] 72 h after agroinfiltration. The samples were collected at 0.5 and 2 h, and non-treated tissues were used for control. For hormone treatments, 7-day-old soybean roots were submerged in different hormones for 3 h, and the concentration of hormones is jasmonic acid (100 µM), salicylic acid (100 µM), abscisic acid (100 µM), ethephon (100 µM), gibberellic acid (100 µM), respectively. The control plants were immersed in water. For each treatment and time point, three leaves or roots from three individual plants were collected for duplications.

Construction of the transformation vector
Genomic DNA was extracted from 6-7-day-old soybean roots, following the cetyltrimethylammonium bromide (CTAB) isolation procedure [58]. The GmaPPO12 and GmaPR1a promoters were isolated with the designed primers (Table. S1). The obtained promoters cover a sequence 1,500-bp in length upstream of the transcription start site. Five 5' deletions, five 3' deletions, and two 5' and 3' deletions of the GmaPPO12 promoter region were generated by PCR, with forward primers containing a KpnI restriction site and a reverse primer containing a BglII restriction site. The sequences of promoters used for the amplification of the deletion promoter fragments are summarized in Figure S3. All of the promoter fragments were digested with KpnI and BglII and cloned into the KpnI-BglII sites of pMDC162 [59], a plant expression vector fused with the CaMV 35S minimal promoter [60] and GUS reporter gene. A full description of plasmid construction can be found in Figure 3A. The pMDC162 vector containing the CaMV 35S minimal promoter lacking cis-acting elements was constructed and used as a negative control. PBI121 contains the GUS (or uidA) gene under the control of the CaMV 35S promoter as the positive control. Both plasmids contain the selectable marker gene nptII, which confers resistance to kanamycin. All primers used in this study are listed in Supplemental Table. 1. All plasmids containing expression cassettes were confirmed by sequence analysis and introduced into Agrobacterium strains by electroporation for transformation.

Agroinfiltration of N benthamiana leaves
All the constructs were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation, as described by Ainsworth et al [61]. Agroinfiltration of N. benthamiana leaves was based on the methods described in Llave [62] with the following modifications. Individual agrobacteria colonies were grown on Luria-Bertani plates with kanamycin (50 µg mL -1 ) for 48 h at 28°C. A single positive colony was used to inoculate a 5 mL culture (LB with 50 µg mL -1 kanamycin). Bacteria were pelleted, resuspended in infiltration medium [10 mM MgCl 2 , 10 mM MES, 150 µM acetosyringone (pH 5.6)] to an OD 600 of 0.5-0.6, then incubated at room temperature for 3 h. The bacterial suspension was infiltrated into the abaxial side of fully expanded 6-week-old N. benthamiana leaves using a needleless 1-mL syringe. For each experiment, the positive control (PBI121 intron with 35S-GUS), the negative control (TATA-GUS in pMDC162 intron), and the constructs under investigation were infiltrated in areas of the same leaf or different leaves. After infiltration, the plants were kept in the greenhouse 72 h for inoculation.

Soybean hairy root transformation
The introduction of expression constructs into soybean hairy roots using the Agrobacterium rhizogene K599 cucumopine strain [63] was by electroporation as described by Ainsworth et al [61]. Individual colonies were picked and verified by enzyme restriction analysis of DNA extracted from randomly selected clones. Plant inoculation was conducted according to Savka [63] and modified as follows: soybean seeds were surfacesterilized with chlorine gas and germinated on Murashige and Skoog (MS) culture medium for 5-6 days in a 16: 8-h light/dark cycle incubator at 25°C. Cotyledons were wounded on the abaxial side with scalpel blades dipped in a culture of Agrobacterium rhizogenes K599 harboring the different constructs and cultured abaxial-side-up on MS culture medium. Carbenicillin and Cefotetan 250 µg/mL were added to inhibit the growth of A. rhizogenes and incubated at 25°C in the dark. The putative transformed hairy roots about 18-20 days after root emergence and at lengths of 4-to 6-cm were used for inoculations.
For fluorometric assays, the plant tissues were homogenized in 1 mL extraction buffer [50 mM NaH 2 PO 4 , pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1 (w/v) sodium laurylsarcosine, 10 mM β-mercaptoethanol], and centrifuged at 12,000g for 10 min at 4°C. Total protein in tissue homogenates was quantified by the Bradford [66] method, using bovine serum albumin (BSA) as a standard. The supernatant (40 µl) was mixed with 400 µL 2 mM 4-methylumbelliferyl-β-d-glucuronide (MUG) on ice and 100 µL was transferred immediately to a fresh tube containing 900 µL of GUS stop buffer (0.2 M Na 2 CO 3 ) to serve as a control. GUS assays were performed at 37°C for 30 min. Stop buffer (0.2 M Na 2 CO 3 ) and 50 nM to 1 mM 4methylumbelliferone (4-MU) were used for calibration and standardization. Fluorescence (excitation 365 nm, emission 455 nm) of each sample was determined in a SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA), and the GUS activity calculated from the slope of the best fit line through the three points to give the increase in fluorescence per minute, and expressed as nanomoles of 4-methyl-umbelliferone (4-MU) produced per minute per milligram of soluble protein. Duncan's multiple range tests were used for statistical analysis (p < 0.05) ( Figure 6A).

Quantitative RT-PCR
Total RNA was extracted by using the PureLink RNA mini kit (Invitrogen), and first strand cDNA was synthesized using an iScript cDNA Synthesis kit (Takara Bio DRR036A). cDNAs were diluted to 100 ng/µL and combined with SYBR master mix. Expression of each gene was normalized to a reference gene of either the soybean elongation factor gene (Genbank accession no. XM_003531518.1) or Nicotiana tabacum elongation factor 1-alpha mRNA (Genbank accession no. AF120093.1), and the specific primers named ACT20-F, R, or EF1a-F, R (see Supplemental Table 1). PCR was performed in triplicate using 96-well optical reaction plates. Quantitative RT-PCR thermal cycler conditions and reaction mixtures were according to the manufacturer's instructions (SYBR Premix Ex Taq TM ). The fluorescence threshold value and gene expression data were calculated using the (ABI 7500 SDS) system software. The endpoint was used in the real-time PCR quantification [67], Ct is defined as the PCR cycle number that crosses an arbitrarily placed signal threshold. The level of transcript abundance relative to the reference gene (termed △Ct) was determined according to the function △Ct =Ct (test gene) -Ct (reference gene). To compare untreated and treated expression levels, the function △△Ct was first determined using the equation △△Ct = △Ct (treatment) -△Ct (control) (where control represented mock-treated plants). The induction ratio of treatment/control was then calculated by the equation 2^(-△△Ct). The primer sequences are listed in Supplemental Table 1. Student's t-tests (p < 0.01) (Figure 2A) or Duncan's multiple range tests (p < 0.05) ( Figure 2B) were used for statistical analysis.

Phylogenetic tree and promoter analysis
A Phylogenetic tree of soybean PPO proteins was constructed by the neighbor-joining method using MEGA 4.0 [68,69]. Genetic distances were estimated using the Dayhoff amino acid substitution matrix. Positions in the alignment lacking amino acid residues were excluded from the pairwise distance estimates. Bootstrap replicates (1000) were used to indicate the level of support from the data for each node of the tree. Predictions pertaining to the types of introns were independently checked using CIWOG [70]. N-terminal transit peptide sequences were predicted using ChloroP 1.1 and TargetP 1.1 [71,72]. The PPO protein conserved regions were analyzed using a combination of NCBI BLASTP and SMART (Simple Modular Architecture Research Tool, http://smart.emblheidelberg.de/).
The cis-regulatory elements in the promoters were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html) [28]. Table S1. Polymerase chain reaction (PCR) primers used in this. study. (DOC) Figure S1. Neighbor-joining phylogenetic tree of soybean PPOS, together with corresponding visual representation of conserved regions, functional motifs, and relative intron positions. The ClustalW multiple sequence alignment was formed using the deduced soybean PPO proteins. The tree was constructed from the ClustalW alignment using the neighbor-joining method of the MEGA program. The scale bar represents 0.05 substitutions per site and the numbers next to the nodes are bootstrap values from 1,000 replicates. Predicted targeting sequences are colored green (chloroplast transit peptide), black (signal peptide), or gray (unknown). The CuA and CuB domains are colored blue, and C-terminal conserved areas are dark gray. Approximate intron positions are shown as vertical bars, mapped onto the predicted protein.
Nucleotide sequence of the soybean GmaPPO12 gene. The translational start sites (+1) are shown in red. Upstream of the translation start sites (+1) is the promoter sequence. The promoter motifs with significant similarity to the previously identified cis-acting elements are shaded and the names are given under the elements. The translation initiation codon (M) downstream amino acid sequence contains PPO conserved regions: the N-terminal chloroplast transit peptide sequence is colored green, CuA and CuB domain sequences are colored blue, C-terminal DWL and KFDV domains are colored red, and unknown sequences are colored gray. The nucleotide sequences colored blue-green are microarray EST. The sequence in purple boxes indicates the primers for quantitative RT-PCR to detect the inducibility of