• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of plntphysLink to Publisher's site
Plant Physiol. Jan 2004; 134(1): 420–431.
PMCID: PMC316321

Transcriptional Regulation of Sorghum Defense Determinants against a Phloem-Feeding Aphid1

Abstract

When attacked by a phloem-feeding greenbug aphid (Schizaphis graminum), sorghum (Sorghum bicolor) activates jasmonic acid (JA)- and salicylic acid (SA)-regulated genes, as well as genes outside known wounding and SA signaling pathways. A collection of 672 cDNAs was obtained by differential subtraction with cDNAs prepared from sorghum seedlings infested by greenbug aphids and those from uninfested seedlings. Subsequent expression profiling using DNA microarray and northern-blot analyses identified 82 transcript types from this collection responsive to greenbug feeding, methyl jasmonate (MeJA), or SA application. DNA sequencing analyses indicated that these encoded proteins functioning in direct defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell maintenance, and photosynthesis, as well as proteins of unknown function. In response to insect feeding, sorghum increased transcript abundance of numerous defense genes, with some SA-dependent pathogenesis-related genes responding to greenbug more strongly than to SA. In contrast, only weak induction of MeJA-regulated defense genes was observed after greenbug treatment. However, infestation tests confirmed that JA-regulated pathways were effective in plant defense against greenbugs. Activation of certain transcripts exclusively by greenbug infestation was observed, and may represent unique signal transduction events independent of JA- and SA-regulated pathways. Results indicate that plants coordinately regulate defense gene expression when attacked by phloem-feeding aphids, but also suggest that aphids are able to avoid triggering activation of some otherwise potentially effective plant defensive machinery, possibly through their particular mode of feeding.

The greenbug (Schizaphis graminum) has been a serious aphid pest on small grains in North America since its introduction over a century ago (Porter et al., 1997). Like many other aphids, greenbug inserts its stylet into phloem sieve elements and consumes phloem sap as its food source (Miles, 1999; Burd, 2002). It initially causes red or necrotic spots on crops such as sorghum (Sorghum bicolor) and wheat (Triticum aestivum), ultimately followed by general necrosis and plant death (Porter et al., 1997; Miles, 1999). The use of resistant hybrids has been the major integrated pest management strategy for managing greenbug on sorghum, however, very little is known about the molecular mechanisms of this resistance.

It has been established that plants respond to insect herbivory by activating an array of defense genes to mount resistance to insects. Much of the information regarding plant defense gene regulation has been derived from studies on chewing insects that crush and devour plant cells and tissues, as well as from the well-established wound signal transduction pathway (Ryan, 2000; de Bruxelles and Roberts, 2001; Kessler and Baldwin, 2002). Damage to cell membranes by mechanical wounding or insect feeding triggers release of linolenic acid from membrane phospholipids (Narvaez-Vasquez et al., 1999). Linolenic acid is converted, via the octadecanoid pathway, to jasmonic acid (JA), a key intracellular defense signal. Accumulation of endogenous JA mediates expression of various early defense signals and late functioning defense genes (Ryan, 2000). The systemic defense response is mediated by transport of wound signals from wounding sites to distant parts of the plant through vascular tissues (de Bruxelles and Roberts, 2001; Orozco-Cardenas et al., 2001). The best-characterized long-distance signal systemin, a small polypeptide, is transmitted via phloem in tomato (Lycopersicon esculentum; Ryan and Pearce, 1998). Perception of systemin by plant cells activates a defense signal transduction pathway leading to biosynthesis of JA and expression of defense genes in systemic tissues.

Despite the commonality between mechanical wounding and insect feeding damage, differential plant responses toward different wound stimuli led to the conclusion that plants can distinguish damage done mechanically from that done by insects. A DNA microarray study with 150 preselected defense-related Arabidopsis genes showed that caterpillar-elicited transcriptional changes differed from those of wounding by forceps. In particular, water stress-induced genes were less responsive to caterpillar (Pieris rapae) feeding (Reymond et al., 2000). Insect chewing also accelerated accumulation of wound-induced transcripts in potato (Solanum tuberosum) leaves (Korth and Dixon, 1997). Furthermore, unique to insect feeding, plants are able to initiate indirect defense by synthesizing specific blends of volatiles that attract natural enemies of the herbivores (Alborn et al., 1997; Pare and Tumlinson, 1997; Schittko et al., 2001), and by forming neoplasmic tissues that impede larval entry into the plant tissue (Doss et al., 2000). Discrimination between mechanical wounding and insect herbivory is thought to be due to recognition of insect-derived elicitors by plant cells (Alborn et al., 1997; Korth and Dixon, 1997; Halitschke et al., 2001).

Considerably less information is available on molecular mechanisms of plant response to aphids, the largest group of phloem-feeding insects. A common feature among reported studies on plant-aphid interactions is that plants activate genes known to mount defenses against bacterial and fungal pathogens (Fidantsef et al., 1999; Moran and Thompson, 2001; Moran et al., 2002). Such a response is generally not stimulated by chewing insects (Reymond et al., 2000). The defense response against pathogens is accompanied by elevated cellular salicylic acid (SA) concentrations and increased expression of pathogenesis-related (PR) proteins that confer disease resistance. Production of reactive oxygen species (ROS), including H2O2, is one of the earliest plant responses in incompatible interactions between pathogens and plants. ROS induce the accumulation of SA and trigger PR protein expression (Wu et al., 1997; Chamnongpol et al., 1998). SA is an essential signaling hormone for activation of local and systemic defenses against pathogens in many plant species (Cao et al., 1998; Dempsey et al., 1999; Zhang et al., 1999). Arabidopsis mutants deficient in SA production or signal transduction exhibit altered disease resistance. NPR1 (nonexpresser of PR genes) is a key regulator in transducing the SA signal (Cao et al., 1997). It contains an ankyrin-repeat domain involved in protein-protein interactions. Mutations in this domain result in loss of resistance, whereas overexpression of NPR1 gene leads to broad-spectrum disease resistance (Cao et al., 1998). Lipase-like proteins, EDS1 and PAD4, directly interact with each other to control SA accumulation, and EDS1 is also engaged early in the hypersensitive response (Feys et al., 2001). Mutant plants eds1-2 and pad4-2 showed reduced SA and decreased EDS1/PAD4-dependent R gene-mediated response. SA has been shown to suppress wound signaling and JA function (Doares et al., 1995). However, SA is not the essential mobile signal transmitted through plants to initiate systemic defense (Alvarez et al., 1998; Wildermuth et al., 2001). Recent identification of a lipid transfer protein implies the nature of the long distant signaling molecule in Arabidopsis may be lipid derived (Maldonado et al., 2002). Transmission of the mobile signal is most likely carried out via the phloem tissue (de Bruxelles and Roberts, 2001).

Similarity in plant responses stimulated by phloem-feeding aphids and by pathogens could be due to the similar effects insect stylets and fungal hyphae have on their hosts during insect feeding and pathogen infection (Fidantsef et al., 1999). The paths of aphid stylets penetrating plant epidermal and parenchymal cells to reach phloem sieves are mainly intercellular or intramural (Miles, 1999). Such a feeding style results in limited plant damage that is distinct from that of chewing insects. Thus, it is not unexpected that plants activate disparate responses when attacked by chewing versus phloem-feeding insects. However, using the Arabidopsis JA-insensitive mutant coi1-1, Moran and Thompson (2001) demonstrated involvement of the wounding pathway as well as SA-regulated genes in response to aphids. Furthermore, the whitefly (Bemisia argentifolii)-induced SLW3 gene from squash did not respond to any known defense-signaling molecules, implying that novel defense regulation could be elicited by phloem-feeding insects (van de Ven et al., 2000; Walling, 2000).

In this study, we aimed at understanding the transcriptional response of sorghum to infestation by the greenbug aphid on a broad scale, and at comparing and contrasting this regulation with JA- and SA-regulated gene expression. We first generated a cDNA collection enriched in greenbug-responsive sorghum genes by subtractive hybridization. We then performed high-throughput DNA microarray analyses to identify greenbug-regulated genes, and compared greenbug-responsive transcript profiles with those after treatments by methyl jasmonate (MeJA) and SA. We also evaluated the effect of MeJA-regulated defense on infestation by greenbug aphids. To our knowledge, this is the first targeted microarray analysis of monocot response against phloem-feeding insects. Our results suggest that greenbug modulates MeJA- and SA-regulated gene expression, an ability most likely linked to its special mode of feeding.

RESULTS

Isolation of Defense-Related Genes in Sorghum

To obtain molecular profiles of plant response to phloem-feeding aphids, we used a combination of subtractive hybridization and cDNA microarray techniques to detect greenbug-responsive genes in sorghum. After forward and reverse subtractions of cDNAs from seedlings infested and uninfested by greenbug, a total of 672 cDNA fragments enriched in greenbug-induced and -suppressed genes were obtained, and their PCR-amplified products were arrayed onto glass slides. To determine the roles of SA and JA pathways in regulation of aphid-responsive genes, the cDNA microarrays were hybridized with probe pairs prepared from total RNAs of greenbug-infested and -uninfested seedlings, as well as from seedlings with or without MeJA or SA treatment. A validated expression fold-change ratio cutoff of 1.5 was applied to determine the cDNA populations that were differentially regulated by the various treatments (Fig. 1). When this threshold was used in control versus control validation experiments (i.e. Cy3- and Cy5-labeled probes were made from the same input RNA and were cohybridized to arrays), 3.2% of the array spots showed 1.5-fold or greater signal differences (Fig. 1), i.e. about 20 cDNAs in the total collection could be potentially miscategorized. To ensure reproducibility, replicate microarrays for each treatment were conducted with separate RNA preparations. Furthermore, selected clones showing altered expression on microarrays were subjected to northern-blot analysis (see below). A total of 128 cDNA clones exhibiting 1.5-fold or greater induction or suppression by greenbug aphid, MeJA, or SA treatment were analyzed by DNA sequencing, and were found to form 82 contigs (Table I).

Figure 1.
Signal intensity patterns of 672 cDNA fragments after microarray with cDNA probes from mRNA of untreated versus treated (greenbug, MeJA, or SA) sorghum seedlings. The self versus self (control versus control RNA) array was carried out by cohybridizing ...
Table 1.
Genes differentially expressed in response to greenbug, Meja and SAa

Coregulation of Defense-Related Genes

As shown in the Venn diagram, 23, 28, and 27 genes were induced, respectively, by greenbug, MeJA, and SA (Fig. 2A; Table I). Among five genes induced under all three treatments were a number of well-known defense-related genes such as various protease inhibitor genes. Three genes were induced only by aphid and MeJA, seven by aphid and SA, and seven by MeJA and SA, respectively (Fig. 2A). Likewise, 10, 29, and 13 genes were down-regulated by greenbug, MeJA, and SA, respectively. Three genes, two photosynthesis-related and one of unknown function, were suppressed by all treatments (Fig. 2B). Commonality observed in gene expression profiles among different treatments suggests that response of sorghum to aphid infestation is mediated, at least partly, by JA and SA signaling processes.

Figure 2.
Venn diagrams showing numbers of overlapping and unique genes induced (A) and suppressed (B) by greenbug (GB), MeJA, or SA at 1.5-fold or higher. Results were based on the mean inductions of two experimental replicates.

Characterization of Greenbug-Responsive Genes in Sorghum

The 82 greenbug-, MeJA-, and SA-responsive cDNA contigs can be categorized into eight groups encoding proteins functioning in defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell maintenance, and photosynthesis, as well as proteins of unknown function (Table I; Figs. Figs.3,3, ,4,4, ,55).

Figure 3.
Greenbug modulates SA-induced PR genes. Total RNA was extracted from untreated and mechanical wounding-, MeJA-, greenbug-, or SA-treated sorghum seedlings at various time points indicated. cDNA fragments encoding thaumatin-like proteins (THAU1 and THAU2), ...
Figure 4.
Greenbug avoids activation of MeJA-induced defense genes. Total RNA was extracted from untreated and greenbug-, mechanical wounding-, MeJA-, or SA-treated sorghum seedlings at various time points indicated. cDNA fragments encoding a lipooxygenase (LOX), ...
Figure 5.
Transcripts of a defense-related protein (DRP) and an LRR-containing protein (SLRR) respond specifically to greenbug infestation. Total RNA was extracted from untreated and greenbug-, mechanical wounding-, MeJA-, or SA-treated sorghum seedlings at various ...

Defense and Defense-Signaling Genes

The first four groups contain genes known to be involved in plant defense responses. Members encoding defense proteins such as various PRs and protease inhibitors, and genes in biosynthetic pathways for phenolics such as flavonone 3′-hydroxylase and O-methyltransferase (Lo and Nicholson, 1998; Frick and Kutchan, 1999), were up-regulated by greenbug feeding. Most of them were also positively regulated by MeJA or SA (Table I). The cyanogenic glucoside dhurrin is a secondary metabolite whose degradation products are known to be potent toxins to herbivore insects (Tattersall et al., 2001). CYP71E1, the P450 gene functioning in the dhurrin biosynthetic pathway, was induced only at earlier time points after greenbug treatment (Fig. 4), and DHUR encoding a dhurrin-degrading enzyme, although present in the subtracted collection, did not respond to greenbug feeding as judged by microarray and northern-blot analyses (Table I; Fig. 4).

Genes encoding antioxidant proteins, such as glutathione S-transferase (GST), lactoylglutathione lyase, and catalase (CAT), were up- or down-regulated by greenbug infestation, suggesting that accumulation and detoxification of ROS simultaneously occur in greenbug-stressed sorghum seedlings. Interestingly, CAT was suppressed by greenbug and MeJA, but was induced by SA, indicating a divergence of SA- and aphid-responsive pathways.

Abiotic Stress-Induced Genes

Greenbug infestation induced a drought-, salt-, and low temperature-responsive gene (DRT) and an aldehyde oxidase gene involved in abscisic acid (ABA) biosynthesis (Table I; Sekimoto et al., 1997). This implies that ABA and ABA-regulated genes also-played a role in sorghum response to greenbug feeding. It has been established that ABA does not directly function in wound signal transduction but may help to maintain healthy physiological conditions near wound sites to ensure wound response (Birkenmeier and Ryan, 1998). Aphid feeding is known to decrease leaf water potential (Cabrera et al., 1994), thus dehydration resulting from greenbug feeding may be the driving force for induction of DRT. A microarray study showing that many wounding-inducible genes were also drought-inducible in Arabidopsis further supports that the role of wound-induced ABA is to respond to water stress (Reymond et al., 2000). SA increased mRNA levels of aldehyde oxidase (Table I), but it did not activate DRT, the drought-responsive gene, further evidencing that aphid and SA differentially regulate subsets of stress-related genes.

Cell Maintenance Genes

Among numerous cell maintenance genes, a nitrite reductase gene (NiR) functioning in nitrogen-assimilation was significantly induced by greenbug infestation (Table I). Feeding directly from phloem sap allows aphids to get access to free amino acids that do not require protein digestion before assimilation. This withdrawal could signal depletion of the nitrogen metabolites Asn and Gln (Sivasankar et al., 1997), leading to the observed induction of NiR.

Genes Involved in Photosynthesis

Photosynthesis-related genes were suppressed strongly by MeJA, and to a lesser extent by SA and aphids (Table I). Suppression of photosynthesis by JA has been established (Creelman and Mullet, 1997), and an inverse correlation between photosynthesis- and defense-related gene regulation has also been observed in plants subjected to fungal infection, elicitor treatment, or insect herbivory (Kombrink and Hahlbrock, 1990; Hermsmeier et al., 2001). This response presumably allows energy reallocation to defense responses, with suppression of less important functions, upon attack by insects and pathogens.

Genes of Unknown Function

Nineteen cDNAs did not match any sequences in GenBank, or matched genes encoding proteins with unknown function. Among the seven greenbug-responsive cDNAs, 2C1 (induced) and 4H3 (suppressed) were greenbug specific. Others were co-regulated with MeJA and/or SA.

Greenbug Infestation Activates SA-Induced Defense Genes Surpassing SA

Strong induction of SA-regulated PR genes, including chitinase (CHIT), glucanase (BGL), thaumatin-like proteins (THAU), and PR10 by greenbug infestation suggested the involvement of an SA signaling pathway. To determine dynamics of PR gene expression in response to mechanical wounding, greenbug infestation, and MeJA and SA signals, selected cDNAs were used as probes for northern-blot analyses at multiple time points. Transcripts of THAUs were induced as soon as 6 h after greenbug challenge, compared with 72 h after SA treatment. However, a chitinase mRNA reached a peak sooner under SA treatment than under greenbug infestation, and PR10 showed a similar expression pattern over time in both treatments (Fig. 3). The stronger, and in some cases, more rapid response of PR genes to greenbug than to SA indicated that greenbug was able to uniquely modulate some genes in the SA-response pathway.

Limited Induction of JA-Regulated Defense Genes by Greenbug Aphid Infestation

A time-course northern-blot analysis of gene expression was also conducted for MeJA up-regulated genes encoding a LOX, a cytochrome P-450 (CYP71E1), DHUR, and a protease inhibitor (BBPI2; Fig. 4). In contrast to the high induction of PR genes by greenbug, the aphid only marginally and temporarily induced JA-regulated defense genes. It is possible that the SA signaling pathway elicited by greenbug suppressed MeJA-responding genes, as in the case of CYP71E1 and DHUR (Fig. 4). However, it should be noted that SA also induced the two JA-induced genes, LOX and BBPI2 (Fig. 4), suggesting that the two pathways are not always exclusive. An alternative explanation for limited expression of JA-induced genes is that tissue damage caused by aphids may not generate sufficiently high endogenous JA levels. Interestingly, however, extensive mechanical wounding also did not result in significant induction of JA-responsive genes in most cases (Fig. 4).

Genes Specifically Induced by Greenbug Infestation

cDNA microarray analysis identified eight genes solely responsive to greenbug infestation. However, the single time point selected for the greenbug microarray experiment may not always fall within time period of gene induction by other treatments. THAUs, for instance, responded to greenbug and SA, but SA induction of THAUs (beginning at 72 h, as shown by northern blots) was not reflected by SA microarray experiment data at 24 h (Table I; Fig. 3). On the other hand, northern-blot analyses at multiple time points indicated the Leu-rich repeat-containing protein (SLRR) and a DRP gene were greenbug specific over the entire time course (Fig. 5). SLRR has been postulated to be a signaling molecule involved in plant-pathogen signal transduction (Hipskind et al., 1996). Accumulation of this transcript could be detected within several hours after fungal infection (Hipskind et al., 1996) or greenbug infestation (Fig. 5), suggesting fungi and phloem-feeding insects may have activated at least partly overlapping signaling cascades in sorghum. The second greenbug-specific gene encoded a protein homologous to a maize (Zea mays) DRP, also known to be induced by sugar starvation (Chevalier et al., 1995). By feeding on phloem sap that contains high concentrations of sugars, aphids could potentially be disturbing sorghum's source-sink metabolism. This assumption is supported by the observation of a 4-fold decrease of soluble carbohydrates in greenbug-infested barley (Hordeum vulgare) seedlings relative to uninfested control seedlings (Cabrera et al., 1994). Thus, the DRP gene could be regulated by a sugar-signaling pathway that has been shown to crosstalk with defense regulatory pathways (Roitsch, 1999).

MeJA-Induced Plant Defense Response Deters Greenbug Infestation

To directly test whether the MeJA-induced defense response has any impact on greenbug infestation, we conducted a choice test where greenbugs were equally exposed to MeJA-treated sorghum seedlings and untreated control seedlings and were allowed to freely choose which plants to infest. The frequency of greenbug infestation on control plants was significantly higher than that on the MeJA-treated plants (chi square = 343.6, df = 1, P < 0.0001). The mean proportion of aphids observed on control plants was 0.647 ± 0.007 (sd) compared with on MeJA plants, with 0.352 ± 0.007 (Fig. 6). Additional choice testing determined that any residual free MeJA that may have remained associated with the MeJA-treated plants was not responsible for deterring greenbug infestation. Of a total of 941 greenbugs, 444 (47.2%) crossed the MeJA line to infest untreated seedlings beyond the line, compared with 497 (52.8%) that chose untreated seedlings without an interposed MeJA line.

Figure 6.
MeJA-induced plant defense response deters greenbug infestation. MeJA-treated and untreated sorghum seedlings were alternately placed in a radial pattern, equally accessible to greenbugs. Greenbugs were allowed to freely choose which plants to infest. ...

DISCUSSION

Direct Feeding and Salivation into Phloem Tissue by Aphids Elicits Strong and Rapid Plant Defense Gene Expression

When aphids' stylets initially enter phloem sieve cells, they often discharge saliva into plant tissues before ingestion of phloem sap materials, and this process can last 10 min or longer (Prado and Tjallingii, 1997). In addition to nutrient transport, the phloem also serves important roles in long distance transport of endogenous hormones for plant development, RNA elicitors for posttranscriptional gene silencing, and signals for systemic defense responses (de Bruxelles and Roberts, 2001; Ueki and Citovsky, 2001). The polypeptide defense signal systemin was translocated from the wound site throughout the whole plant via phloem within only 1 to 2 h (Ryan and Pearce, 1998). In addition, the systemic accumulation of H2O2, the second messenger for defense gene activation, occurred near the vascular tissues after wounding or pathogenesis (Alvarez et al., 1998; Orozco-Cardenas et al., 2001). Aphids' saliva contains various hydrolytic enzymes (Miles, 1999) that may function as elicitors, as exemplified by the β-glucosidase identified from the oral secretion of a Lepidopteran insect (Mattiacci et al., 1995). The time-frame shift of THAU induction caused by greenbug compared with SA-induced gene expression is very much analogous to the effect of Manduca spp. regurgitants on wound-induced genes (Korth and Dixon, 1997). It is possible that direct release of the salivary compounds into phloem tissues allowed sorghum to perceive aphid invasion, and that this recognition resulted in stronger and more rapid defense gene expression compared with SA treatment.

H2O2 May Play a Role in Plant Defense against Aphids

Production of ROS, particularly H2O2, has repeatedly been associated with diverse plant-pathogen and plant-insect interactions (Alvarez et al., 1998; Orozco-Cardenas and Ryan, 1999; Orozco-Cardenas et al., 2001). H2O2 stimulates SA and ethylene production, induces PR protein expression, and enhances pathogen tolerance (Wu et al., 1997; Chamnongpol et al., 1998). Systemic H2O2 production has also been observed in response to wounding in several plant species including barley and maize (Orozco-Cardenas and Ryan, 1999), and H2O2 has been confirmed as a second messenger in activating defense gene expression (Orozco-Cardenas et al., 2001). Accumulation of H2O2 is triggered by oligo-GalUA released from the cell walls of vascular bundle cells by wound-induced plant polygalacturonases (Bergey et al., 1999). Aphids, although they only cause limited wounding, produce and secrete polygalacturonases into plant tissues (Ma et al., 1990). Existence of this enzyme in aphids' saliva is thought to predigest plant polysaccharides for nutritional purposes (Miles, 1999). Yet, it could also potentially cause release of oligo-GalUA and production of H2O2, especially because aphid-derived polygalacturonases are directly secreted into the phloem and readily distributed throughout the plant. Direct anti-insect activity of ROS has been demonstrated (Bi and Felton, 1995). Therefore, in sorghum, down-regulation of a CAT after greenbug infestation (Table I) may allow the plant to maintain increased H2O2 levels that can cause damage to the insect midgut. By this rationale, the observed induction of GST, encoding xenobiotics-metabolizing and ROS-detoxifying enzymes, would appear to represent an attempt of sorghum to cope with oxidative damage resulting from elevated H2O2 levels. However, the fact that greenbug-induced necrosis continues to spread, leading eventually to plant death, indicates that induction of GSTs is insufficient to prevent widespread oxidative damage to the plant.

Are Plants Misled by Aphids?

It has been proposed that phloem-feeding insects are perceived as pathogens due to similarities in the manner of penetration of plant tissues by fungal hyphae and aphid stylets, and to some extent, by the similar hydrolytic enzymes released during fungal growth and insect feeding (Fidantsef et al., 1999; Walling, 2000). In accordance with this view, we observed a strong induction of SA-regulated PR genes by greenbug feeding, but a weak induction of wounding and JA-regulated genes. Although up-regulation of PR genes by several aphid species has been reported, no direct role has been established for PR proteins in defense against phloem-feeding insects. Higher chitinase and glucanase activities did not occur in greenbug-resistant sorghum (Krishnaveni et al., 1999). Likewise, induction of SA-dependent responses did not confer resistance to green peach aphid in Arabidopsis (Moran and Thompson, 2001). In contrast, control of insect pests including phloem-feeders by JA-regulated genes such as protease inhibitors has been established (Rahbe and Febvay, 1993; Tran et al., 1997). In our study, significantly less greenbug infestation was observed on MeJA-pretreated seedlings, suggesting the effectiveness of plant defense elicited by MeJA against aphid invasion. We also found CYP71E1, one of the sorghum cytochrome P450s involved in dhurrin biosynthesis (a cyanogenic glucoside derived from Tyr), to be highly induced by MeJA (Fig. 4). Upon damage of plant tissue by insect herbivory, dhurrin is hydrolyzed by DHUR to produce toxic hydrogen cyanide. Integration of dhurrin biosynthesis into Arabidopsis conferred resistance to flea beetle (Phyllotreta nemorum; Tattersall et al., 2001). Thus, activation of gene expression involved in dhurrin biosynthesis and degradation could negatively impact aphids. Another effective anti-insect gene, LOX, regulates plant defense by its role in the octadecanoid pathway for JA biosynthesis (Royo et al., 1999). Yet, greenbug only weakly induced the accumulation of these mRNAs compared with the levels of induction by MeJA. Thus, it appears that sorghum tended to activate ineffective, but suppress effective, defense gene sets when attacked by aphids. Supporting this conclusion, some aphids such as Aphis fabae accepted pre-infested wheat as host plants more readily than un-infested wheat (Prado and Tjallingii, 1997). It seems aphid feeding not only avoided provoking plant defense, but also stimulated changes in phloem components that were nutritionally advantageous to aphids themselves.

Several scenarios for inadequate activation of defense gene expression are possible. First, minimal tissue damage by aphid infestation could produce insufficient JA levels for gene induction. Second, strong induction of SA-dependent responses may have suppressed the JA-signaling pathway due to pathway crosstalk (Doares et al., 1995). However, we noticed that mechanically wounded sorghum seedlings in our experiments, which exhibited significant tissue damage, also did not induce JA-responsive genes. Thus, there is a third possibility that wound-induced endogenous ethylene may have suppressed JA-induced genes. This suppression has been observed in numerous plant species (Zhu-Salzman et al., 1998; Rojo et al., 1999; Stotz et al., 2000; Winz and Baldwin, 2001). Even though greenbug infestation entailed minimal wounding, infestation was found to cause ethylene production in barley (Argandona et al., 2001), and this ethylene evolution, elicited by tissue damage or by aphid salivary compounds, could potentially block JA-induced gene expression.

Plant response to herbivore and pathogen attacks often involves the interaction of multiple signal pathways that are controlled by a small number of global signals (Reymond and Farmer, 1998). However, it is questionable whether the outcome of the crosstalk always leads to the most effective plant defense for the specific challenge. Unnecessary SA production and SA-mediated defense responses were also elicited by a nonhost pathogen in Arabidopsis (van Wees and Glazebrook, 2003). In our study, the observed gene regulation profile in response to greenbug infestation builds toward a case for subversion of potentially effective plant defense measures to a misguided anti-microbial defense pathway in sorghum. Aphid-vectored viruses and endosymbionts could be responsible for this elicitation of pathogen-responsive gene expression. Infestation by whiteflies vectoring tomato mottle virus induced much stronger PR protein expression than the virus-free white-flies (McKenzie et al., 2002). Accordingly, higher accumulation of PR proteins did not negatively impact the whitefly reproduction. On the contrary, more eggs and nymphs were associated with high PR-expressing plants (McKenzie et al., 2002). More studies are needed to fully unravel phloem feeding insect-host plant interactions; however, evidence from our study indicates the phloem-feeding insects may currently hold an important advantage.

MATERIALS AND METHODS

Growing Sorghum (Sorghum bicolor) Seedlings and Rearing Greenbugs (Schizaphis graminum)

Sorghum seeds (ATx399 × RTx430) were planted in potting soil in plastic pots (4.5-inch diameter and 3.5-inch depth), and seedlings were grown in an insect-free growth chamber (30°C, 60% relative humidity, and a 13-h light/11-h dark photoperiod). Two-week-old sorghum seedlings were used for greenbug culturing, and infested plants were maintained in a separate chamber under the same growth conditions.

Plant Treatments with Mechanical Wounding, MeJA, SA, and Greenbug Infestation

One-week-old seedlings were used for all treatments. Wounding was done by pressing a rounded file against seedling leaves and stems until the whole area above the soil of each seedling was covered by wounding spots. MeJA solution (200 μm) was sprayed onto seedling leaves to run-off with a spray bottle. The plants were then grown under constant light in a sealed container. Greenbugs were brushed onto seedlings at a density of approximately 20 per seedling. Before harvest, greenbugs were removed from greenbug-infesting seedlings by flushing the plants with deionized water. Seedlings were gently dried using paper towels before freezing in liquid nitrogen. Plant materials above the soil from wounding, MeJA, and greenbug treatments were harvested after 0-, 6-, 12-, 24-, and 48-h time points, wrapped in aluminum foil, frozen in liquid nitrogen, and stored at -80°C until use. For the SA treatment, sorghum seedlings were grown hydroponically. Briefly, sorghum seeds were germinated for 3 d as described by Finlayson et al. (1998). The seedlings were then transferred into truncated pipette tips placed in plastic pegboard sheets that were suspended over plastic buckets filled with 0.5× Hoagland solution. An airstone was used to aerate the growth medium. After an additional 5-d growth period with one medium exchange, SA was added to a final concentration of 2 mm. Tissues above the roots were harvested at 0, 6, 24, 48, and 72 h, as described above. Approximately 20 seedlings were collected at each time point for each treatment. Control plants not subjected to treatments were also harvested at each time point for each treatment.

Construction of the Subtractive cDNA Library

Sorghum seedlings infested with greenbugs for 48 h and control seedlings (respectively) were ground into fine powder in liquid nitrogen. mRNAs from each sample were isolated using a QuickPrep micro-mRNA purification kit (Amersham Pharmacia Biotech, Piscataway, NJ). The PCR-select cDNA subtraction kit (Clontech, Palo Alto, CA) was used to obtain cDNAs corresponding to genes differentially regulated by greenbug infestation. Forward and reverse subtractive hybridizations were performed. The forward subtraction used tester cDNA obtained from mRNA of greenbug-infested seedlings and driver cDNA from control seedlings, and vice versa in the reverse subtraction. Subtracted cDNAs were subjected to two rounds of PCR amplification to normalize cDNA populations. The PCR products were ligated into pT-Adv vector (Clontech) and transformed into Escherichia coli cells. Bacterial clones harboring cDNA inserts were inoculated, amplified, and maintained in freezer medium [50 mm potassium phosphate, 2 mm sodium citrate, 0.8 mm MgSO4, and 7 mm (NH4)2SO4, pH 7.5] with carbenicillin (50 μg mL-1) in 96-well microplates.

DNA Microarrays and Probe Preparation

cDNA inserts of the subtracted collections were PCR amplified in 150-μL reactions in microplate format, using M13 forward and M13 reverse universal primers. PCR products confirmed by gel electrophoresis were precipitated by addition of 15 μL of 3 m NaOAc, pH 5.2, and 150 μL of isopropanol reaction-1 at -20°C overnight. Microplates were centrifuged at 3,200 rpm for 2 h at 4°C. Pellets were washed with 70% (w/v) ethanol, vacuum dried, and resuspended in 35 μL of spotting solution (2× SSC and 0.1% [w/v] Sarkosyl) each. DNA clones were printed onto poly-L Lys-coated glass slides (CEL Associates, Houston) using an Affymetrix 417 arrayer. After printing, DNA was UV crosslinked at 550 mJ, and slides were further processed by blocking in 0.2% (w/v) SDS for 10 min at 25°C, followed by DNA denaturation in boiling water for 2 min, and treatment with -20°C ethanol (95%, w/v) for 2 min.

Microarray probes were synthesized from equal amounts of total RNA extracted from sorghum seedlings subjected to MeJA (24 h), SA (24 h), or greenbug infestation (48 h), as well as from their respective controls, using the 3DNA expression array system (Genisphere, Hatfield, PA) according to manufacturer's instructions. Briefly, primers comprising oligo dT and a proprietary “capture sequence” for Cy3 or Cy5, respectively, were used with reverse transcriptase to synthesize cDNA probes. These probes (derived from 5 μg of input RNA channel-1 for each microarray) were then mixed with an equal volume of hybridization buffer (Express-hyb; Clontech), added to the microarray and covered with a coverslip, and the slides were sealed in aluminum hybridization chambers (Monterrey Industries, Monterrey, CA) for hybridization at 65°C overnight. After washing (2× SSC/0.1% [w/v] SDS for 10 min at 65°C, followed by 10 min in 2× SSC and then 10 min in 0.2× SSC, both at 25°C with shaking), a second hybridization was conducted to incorporate Cy3 or Cy5 fluor, respectively, which was coupled to an oligonucleotide complementary to the “capture sequence.” Thus, cDNA probes bound to the microarray were fluor labeled by hybridization.

Data Acquisition and Analysis

Slides were scanned with a four-laser confocal scanner (Packard Scanarray 5000; Packard BioChip Technologies, Billerica, MA) using the Scanarray program. Scanning parameters were adjusted to obtain balanced signals on the two channels using the line-scan function. Image analysis was done with the Quantarray Program (Packard BioScience, Downers Grove, IL). Data from both channels were background subtracted and normalized. Normalization factors were generated using spots composing the middle 75% of signal intensity values. Means and standard deviations of normalized and background-subtracted fold-change values from replicate experiments were derived in Excel (Microsoft, Redmond, WA), and are presented in Table I. For each given cDNA spot, gene expression was considered to be changed by treatments if the spot had an average fold change ratio of ≥1.50 or ≤0.67 over the two treatments, and gave a signal intensity of >3,000 in one or both of the two channels (control or treatment) in at least one replicate of at least one treatment. Fold-cutoffs for valid induction and suppression ratios were established by a series of self versus self microarray experiments in which Cy3- and Cy5-labeled probes made from the same RNA were cohybridized to the DNA arrays. The labeling system used repeatedly gave a false-positive rate of 3% to 4% when using 1.5-fold induction and suppression ratio cutoffs and a signal intensity cutoff of 3,000. This characteristic behavior was also observed in self versus self tests using RNAs from diverse treatments (data not shown). A series of preliminary dye-swap experiments demonstrated that the labeling system used resulted in negligible dye bias, as has been reported elsewhere (Yu et al., 2002). Therefore, we adopted the convention of using Cy5 fluor to label the control probe in all experiments.

DNA Sequencing and Data Analyses

Differentially expressed cDNAs were subjected to dideoxy terminator cycle sequencing using the ABI BigDye sequencing kit (PE Biosystems, Foster City, CA), and were analyzed on an ABI Prism 3100 DNA sequencer (PE Biosystems). Sequencher software (Gene Codes, Ann Arbor, MI) was used to trim the vector sequence from raw sequence data, and to assemble contigs. cDNA identities were determined by sequence comparison with the GenBank database using BLASTX.

Northern-Blot Analyses and Signal Quantitation

Twenty micrograms of total RNA from each selected sample was separated on 1.2% (w/v) agarose formaldehyde gels, transferred to Hybond-N nylon membrane, and hybridized with 32P-labeled cDNA probes. Blots were washed (2× SSC/0.1% [w/v] SDS, 1× SSC/0.1% [w/v] SDS, and 0.1% SSC/0.1% SDS [w/v] at 65°C) and exposed to x-ray film. Ethidium bromide-stained total RNA gels were used to visually ensure even sample loading.

Infestation Choice Test

One-week-old sorghum seedlings were subjected to MeJA vapor treatment in a 10-L air-tight container for 15 h. Twenty microliters of 10× diluted MeJA solution was applied to a 1-× 1-cm filter paper placed in the sealed container. The final concentration of MeJA was 900 nmol L-1 air. After 15 h, the shoots from MeJA-treated and untreated plants were excised and each was placed in an Eppendorf tube filled with distilled water at the cut end. The tube openings were sealed around the stems with parafilm. This system protected the seedling from dehydration for several days. Several hundred greenbug aphids were brushed onto the center of a large piece of filter paper, surrounded in a radial pattern by 10 pairs of excised sorghum seedlings (MeJA-treated and untreated control) several centimeters away. Alternating MeJA-treated and untreated plants were equally accessible to the aphids. After 5 to 8 h, all aphids were able to locate host plants, MeJA-treated, or untreated control. The number of aphids on each seedling was counted at the 8-h time point. The experiment was performed three times. Chi-square tests were used to determine statistical significance of differences between treatments (Sokal and Rohlf, 1995). Count data were transformed to proportions for visual display.

To determine whether any residual MeJA vapor potentially remaining associated with the treated plants could deter greenbug infestation, untreated sorghum shoots were arranged in two straight rows on a piece of filter paper, one row on either side of, and equidistant from, the greenbug introduction zone. A thin line of MeJA (400 μm) was painted on the filter paper in front of one of the two rows of seedlings, so that the insects would need to cross the line to get to that row of plants. The greenbugs were then immediately introduced. Eight hours later, the number of aphids on each seedling was counted, and total greenbugs on each of the two groups were compared.

Distribution of Materials

The expressed sequence tag clones generated in this project will be made available upon request for noncommercial research purposes.

Notes

1This work was partially supported by a grant from USDANRI, proposal no. 2000-02914.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.028324.

References

  • Alborn T, Turlings TCJ, Jones TH, Stenhagen G, Loughrin JH, Tumlinson JH (1997) An elicitor of plant volatiles from beet armyworm oral secretion. Science 276: 945-949.
  • Alvarez ME, Pennell RI, Meijer PJ, Ishikawa A, Dixon RA, Lamb C (1998) Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 92: 773-784. [PubMed]
  • Argandona VH, Chaman M, Cardemil L, Munoz O, Zuniga GE, Corcuera LJ (2001) Ethylene production and peroxidase activity in aphid-infested barley. J Chem Ecol 27: 53-68. [PubMed]
  • Bergey DR, Orozco-Cardenas M, de Moura DS, Ryan CA (1999) A wound- and systemin-inducible polygalacturonase in tomato leaves. Proc Natl Acad Sci USA 96: 1756-1760. [PMC free article] [PubMed]
  • Bi JL, Felton GW (1995) Foliar oxidative stress and insect herbivory: primary compounds, secondary metabolites, and reactive oxygen species as components of induced resistance. J Chem Ecol 21: 1511-1530. [PubMed]
  • Birkenmeier GF, Ryan CA (1998) Wound signaling in tomato plants: evidence that ABA is not a primary signal for defense gene activation. Plant Physiol 117: 687-693. [PMC free article] [PubMed]
  • Burd JD (2002) Physiological modification of the host feeding site by cereal aphids (Homoptera: Aphididae). J Econ Entomol 95: 463-468. [PubMed]
  • Cabrera HM, Argandona VH, Corcuera LJ (1994) Metabolic changes in barley seedlings at different aphid infestation levels. Phytochemistry 35: 317-319.
  • Cao H, Glazebrook J, Clarke JD, Volko S, Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88: 57-63. [PubMed]
  • Cao H, Li X, Dong XN (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proc Natl Acad Sci USA 95: 6531-6536. [PMC free article] [PubMed]
  • Chamnongpol S, Willekens H, Moeder W, Langebartels C, Sandermann H, Van Montagu A, Inze D, Van Camp W (1998) Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco. Proc Natl Acad Sci USA 95: 5818-5823. [PMC free article] [PubMed]
  • Chevalier C, Bourgeois E, Pradet A, Raymond P (1995) Molecular cloning and characterization of 6 cDNAs expressed during glucose starvation in excised maize (Zea mays L.) root tips. Plant Mol Biol 28: 473-485. [PubMed]
  • Creelman RA, Mullet JE (1997) Biosynthesis and action of jasmonates in plants. Annu Rev Plant Physiol Plant Mol Biol 48: 355-381. [PubMed]
  • de Bruxelles GL, Roberts MR (2001) Signals regulating multiple responses to wounding and herbivores. Crit Rev Plant Sci 20: 487-521.
  • Dempsey DA, Shah J, Klessig DF (1999) Salicylic acid and disease resistance in plants. Crit Rev Plant Sci 18: 547-575.
  • Doares SH, Narvaez-Vasquez J, Conconi A, Ryan CA (1995) Salicylic acid inhibits synthesis of proteinase-inhibitors in tomato leaves induced by systemin and jasmonic acid. Plant Physiol 108: 1741-1746. [PMC free article] [PubMed]
  • Doss RP, Oliver JE, Proebsting WM, Potter SW, Kuy SR, Clement SL, Williamson RT, Carney JR, DeVilbiss ED (2000) Bruchins: insect-derived plant regulators that stimulate neoplasm formation. Proc Natl Acad Sci USA 97: 6218-6223. [PMC free article] [PubMed]
  • Feys BJ, Moisan LJ, Newman MA, Parker JE (2001) Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4. EMBO J 20: 5400-5411. [PMC free article] [PubMed]
  • Fidantsef AL, Stout MJ, Thaler JS, Duffey SS, Bostock RM (1999) Signal interactions in pathogen and insect attack: expression of lipoxygenase, proteinase inhibitor II, and pathogenesis-related protein P4 in the tomato, Lycopersicon esculentum. Physiol Mol Plant Pathol 54: 97-114.
  • Finlayson SA, Lee IJ, Morgan PW (1998) Phytochrome B and the regulation of circadian ethylene production in sorghum. Plant Physiol 116: 17-25. [PMC free article]
  • Frick S, Kutchan TM (1999) Molecular cloning and functional expression of O-methyltransferases common to isoquinoline alkaloid and phenylpropanoid biosynthesis. Plant J 17: 329-339. [PubMed]
  • Halitschke R, Schittko U, Pohnert G, Boland W, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses. Plant Physiol 125: 711-717. [PMC free article] [PubMed]
  • Hermsmeier D, Schittko U, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: large-scale changes in the accumulation of growth- and defense-related plant mRNAs. Plant Physiol 125: 683-700. [PMC free article] [PubMed]
  • Hipskind JD, Nicholson RL, Goldsbrough PB (1996) Isolation of a cDNA encoding a novel leucine-rich repeat motif from Sorghum bicolor inoculated with fungi. Mol Plant-Microbe Interact 9: 819-825. [PubMed]
  • Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: the emerging molecular analysis. Annu Rev Plant Biol 53: 299-328. [PubMed]
  • Kombrink E, Hahlbrock K (1990) Rapid, systemic repression of the synthesis of ribulose 1,5-bisphosphate carboxylase small-subunit messenger-RNA in fungus-infected or elicitor-treated potato leaves. Planta 181: 216-219. [PubMed]
  • Korth KL, Dixon RA (1997) Evidence for chewing insect-specific molecular events distinct from a general wound response in leaves. Plant Physiol 115: 1299-1305. [PMC free article] [PubMed]
  • Krishnaveni S, Muthukrishnan S, Liang GH, Wilde G, Manickam A (1999) Induction of chitinases and β-1,3-glucanases in resistant and susceptible cultivars of sorghum in response to insect attack, fungal infection and wounding. Plant Sci 144: 9-16.
  • Lo SCC, Nicholson RL (1998) Reduction of light-induced anthocyanin accumulation in inoculated sorghum mesocotyls: implications for a compensatory role in the defense response. Plant Physiol 116: 979-989. [PMC free article] [PubMed]
  • Ma R, Reese JC, Black WC, Bramel-Cox P (1990) Detection of pectinesterase and polygalacturonase from salivary secretions of living greenbugs, Schizaphis graminum (Homoptera, Aphididae). J Insect Physiol 36: 507-512.
  • Maldonado AM, Doerner P, Dixon RA, Lamb CJ, Cameron RK (2002) A putative lipid transfer protein involved in systemic resistance signalling in Arabidopsis. Nature 419: 399-403. [PubMed]
  • Mattiacci L, Dicke M, Posthumus MA (1995) β-Glucosidase: an elicitor of herbivore-induced plant odor that attracts host-searching parasitic wasps. Proc Natl Acad Sci USA 92: 2036-2040. [PMC free article] [PubMed]
  • McKenzie CL, Shatters RG, Doostdar H, Lee SD, Inbar M, Mayer RT (2002) Effect of geminivirus infection and Bemisia infestation on accumulation of pathogenesis-related proteins in tomato. Arch Insect Biochem Physiol 49: 203-214. [PubMed]
  • Miles PW (1999) Aphid saliva. Biol Rev 74: 41-85.
  • Moran PJ, Cheng YF, Cassell JL, Thompson GA (2002) Gene expression profiling of Arabidopsis thaliana in compatible plant-aphid interactions. Arch Insect Biochem Physiol 51: 182-203. [PubMed]
  • Moran PL, Thompson GA (2001) Molecular responses to aphid feeding in Arabidopsis in relation to plant defense pathways. Plant Physiol 125: 1074-1085. [PMC free article] [PubMed]
  • Narvaez-Vasquez J, Florin-Christensen J, Ryan CA (1999) Positional specificity of a phospholipase A activity induced by wounding, systemin, and oligosaccharide elicitors in tomato leaves. Plant Cell 11: 2249-2260. [PMC free article] [PubMed]
  • Orozco-Cardenas M, Ryan CA (1999) Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway. Proc Natl Acad Sci USA 96: 6553-6557. [PMC free article] [PubMed]
  • Orozco-Cardenas ML, Narvaez-Vasquez J, Ryan CA (2001) Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 13: 179-191. [PMC free article] [PubMed]
  • Pare PW, Tumlinson JH (1997) De novo biosynthesis of volatiles induced by insect herbivory in cotton plants. Plant Physiol 114: 1161-1167. [PMC free article] [PubMed]
  • Porter DR, Burd JD, Shufran KA, Webster JA, Teetes GL (1997) Greenbug (Homoptera: Aphididae) biotypes: selected by resistant cultivars or pre-adapted opportunists? J Econ Entomol 90: 1055-1065.
  • Prado E, Tjallingii WF (1997) Effects of previous plant infestation on sieve element acceptance by two aphids. Entomol Exp Appl 82: 189-200.
  • Rahbe Y, Febvay G (1993) Protein toxicity to aphids: an in vitro test on Acyrthosiphon pisum. Entomol Exp Appl 67: 149-160.
  • Reymond P, Farmer EE (1998) Jasmonate and salicylate as global signals for defense gene expression. Curr Opin Plant Biol 1: 404-411. [PubMed]
  • Reymond P, Weber H, Damond M, Farmer EE (2000) Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 12: 707-720. [PMC free article] [PubMed]
  • Roitsch T (1999) Source-sink regulation by sugar and stress. Curr Opin Plant Biol 2: 198-206. [PubMed]
  • Rojo E, Leon J, Sanchez-Serrano JJ (1999) Cross-talk between wound signalling pathways determines local versus systemic gene expression in Arabidopsis thaliana. Plant J 20: 135-142. [PubMed]
  • Royo J, Leon J, Vancanneyt G, Albar JP, Rosahl S, Ortego F, Castanera P, Sanchez-Serrano JJ (1999) Antisense-mediated depletion of a potato lipoxygenase reduces wound induction of proteinase inhibitors and increases weight gain of insect pests. Proc Natl Acad Sci USA 96: 1146-1151. [PMC free article] [PubMed]
  • Ryan CA (2000) The systemin signaling pathway: differential activation of plant defensive genes. Biochim Biophys Acta 1477: 112-121. [PubMed]
  • Ryan CA, Pearce G (1998) Systemin: a polypeptide signal for plant defensive genes. Annu Rev Cell Dev Biol 14: 1-17. [PubMed]
  • Schittko U, Hermsmeier D, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: accumulation of plant mRNAs in response to insect-derived cues. Plant Physiol 125: 701-710. [PMC free article] [PubMed]
  • Sekimoto H, Seo M, Dohmae N, Takio K, Kamiya Y, Koshiba T (1997) Cloning and molecular characterization of plant aldehyde oxidase. J Biol Chem 272: 15280-15285. [PubMed]
  • Sivasankar S, Rothstein S, Oaks A (1997) Regulation of the accumulation and reduction of nitrate by nitrogen and carbon metabolites in maize seedlings. Plant Physiol 114: 583-589. [PMC free article] [PubMed]
  • Sokal RR, Rohlf FJ (1995) Biometry. The Principles and Practice of Statistics in Biological Research, Ed 3. W. H. Freeman and Company, New York.
  • Stotz HU, Pittendrigh BR, Kroymann J, Weniger K, Fritsche J, Bauke A, Mitchell-Olds T (2000) Induced plant defense responses against chewing insects: Ethylene signaling reduces resistance of Arabidopsis against Egyptian cotton worm but not diamondback moth. Plant Physiol 124: 1007-1017. [PMC free article] [PubMed]
  • Tattersall DB, Bak S, Jones PR, Olsen CE, Nielsen JK, Hansen ML, Hoj PB, Moller BL (2001) Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293: 1826-1828. [PubMed]
  • Tran P, Cheesbrough TM, Keickhefer RW (1997) Plant proteinase inhibitors are potential anticereal aphid compounds. J Econ Entomol 90: 1672-1677.
  • Ueki S, Citovsky V (2001) RNA commutes to work: regulation of plant gene expression by systemically transported RNA molecules. Bioessays 23: 1087-1090. [PubMed]
  • van de Ven WTG, LeVesque CS, Perring TM, Walling LL (2000) Local and systemic changes in squash gene expression in response to silverleaf whitefly feeding. Plant Cell 12: 1409-1423. [PMC free article] [PubMed]
  • van Wees SCM, Glazebrook J (2003) Loss of non-host resistance of Arabidopsis NahG to Pseudomonas syringae pv. phaseolicola is due to degradation products of salicylic acid. Plant J 33: 733-742. [PubMed]
  • Walling LL (2000) The myriad plant responses to herbivores. J Plant Growth Regul 19: 195-216. [PubMed]
  • Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414: 562-565. [PubMed]
  • Winz RA, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata: Insect-induced ethylene reduces jasmonate-induced nicotine accumulation by regulating putrescine N-methyltransferase transcripts. Plant Physiol 125: 2189-2202. [PMC free article] [PubMed]
  • Wu GS, Shortt BJ, Lawrence EB, Leon J, Fitzsimmons KC, Levine EB, Raskin I, Shah DM (1997) Activation of host defense mechanisms by elevated production of H2O2 in transgenic plants. Plant Physiol 115: 427-435. [PMC free article] [PubMed]
  • Yu J, Othman M, Farjo R, Zareparsi S, MacNee S, Yoshida S, Swaroop A (2002) Evaluation and optimization of procedures for target labeling and hybridization of cDNA microarrays. Mol Vision 8: 130-137. [PubMed]
  • Zhang YL, Fan WH, Kinkema M, Li X, Dong XN (1999) Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proc Natl Acad Sci USA 96: 6523-6528. [PMC free article] [PubMed]
  • Zhu-Salzman K, Salzman RA, Koiwa H, Murdock LL, Bressan RA, Hasegawa PM (1998) Ethylene negatively regulates local expression of plant defense lectin genes. Physiol Plant 104: 365-372.

Articles from Plant Physiology are provided here courtesy of American Society of Plant Biologists

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...