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First published online February 1, 2008; 10.1104/pp.107.111484 Plant Physiology 146:1810-1820 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Herbivore-Induced Callose Deposition on the Sieve Plates of Rice: An Important Mechanism for Host Resistance1,[W],[OA]Key Laboratory of Ministry of Education for Plant Development Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People's Republic of China
The brown planthopper (Nilaparvata lugens Stål; BPH) is a specialist herbivore on rice (Oryza sativa) that ingests phloem sap from the plant through its stylet mouthparts. Electronic penetration graphs revealed that BPH insects spent more time wandering over plants carrying the resistance genes Bph14 and Bph15, but less time ingesting phloem than they did on susceptible plants. They also showed that their feeding was frequently interrupted. Tests with [14C]sucrose showed that insects ingested much less phloem sap from the resistant than the susceptible plants. BPH feeding up-regulated callose synthase genes and induced callose deposition in the sieve tubes at the point where the stylet was inserted. The compact callose remained intact in the resistant plants, but genes encoding β-1,3-glucanases were activated, causing unplugging of the sieve tube occlusions in susceptible plants. Continuing ingestion led to a remarkable reduction in the susceptible plants' sucrose content and activation of the RAmy3D gene, leading to starch hydrolysis and ultimately carbohydrate deprivation in the plants. Our results demonstrate that BPH feeding induces the deposition of callose on sieve plates in rice and that this is an important defense mechanism that prevents insects from ingesting phloem sap. In response, however, the BPH can unplug sieve tube occlusions by activating β-1,3-glucanase genes in rice plants.
Globally there is an enormous diversity of herbivorous insects that attack and damage plants. However, long-term coevolution of herbivorous insects and plants has led to the development of an array of constitutive and induced defenses that enable plants to protect themselves from herbivore attack (Rausher, 2001  ; Becerra, 2007). Constitutive defense mechanisms include physical barriers (such as cuticles, thorns, trichomes, and cell walls) and preexisting metabolites that are harmful or even toxic to insects (Mithöfer et al., 2005). Induced plant defenses involve the activation of mechanisms that directly or indirectly deter herbivores (Walling, 2000). Some secondary metabolites (Baldwin et al., 2001; Benderoth et al., 2006) and induced proteins, such as lectins, chitinases, proteinase inhibitors, and enzymes (Zavala et al., 2004; Chen et al., 2005; Harfouche et al., 2006), often play direct defense roles by, for example, repelling insects, inhibiting their feeding, or damaging their digestive or neural systems. Indirect defenses include the release of volatiles that signal the location of insects on infested plants to parasitoids and predators (De Moraes et al., 2001; Schnee et al., 2006; Kempema et al., 2007). When attacked by insects, plants can also produce endogenous signal molecules, such as stress hormones, including jasmonic acid (JA), ethylene, abscisic acid (ABA), and salicylic acid (SA) that regulate signal transduction cascades in plant cells, leading to the activation and modulation of defense-related genes (Li et al., 2002; Schmelz et al., 2006).
Plants show varied responses to herbivores that are strongly correlated with the mode of herbivore feeding. Chewing insects and cell-content feeders, such as caterpillars and beetles, cause extensive tissue damage and activate wound-signaling pathways in which JA plays a central role (Kandoth et al., 2007
The brown planthopper (Nilaparvata lugens Stål; BPH) is an insect that feeds on the leaf sheath of rice (Oryza sativa) plants, ingesting nutrients specifically from the rice phloem using its piercing mouthparts (stylet), forming a stylet sheath during the feeding process. Feeding by numerous BPHs on a single plant generally results in the susceptible plants yellowing, browning, and drying. In the last decade, the BPH has frequently caused widespread destruction of rice crops and heavy losses of yields (Shi et al., 2003
The main methods used to control BPH pests are to apply chemical insecticides and/or develop and grow resistant varieties in an integrated pest management strategy. However, the cost of chemical control is often very high and the chemicals can destroy the natural balance of BPH predators that help to keep the BPH population in check. The misuse of chemical pesticides may also cause a resurgence of the insect. Therefore, the most economic and efficient method for controlling the BPH is to exploit the host resistance to attack (Renganayaki et al., 2002 The aim of this study was to further explore the interactions between the BPH insects and rice plants in an attempt to elucidate the mechanisms involved in rice resistance to the BPH. We used a susceptible rice plant variety (TN1) as a control and first studied the feeding behavior of the BPH on rice plants carrying the BPH resistance genes Bph14 and Bph15 using the electronic penetration graph (EPG) technique. We then examined the anatomical features of the punctured phloem cells, especially the induced callose, by observing and counting the number of sieve plates with callose deposition. Real-time PCR was performed to examine the expression of genes coding for callose synthases and degrading enzymes. To our knowledge, this is the first targeted callose analysis of rice resistance to BPH feeding. Our results suggest that the induced callose sealing in sieve tubes plays an important role in the inhibition of BPH feeding. However, the BPH can unplug the sieve tube occlusions by activating β-1,3-glucanase genes in rice plants.
EPG Monitoring the Feeding Behavior of BPH
BPHs ingest phloem sap from rice plants through their narrow piercing-sucking mouthparts, which are called stylets. During the feeding process, the stylet transiently punctures the epidermis, making the first probing, and then penetrates the plant cell walls; the insect subsequently salivates into the cells and ingests the phloem sap. In this study, the feeding activities of the BPH on different varieties of rice, with varying levels of resistance, were monitored electrically using a real-time EPG technique (Tjallingii, 2006
Waveform type 1 appeared when the stylet was outside the plant tissue and the electronic circuit remained open. Type 1 behavior can be divided into two subtypes: (1) BPH insects resting on plants; and (2) BPH insects walking or searching for feeding sites (Supplemental Fig. S1A). In an 8-h recording period, the total duration of type 1 behavior on the resistant variety B5 (carrying Bph14 and Bph15 genes) was 168.2 min, much longer than that on the susceptible rice variety TN1 (19.7 min, carrying no BPH-resistance gene). Two moderately resistant varieties, RI35 (carrying Bph14) and YHY15 (carrying Bph15), showed similar results to B5, with total durations of type 1 behavior of 110.4 min and 120.5 min, respectively (Table I , total durations). Type 1 behavior occurred 35 times on a resistant B5 plant and only 14 times on a susceptible TN1 plant (Table I, no. of times). The mean duration for type 1 behavior per feeding event on the three resistant varieties (5.5–8.7 min) was significantly longer than on the susceptible TN1 variety (1.5 min; Table I, mean durations). These findings indicate that BPHs spent more time resting or moving about on resistant plants than on susceptible ones, probing more frequently on resistant plants; the data also show that feeding was often interrupted on resistant plants.
Waveform type 2 occurred when the BPH insects used their stylets to search for the target cells in plant tissues in a series of activities, including penetrating plant cells, salivating, tasting, and forming branches of the stylet sheath (Supplemental Fig. S1A). The total duration of this waveform type on the resistant B5 plant was 114.6 min, significantly longer than that on the susceptible TN1 plant (33.3 min). This suggests that the insects spend more time searching for suitable target feeding cells in the resistant plant tissue (Table I, total durations). The frequency of this behavior over the 8-h recording period showed a general tendency to increase with higher levels of plant resistance (Table I, no. of times). Waveform type 3 occurred when the stylet penetrated the vascular bundle of the rice plant (Supplemental Fig. S1B). Overall, the total duration of waveform type 3 was correlated with the plants' level of resistance, but there was a significant difference between the two moderately resistant varieties (RI35 and YHY15) and the difference between TN1 and YHY15 was insignificant (Table I, total durations). Such differences in type 3 behavior of BPHs on these moderately resistant varieties might be attributable to the differences in resistance genes of the plants. Waveform type 4 represented xylem ingestion by the BPHs (Supplemental Fig. S1C). We assumed that the xylem was not the resistant element within the plant because of the irregular duration of this behavior on the B5 (75.5 min), YHY15 (125.9 min), RI35 (123.9 min), and TN1 (58.2 min) varieties. There was no clear relationship between resistance level and total duration of this type of behavior (Table I, total durations). Waveform type 5, representing phloem ingestion, gave a better indication of resistance because it reflected the relative quantity of phloem sap ingested by the BPHs (Supplemental Fig. S1D). During the 8-h recording period, the total duration of type 5 behavior on the resistant variety B5 was 33 min, approximately one-tenth of that on the susceptible variety TN1 (340.2 min; Table I, total durations). Moreover, the mean duration of each period of phloem sap ingestion was much shorter on the resistant varieties than on the susceptible control TN1 (Table I). These findings suggest that the BPHs spend less time ingesting phloem sap from resistant plants than from susceptible plants.
Suc is the main carbohydrate that is transported long distances through the phloem and ingested by the BPH. By culturing rice plants in a [14C]Suc solution, 14C can easily be introduced into the phloem. The quantity of phloem sap ingested by the BPHs can then be estimated by monitoring the radioactivity of 14C in the insects. We used the ratio of radioactivity (14C) in the insect to that in the plant, designated the I:P index, as an indicator of the relative quantity of phloem sap ingested by BPHs. The results showed that the I:P ratio for BPHs that had fed on resistant B5 plants was very low (0.02, compared with 0.85 for insects feeding on susceptible TN1 plants) over a 20-h period (Fig. 2 ). The I:P ratios for BPHs that fed on the moderately resistant varieties YHY15 and RI35 were 0.54 and 0.28, respectively. These results strongly indicate that the BPH insects ingested less phloem sap from the resistant rice plants than they did from the susceptible control (TN1).
Callose Deposition on the Sieve Plates of BPH-Infested Plants To investigate the mechanisms that prevent BPHs from continuously ingesting phloem sap from resistant rice plants, the leaf sheaths of BPH-infested and BPH-free resistant and susceptible plants were sectioned and examined histopathologically. The sections were stained with 0.1% aniline blue and examined under a fluorescence microscope. In the BPH-free untreated rice plants, there was little or no callose deposition on the sieve plates in the leaf sheaths (Fig. 3, E and F ). When the plants were infested with the BPH, more callose was deposited on the sieve plates of the target sieve tubes, where the stylets had been inserted—the sieve plates were obviously thickened and emitted strong fluorescence (Fig. 3, A–D). Counts of the bright callose plugs revealed that callose deposition increased during the first 3 d of infestation in both B5 and TN1 plants, but there were more callosic sieve plates in the former (13.7 callosic sieve plates in 50 sections) than the latter (5.8). Moreover, with prolonged BPH feeding, the callose deposition decreased quickly in TN1 plants to only 2.4 callosic sieve plates in 50 sections after 4 d, but a high level of callose deposition remained in the B5 plants (12.7; Fig. 3G). Further analysis revealed that there were strong fluorescence signals from callose in almost all the sieve tubes penetrated by stylet sheaths in the resistant B5 plants, but a large proportion of the target sieve tubes did not have bright callose depositions in the susceptible TN1 plants (Fig. 4, D and H ).
Expression of Callose Synthase and β-1,3-Glucanase in Rice Plants Callose deposition is a dynamic process coordinated through the activities of callose synthase and the callose-hydrolyzing enzyme β-1,3-glucanase. To investigate the mechanisms responsible for the differential callose deposition in the resistant and susceptible rice plants, the expression of 10 callose synthase-encoding genes were investigated using semiquantitative reverse transcription (RT)-PCR. We detected transcripts of four of these genes, namely, OsGSL1, OsGSL3, OsGSL5, and OsGSL7 (Supplemental Fig. S2). Three of the detected genes, OsGSL1, OsGSL3, and OSGS5, were further analyzed by real-time PCR. These genes were clearly up-regulated after the B5 and TN1 plants were treated with BPH for 6 h, reaching high levels after 12 h (2- to 4-fold of uninfested plants). The expression levels remained high in the following 72 to 96 h, generally over 3-fold higher than those in uninfested plants (Fig. 3, H–J). These observations suggest that the callose synthase genes were up-regulated and consequently callose synthesis was enhanced in both the resistant and susceptible plants attacked by the BPH. The expression patterns of six β-1,3-glucanase genes were also investigated. The patterns of four of them were found to differ between BPH-infested B5 and TN1 plants. Osg1 was up-regulated by 1.2-fold within 3 h in susceptible TN1 plants and kept increasing from 6 to 48 h, with a decline after 72 h. The highest expression level occurred after treatment for 48 h, about 5-fold increased in comparison to the untreated control (Fig. 4J). Gns5 showed an increase both in TN1 and B5 plants, but the increase in TN1 was much higher than in B5 plants. The greatest increase in TN1 was >9-fold relative to the uninfested control (Fig. 4I). Gns4 was constitutively expressed in B5, but was induced in TN1 plants. Gns6 shared similar expression patterns with Gns5. Little or no expression of either Gns2 or Gns3 was detected in the leaf sheath of the rice plants (Supplemental Fig. S2). The expression of β-1,3-glucanase genes, such as Osg1 and Gns5, was clearly up-regulated in the susceptible rice plants; in contrast, the expression level of these genes was up-regulated much less (Gns5) or even absent at a detectable level (Osg1) in resistant ones.
To investigate the anatomical effects of BPH feeding on rice plants, leaf sheaths of plants representing the most resistant and most susceptible varieties (B5 and TN1, respectively) were sectioned, stained in 3% KI-1% I2 solution, and examined under a microscope. We found abundant starch granules in the leaf sheaths of the uninfested plants and there appeared to be more in the B5 than in the TN1 plants (Fig. 5A , top two sections). In the BPH-infested plants, starch granules were rapidly consumed after 1 d in the susceptible TN1 variety and most were exhausted after 3 d of infestation (Fig. 5A, right). In contrast, the starch granules disappeared much more slowly in the resistant variety (B5; Fig. 5A, left).
The starch content of leaf sheaths was also determined. Results showed that the starch content decreased much more quickly in TN1 than in B5 under the stress of BPH infestation. However, it should be noted that there was much more starch in B5 (22.7 mg g–1 fresh weight) than in TN1 (16.7 mg g–1 fresh weight) plants that were not infested by BPHs (Fig. 5B, bottom). The Suc content varied in a similar way to that of the starch content (Fig. 5B, top). In the BPH-infested TN1 plants, Suc content fell to approximately 56% and 30% of the untreated control plant levels after 1 and 4 d, respectively (Fig. 5B), showing that susceptible TN1 plants can be rapidly and seriously deprived of carbohydrates during infestation by these insects.
The expression of nine
Enhancing host resistance is an important component of integrated pest management. However, the mechanism of rice's resistance to BPH is still uncertain. In the past, researchers considered that it might be governed by the presence of chemicals confined to the phloem (Sogawa and Pathak, 1970 ). Bph1 was the first BPH resistance gene identified and is associated with the production of flavonoids, including SA; amino acids, such as Asp, Glu, Ala, Ser, Leu, Asn, and Val; and organic acids, such as succinic acid, malic acid, oxalic acid, and transaconitic acid (Sogawa and Pathak, 1970; Sogawa, 1976). There is now considerable evidence to suggest that induced defenses are effective and have low fitness costs. Several studies have revealed that gene expression profiles in rice are reorganized in response to BPH feeding (Zhang et al., 2004; Yuan et al., 2005; Park et al., 2007; Wang et al., 2008). Such reorganization has been well documented in other plant-herbivore systems, supporting the hypothesis that inducible defenses contribute to rice resistance to the BPH. The data gathered in this experiment show that BPH feeding affects the expression of genes associated with the synthesis and hydrolysis of callose and starch decomposition in rice plants, resulting in the deposition of callose plugs on the sieve plates. This prevents BPH insects from continuously ingesting phloem sap and allows normal carbohydrate levels to be maintained in resistant plants, as described below.
Phloem, the target of BPH feeding, mainly consists of sieve tubes and companion cells. The functional units of sieve tubes are series of sieve elements that have porous sieve plates at their abutting ends, allowing the phloem sap to flow continuously (Will et al., 2007
It has been reported that callose synthesis is Ca2+ dependent (King and Zeevaart, 1974
Because callose synthase genes were up-regulated and callose deposition occurred in both the resistant and susceptible rice plants a short time after BPH feeding commenced (Fig. 3), the insects had to overcome the physical barriers imposed by callose to obtain sufficient food even from susceptible varieties. In the aphid/broad bean system, aphid saliva can prevent sieve tube plugging by forisomes (Will et al., 2007
Starch is a major end product of photosynthesis; it is produced in chloroplasts and is the main energy storage substance in cereal grains and leaf sheaths. In the chloroplast and amyloplast, starch metabolism is closely related to other metabolic processes in the cytosol, such as Suc metabolism, glycolysis, and glyconeogenesis, so starch content is indicative of the dynamic transformation of carbohydrates. In treated susceptible TN1 plants, a large amount of phloem sap was ingested by BPHs and plant Suc content rapidly declined. In contrast, photosynthesis was suppressed in the BPH-infested susceptible plants (Yuan et al., 2005 Our understanding of the resistance mechanism can be encapsulated in the following model of interactions between the BPH and the rice plant (Fig. 6 ). First, the BPH acts on the plant by penetrating its tissues, ejecting saliva into its cells, and sucking up phloem sap. In response to BPH feeding, the plant up-regulates expression of its callose synthase and β-1,3-glucanase genes. Consequently, callose deposition occludes the sieve tubes and prevents the BPH from ingesting the phloem sap. However, β-1,3-glucanases that decompose the deposited callose and thereby facilitate the BPH's continued feeding from the phloem are strongly induced in susceptible plants, but much more weakly induced in resistant plants. Thus, differential expression of β-1,3-glucanases can account for between-plant differences in resistance levels.
We have demonstrated that feeding by the BPH can induce callose synthesis and deposition on the sieve plates of rice plants. Callose deposition affects phloem transportation and plays an important role in preventing the BPH from ingesting the phloem sap. Our results show not only that callose deposition is sufficient for resistant plants to defend themselves against the BPH, but also that some specific β-1,3-glucanases are active callose-decomposing enzymes, induced by BPH activity and responsible for the susceptibility of TN1 plants. The differential expression of these enzymes may result in different resistance levels in rice plants.
Plants and Insects
Five rice (Oryza sativa) varieties were used in this study. B5 is a line carrying BPH resistance genes Bph14 and Bph15 from wild rice (Oryza officinalis) and exhibits high resistance to BPH with the severity score below 3.0 in the seedling bulk test (Huang et al., 2001 Unless otherwise stated, the brown planthopper (Nilaparvata lugens Stål; BPH) insects were three- to four-instar nymphae, and the insects were maintained on TN1 plants in the Genetics Institute, Wuhan University.
To link the EPG waveforms with the feeding behavior patterns of the BPH, a microscope was coupled to EPG equipment, as follows. Special plastic slides, each with a 1-cm-diameter hole in the center, were prepared and covered with stretched parafilm. Suc solution or tap water (each with a small amount of active carbon powder to trace the water flow) was dropped onto the parafilm to serve as an artificial food source and mounted under a coverslip. BPH insects with a gold wire (length 3–5 cm, diameter 20 µm) attached to the dorsum by conductive silver glue were then allowed to probe the food through the parafilm. The gold wire from each insect and a copper wire (diameter 0.1 mm) immersed in the food were linked to a Giga 4 model DC-EPG amplifier (Wageningen University). The EPG setup was housed in a climate-controlled room (25°C ± 2°C) and shielded from electrical noise by an earthed Faraday cage. The EPG was also linked to a computer running PROBE 3.1 software (attached to the EPG equipment). The electronic signals from the different channels were converted into digital data using a DI-710 data logger (DATAQ) and transformed into waveforms displayed on the computer screen in real time. By relating the feeding behavior of BPH insects under the microscope with the real-time EPG waveforms displayed on the screen, we were able to categorize the waveform types (Supplemental Fig. S1). For EPG recordings of BPH insects feeding on rice plants, adult brachypterous females (2 d after the final molt) were collected at 9 AM, and attached to a gold wire, as described above. After being starved (but provided with water) for 1 h, each insect was placed on the leaf sheath of the plant to be tested and the gold wire from its dorsum was connected to the EPG. Before acquiring and processing data, WINDAQ Waveform Browser software (DATAQ) was run for 30 min to pretest the activity of the insect. Data were acquired at 100-Hz sample frequency, stored on the computer's hard disc, and simultaneously displayed on a screen. The data were analyzed using ANA3.0 software (Wageningen University). EPG recordings were carried out for 8 h/insect/plant, with at least seven replicates for each variety, using fresh seedlings and insects in each case. In a single experiment, all four genotypes were examined simultaneously, one channel for each genotype, and then the experiment was repeated. Data were compared using Kruskal-Wallis one-way ANOVA ranking and Scheffe's post-hoc pairwise comparisons (P < 0.05).
For 14C-labeling, rice seedlings at the three-leaf stage were transferred to a vial containing a 1-mL solution of [14C]Suc from Sigma and nonlabeled Suc (4 µCi and 15 mg/vial, respectively). Each seedling was held in place with a sponge and pushed into the neck of the vial so that the root was immersed in the solution. Each plant, together with 10 insects, was placed in a test tube (30 mm x 200 mm) and covered with gauze to prevent the insects from escaping. Seedlings were allowed to take up the Suc solution at 25°C ± 2°C in darkness. Twenty hours later, the seedlings were removed from the tubes, their roots were discarded, and their remaining parts were cut into 1-cm-long segments. Insects and plant segments were then plunged into 5 mL of 80% ethanol-water solution (v/v), boiled for 10 min, and then centrifuged at 4,000g for 5 min at 4°C. The supernatants containing the extracted soluble fractions were collected and concentrated to 500 µL, 100 µL of which was used to determine their 14C content, using a Beckman LS6500 liquid scintillation spectrometer (Beckman). The I:P index was used to evaluate the distribution of 14C between the insects and plants, reflecting the proportion of soluble 14C ingested by the insects from the phloem.
Rice plants were each infested with 10 BPHs. Leaf sheaths were collected, fixed in FAE (formaldehyde:acetic acid:70% ethanol, 5:5:90 [v/v/v]), dehydrated, embedded in paraffin, and cut into 10-µm-thick sections using a microtome. The sections were mounted on microscope slides, dewaxed, and rehydrated for staining at room temperature.
To highlight starch and saliva sheaths, the rehydrated sections were stained in 3% (w/v) KI-1% I2 solution for 1 min, then examined under a light microscope. For callose observations, 10-µm-thick sections were mounted on glass slides (50 sections/slide). Callose staining was performed as described by Dietrich et al. (1994)
Fresh leaf sheaths (approximately 1 g) were powdered in liquid nitrogen, homogenized in 4 mL of 80% (v/v) ethanol, heated in a water bath at 80°C for 40 min, and centrifuged at 4,000g for 10 min. The supernatant fraction was collected and the solid fraction was washed with 80% ethanol and centrifuged; this procedure was repeated twice. The supernatants collected from each sample were combined and then active carbon was added and filtered for Suc analysis, whereas the pellet was dried for starch determination.
Suc was measured using the anthrone-sulfuric acid method (Trevelyan and Harrison, 1952 The dried pellet was added to 5 mL of 80% Ca(NO3)2 (w/v), placed in a 100°C water bath for 10 min, and then centrifuged at 4,000g for 4 min. The supernatant fraction was collected and the solid fraction was washed with 80% Ca(NO3)2 and centrifuged; this procedure was repeated twice. All the supernatants from each sample collected were combined and added to 20 mL of water to prepare the starch solution. One milliliter of starch solution was mixed with 2 mL of 80% Ca(NO3)2 and 100-µL solution of 0.01 N I2-KI (1.3 g I2 and 4.0 g KI in water, final volume 1 L). The mixture was transferred by pipette into a 1-cm-diameter spectrophotometer cup, and its A620 was measured using a UV-1601 spectrophotometer.
Total RNA was extracted from the leaf sheaths (approximately 200 mg fresh weight) with TRIzol reagent (Invitrogen), and the remaining DNA was degraded using a Turbo DNA-free kit (Ambion). cDNA was synthesized from the total RNA (4 µg) using a Thermoscript RT-PCR system (Invitrogen), with oligo(dT)20 primers, following the manufacturer's instructions. RT-PCR was performed using Taq DNA polymerase (MBI Fermentas) in 10-µL reaction mixtures with the gene-specific primers listed in Supplemental Table S1, which were either directly synthesized according to previously published information (Tomoya et al., 2002
Genes for real-time PCR analysis were screened based on the results of semiquantitative RT-PCR; the genes that showed obvious variation were chosen for real-time PCR, whereas the genes that could not be detected or showed no obvious variation were not chosen for further study. The primers (Table II
) for real-time PCR were redesigned using primer premier 5.0 software according to cDNA sequences obtained from NCBI GenBank (see above). Reactions were carried out on the ABI PRISM 7300 real-time PCR system (Applied Biosystems) using three-step cycling conditions of 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, 55°C to 60°C for 15 s, and 72°C for 28 s. After the amplification steps, the melting curve was determined for each primer pair at a final stage of 15 s at 95°C, 15 s at 60°C, and 15 s at 95°C to verify the presence of only one specific product. The reaction mixture (20 µL) contained 2 µL of cDNA solution, 10 µL SYBR Green real-time PCR master mix (QPK-201; TOYOBO), and about 5 pmol each primer. The reactions were performed in triplicate and the results were averaged. A standard curve was prepared using 5 µL of cDNA solutions in which serially diluted samples (original, 5-, 25-, 125-diluted) were included. The slopes of Ct and
Supplemental Data The following materials are available in the online version of this article.
We thank Freddy Tjallingii (Wageningen University) for valuable technical advice about EPG, and Jie Zhao and Yingtang Lu (Whan University) for valuable help with the epifluorescence microscope and liquid scintillation spectrometer. We also thank Hongyu Yuan (Xinyang Normal University) for valuable advice and assistance with the real-time PCR system. We acknowledge Yanchang Wang (Wuhan Institute of Botany, Chinese Academy of Sciences) and members of our laboratory for their assistance with this project. Received October 24, 2007; accepted January 28, 2008; published February 1, 2008.
1 This work was supported by the National Natural Science Foundation of China (grant nos. 30730062 and 30671287) and the National Special Key Project on Functional Genomics and Biochips (grant no. 2006AA10A103).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Guangcun He (gche{at}whu.edu.cn).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.111484 * Corresponding author; e-mail gche{at}whu.edu.cn.
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TOP
ABSTRACT
RESULTS
-amylase and three β-amylase genes in the leaf sheaths of rice plants was investigated using semiquantitative RT-PCR. We found that RAmy3D (Os08g0473900), one of the genes examined, was strongly induced in the BPH-infested TN1 plants, but was only weakly expressed in the B5 plants (Supplemental Fig. S2). This indicates that RAmy3D plays a role in the response of susceptible rice plants to BPH feeding by breaking down the starch. 
Ct (target gene) – (reference gene) and R2 values of each sample were calculated by the ABI PRISM 7300 SDS vl.X and Microsoft Excel 2003. Relative quantification was performed with the 2–