Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae
- Chunling Zhang†1,
- Haojie Shi†1,
- Lei Chen†1, 2,
- Xiaomeng Wang†1,
- Beibei Lü1,
- Shuping Zhang1,
- Yuan Liang1,
- Ruoxue Liu1,
- Jun Qian1,
- Weiwei Sun1,
- Zhenzhen You1 and
- Hansong Dong1Email author
© Zhang et al; licensee BioMed Central Ltd. 2011
Received: 20 August 2010
Accepted: 13 January 2011
Published: 13 January 2011
Treatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2), one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis) PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1.
The electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT) Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana) plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants.
The repression in phloem-feeding activities of M. persicae as a result of AtPP2-A1 overexpression, and as a deterrent effect of HrpNEa treatment in WT Arabidopsis rather than the atpp2-a1/E/142 mutant suggest that AtPP2-A1 plays a role in plant resistance to the insect, particularly at the phloem-feeding stage. The accompanied change of aphid population in leaf colonies suggests that the function of AtPP2-A1 is related to colonization of the plant.
Harpins are multifunctional proteins produced by Gram-negative plant pathogenic bacteria [1, 2]. The first-characterized  and well-studied harpin [2–7], HrpNEa, is secreted by Erwinia amylovora, the bacterial pathogen that causes fire blight disease in rosaceous plants . Multiple functions of harpin proteins, especially in eliciting plant defense responses, were also elucidated initially by studies using HrpNEa as a paradigm [1–3]. Early studies demonstrated that the external application of HrpNEa was able to induce resistance in a variety of plant species [3–7], and that the induced resistance effectively protected plants from attacks by insect herbivores [2, 7–9]. HrpNEa-induced resistance to insects first was suggested based on observations of field-grown peppers. Plants that had been treated with HrpNEa incurred fewer injuries from the European corn borer than comparable untreated plants . A deterrent effect on striped cucumber beetles was observed in HrpNEa-treated cucumber; striped cucumber beetles preferred to colonize untreated control plants rather than HrpNEa-treated plants . HrpNEa-induced resistance was also effective in impeding infestations of aphids, an important type of phloem-feeding herbivores [9, 10]. In cucumbers grown under environmentally controlled conditions, HrpNEa treatment had a deterrent effect on colonization by the muskmelon aphid Aphis gossypii (Glover), which preferred to colonize control plants rather than HrpNEa-treated plants . In Arabidopsis thaliana (Arabidopsis), moreover, HrpNEa-induced resistance was shown to repress infestation of the green peach aphid Myzus persicae (Sulzer), a generalist phloem-feeding insect .
Phloem-feeding insects are highly specialized in their mode of feeding  and present a unique stress on plant fitness [12–15]. These insects use their slender stylets to feed from a single-cell type, the phloem sieve element [8, 16]. The feeding process can be monitored by the electrical penetration graph (EPG) technique . Pivotally, a stylet puncturing of the host plant cell, shown as a probe in the EPG, may lead to uptake of the phloem sap. In order to prevent protein clogging inside the sieve element, ejection of watery saliva is essential in feeding from the phloem [13, 16]. This ejection is detected in the EPG as E1 salivation and always precedes phloem sap ingestion . During ingestion from the sieve element, the watery E2 salivation occurs, and this E2 saliva is added to the ingested sap, thought to prevent phloem proteins from clogging inside the capillary food canal . Therefore, salivation is a crucial event during the phloem-feeding process for insects to overcome a number of phloem-related plant properties and reactions [13–19].
In response to the phloem-feeding stress, plants defend themselves specifically using the phloem-based defense (PBD) mechanism [14–16], which can be also activated by other cues, such as wounding [20–22], besides insect attacks [14, 20–22]. Proposed components of PBD include the phloem protein 1 (PP1) and phloem protein 2 (PP2), which represent a type of the most abundant proteins in the phloem sap . PP2 is a phloem lectin conserved in plants [23, 24] and is believed to play a role in the establishment of PBD induced by insect attacks [21, 25, 26] and other stresses, such as wounding [16, 21, 22, 26] and oxidative conditions . In pumpkin, PP1 monomers and PP2 dimers are covalently cross-linked via disulphide bonds, forming high molecular weight polymers that close the sieve pores [21, 25, 26]. This response is induced by oxidative stress  but normally accompanies the synthesis of the β-1,3-glucan callose by callose synthase  that accumulates on sieve plates after different stress treatments . Phloem protein plugging and callose closure of sieve pores, and callose coagulation on sieve plates as well, is hypothesized to serve as a physical barrier to prevent the insect from phloem-feeding activity . Nevertheless, evidence for the function of phloem proteins in insect defense has been in paucity.
In the completely sequenced Arabidopsis genome, PP2 (previously PP2-like) genes were identified as a large multigene family constituted of 30 members [23, 27], AtPP2-A1 to AtPP2A-15 and AtPP2-B1 to AtPP2-B15 . To our knowledge, however, little has been known about bioprocesses affected by these genes and properties of the encoded proteins. Although Arabidopsis mutants that represent multiple mutation alleles of AtPP2 have been generated [27, 28], subsequent biological effects have not been studied, and especially, effects of AtPP2 mutations on the plant resistance to insects are unclear. For example, different types of Arabidopsis mutants were generated by T-DNA insertion at distinct locations in the AtPP2-A1 DNA sequence; atpp2-a1/P/-210 resulted from the insertion at nucleotide residue -210 in the promoter region. When grown on an artificial medium, the atpp2-a1/P/-210 mutant performs as the wild-type (WT) plant in response to infestations of M. persicae adults and newborn nymphs in 24 hours after colonization by the adults . There is as yet no evidence to show if atpp2-a1/P/-210 impacts longer behaviors and feeding activities of the insect and if other mutation alleles of AtPP2-A1 have biological effects [27, 28].
The purpose of this study was to obtain genetic evidence that could elucidate a function of AtPP2-A1 in Arabidopsis resistance to M. persicae. We began with determining the effect of AtPP2-A1 on phloem feeding of aphids that colonized the plants treated with HrpNEa according to previous evidence that the HrpNEa treatment and M. persicae infestation had some degrees of overlapping effects on the induction of plant responses. For example, formation of the PP2-PP1 complex needs reactive oxygen burst in cucurbit  while reactive oxygen burst is a conserved response in Arabidopsis treated with any harpins [30, 31]. M. persicae infestation induces an elevation of the ethylene level  and triggers modest induction of ethylene-dependent responses [32, 33], whereas, HrpNEa induces resistance to M. persicae by activating the ethylene-signaling pathway [4, 34]. Therefore, we devised to determine the possibility that HrpNEa-induced resistance involves the PBD mechanism to encounter with M. persicae infestation. In order to further test this hypothesis, we generated AtPP2-A1-overexpression plants and investigated them to elucidate the supposed function of AtPP2-A1. In this article, we report evidence that harpin-induced expression and transgenic overexpression of AtPP2-A1 induce a repression in the phloem-feeding activity of M. persicae.
HrpNEa treatment in Arabidopsis induces a repression in phloem feeding and colonization by M. persicae
Four-hour electrical penetration graph (EPG) analyses of the green peach aphid Myzus persicae feeding from wild-type (WT) Arabidopsis plants
Control group mean (SD*)
HrpNEatreatment group mean (SD*)
Student's t-test (n = 20)
Number of nonpuncturing phase
p < 0.01
p < 0.01
Duration of nonpuncturing, min
p < 0.01
p < 0.01
p < 0.01
Time to 1st cell puncturing, min
Time to 1st pathway, min
Number of pathway phase
p < 0.01
p < 0.01
p < 0.01
Duration of pathway phase, min
p < 0.01
p < 0.01
p < 0.05
p < 0.01
p < 0.01
Time to 1st phloem phase, min
p < 0.01
Number of cell puncturing after 1st phloem phase
p < 0.01
Number of phloem phase
p < 0.01
p < 0.01
p < 0.01
Duration of phloem phase, min
p < 0.01
p < 0.01
p < 0.01
p < 0.01
Duration of phloem feeding, min
p < 0.01
p < 0.01
p < 0.01
Based on the four-hour EPG record, the proportions of times within the pathway phase and time to the first phloem phase were much longer, suggesting the impediment to aphids in locating the ingestion site within the vascular tissue, in HrpNEa-treated plants compared with control plants (Figure 1a; Table 1). On HrpNEa-treated plants, moreover, aphids took fewer actions to puncture cells (Table 1, Number of cell puncturing) and to enter the phloem phase (Table 1, Number of phloem phase) after the first entry of phloem phase. These results suggested that phloem properties of HrpNEa-treated plants were changed as unfavourable to aphid feeding. In consistence with this notion, total duration of the phloem phase was markedly shorter in HrpNEa-treated plants than in control plants (Table 1). Noticeably, duration of the phloem phase in the second hour of the EPG monitoring, being 30 in HrpNEa-treated plants and 14 min in control plants, on average, strongly suggested the deterrent effect of the HrpNEa treatment on the phloem-feeding activity of M. persicae.
In the phloem phase, E1 and E2 salivations were recognized by dissecting the EPG waveform patters (Figure 1b). Compounds of E1 and E2 saliva produced by aphids after stylet entry of the phloem are believed to function in preventing protein clogging inside the sieve element and preventing phloem proteins from clogging inside the capillary food canal, respectively . Thus, E1 and E2 salivations play an important role in ingestion of the phloem sap by the insects [13, 16]. As shown in Table 1, durations of both E1 and E2 salivations were much shorter in HrpNEa-treated plants than control plants, confirming the deterrent effect of the HrpNEa treatment on the phloem-feeding activity of M. persicae.
Arabidopsis atpp2-a1/E/142 mutant pampers M. persicaein phloem feeding
Information on AtPP2-A-defected Arabidopsis mutants investigated in this study
T-DNA insertion site
Mutant seed stock no.a
T-DNA insertion lines; a modified approach of thermal asymmetric interlaced-PCR was used to amplify DNA fragments flanking the T-DNA left border from the transformed lines; no phenotype information available at this time.
Sequence-indexed T-DNA insertion lines; presence of the insertion was analyzed by PCR; kanamycin resistance gene may be silenced; PCR- or hybridization-based segregation analysis is required to confirm presence and homozygosity of insertion; may be segregating for phenotypes that are not linked to the insertion; may have additional insertions potentially segregating; no phenotype information available at this time.
Parallel RT-PCR analyses of RNA samples isolated at 0 and 24 hpt revealed that the AtPP2-A genes performed differently in corresponding mutants compared with the WT plant (Figure 3c). Both the basal expression (0 hpt) and HrpNEa-induced expression (24 hpt) of AtPP2-A1 was detected in the atpp2-a1/P/-210 mutant as in WT but not in the atpp2-a1/E/142 mutant (Figure 3c). This result was confirmed by northern blot hybridization (Figure 3d). And this result conformed to the PLACE Web Signal Scan , which revealed 37 types of cis-acting regulatory DNA elements present in the predicted 344-bp promoter of AtPP2-A1. Eighteen elements exist as a single copy and 19 elements have multiple copies, located at distant 83 sites in the promoter sequence. However, none of the elements was disrupted by T-DNA insertion and this might account for AtPP2-A1 expression in atpp2-a1/P/-210. Similarly, none of 35 types of cis-acting regulatory DNA elements scanned in the upstream -370 region of the AtPP2-A14 DNA sequence was disrupted in atpp2-a14/P/-320. This mutant performed as WT in both the basal expression and HrpNEa-induced expression of AtPP2-A14 (Figure 3c). The other eight mutants behaved differently in expression of the corresponding AtPP2-A genes. AtPP2-A3, -A11, -A13, and -A15 were not expressed in their corresponding mutants atpp2-a3/I/1650, -a11/E/177, -a13/E/1872, and -a15/E/312. In contrast, atpp2-a10/P/-157, a11/P/-394, a12/P/-293, a14/P/-320 performed as WT in the expression of the corresponding AtPP2-A genes. In atpp2-a12/P/-293 and atpp2-a12/P/-293, T-DNA insert did not disrupt any DNA regulatory motifs present in AtPP2-A11 and AtPP2-A12 promoters. In atpp2-a10/P/-157, T-DNA insert disrupted the pollen-specific transcription activator element AGAAA (#S000245) [40, 41] located between -159 and -155 in the AtPP2-A10 sequence. In atpp2-a11/P/-394, the MYB recognition site TGGTTT (#S000408)  located between -398 and -393 in the AtPP2-A11 sequence was disjoined by T-DNA insertion. However, both mutations did not affect basal expression of the genes (Figure 3c). In the ten mutants, therefore, only atpp2-a1/E/142 represents an effective mutation allele, which may be responsible for a transcriptional stop of AtPP2-A1 in the plant and result in experimental compromises in both the basal expression and HrpNEa-induced expression of the gene.
Four-hour EPG analysis of aphid feeding from WT Arabidopsis and the atpp2-a 1/E/142 mutant
EVP treatment mean (SD)
HrpNEatreatment mean (SD)
Student's t-test (n = 20)
EVP treatment mean (SD)
Student's t-test (n = 20)
Total duration of nonpuncturing, min
p > 0.05
Duration of pathway phase, min
p < 0.05*
Total duration of phloem phase, min
p < 0.005*
To gain information about the general function of AtPP2-A1 in Arabidopsis resistance to M. persicae, we compared atpp2-a1/E/142 with the other nine mutants and with WT as well in the effects on multiplication of the insect and subsequent nymph activities. The reproduction rate was scored as the ratio between total numbers of newborn nymphs and total numbers of aphid adults that stayed on leaves in five days after colonization. As shown in Figure 4d, reproduction rates were much smaller under the condition of HrpNEa treatment vs. control (Student's t-test, p < 0.01) irrespective of the plant genotypes, suggesting that HrpNEa-induced repression of M. persicae multiplication  was not related to the AtPP2-A1 gene. The gene, however, showed a repressive effect on plant colonization by newborn nymphs. Nymph colonies were more stable on atpp2-a1/E/142 with a smaller proportion of the population decrease than the other mutants or WT (Figure 4e; ANOVA test, p < 0.01). In atpp2-a1/E/142, the deterrent effect of HrpNEa on colonization by nymphs was little, but the effect was evident in the other mutants as in the WT plant (Figure 4e). Evidently, AtPP2-A1 does not affect aphid reproduction, but instead, the gene plays a role in repressing plant colonization by nymphs as by adults.
AtPP2-A1-overexpression confers repressed phloem feeding of M. persicae
Real-time RT-PCR was conducted with RNA samples from leaves and primers specific to AtPP2-A1. As shown in Figure 5b, levels of the AtPP2-A1 transcript varied with the different PP2OETAt lines, and levels of the transcript were greater in all the PP2OETAt lines than the transgenic control plant. Compared with the transgenic control plant, PP2OETAt lines seemed more resistant to colonization and feeding by aphids. Smaller populations of aphids were able to stay for 24 hours on leaf colonies of PP2OETAt than the transgenic control plant (Figure 5c). Consistently, aphids preferred to feed from the transgenic control plant rather than PP2OETAt (Figure 5d). Total duration of the phloem phase in the four-hour EPG record was much shorter in PP2OETAt than in the control plant (Figure 5d).
Based on statistical analyses (ANOVA test, p < 0.01), the ten PP2OETAt lines differed significantly from the transgenic control plant in levels of AtPP2-A1 expression (Figure 5b), the number of aphids that were able to stay for 24 hours on leaf colonies (Figure 5c), and durations of the phloem phase (Figure 5d). In the ten PP2OETAt lines, the number of aphids that were able to stay for 24 hours on leaf colonies was increased (Figure 5c), but durations of the phloem phase was decreased (Figure 5d), with increases in levels of AtPP2-A1 expression (Figure 5b). The PP2OETAt1 line showed as the greatest expresser of AtPP2-A1 and the greatest repressor of colonization and feeding by M. persicae. In addition, a greater repression of phloem feeding by aphids was observed in the presence than the absence of HrpNEa treatment (not shown), suggesting that original and introduced versions of the AtPP2-A1 gene might be able coordinate their functions and might function simultaneously, in PP2OXTA1.
AtPP2-A1 expression in different organs of PP2OETAt1 is consistent with repression of phloem feeding by M. persicae
Real-time RT-PCR analyses revealed that AtPP2-A1 expression varied greatly in different organs of PP2OETAt1. The expression of AtPP2-A1 was conspicuous in leaves, stems, calyces, and petals but little transcript was detected from flower stalks (Figure 6c). Amounts of the AtPP2-A1 transcript were much greater in leaves, stems, calyces, and petals of PP2OETAt1 than the transgenic control plant. However, close amounts of the transcript were detected from flower stalks of both plants. This result suggested the overexpression of AtPP2-A1 in all the organs except flower stalks of PP2OETAt1. Levels of the gene overexpression were higher in leaves, calyces, and petals compared with stems (Figure 6c; ANOVA test, p < 0.01).
The organ-differential levels of AtPP2-A1 overexpression were negatively correlated with the extents by which apterous agamic M. persicae females fed from the different organs. Based on total duration of the phloem phase in the four-hour EPG record (Figure 6d), aphids preferred to feed from leaves, calyces, and petals, but aphids were also able to feed from stems and flower stalks. However, durations of the phloem phase were much shorter when aphids were feeding from leaves, stems, calyces, and petals of PP2OETAt1 compared with the transgenic control plant (Student's t-test, p < 0.01), suggesting that the phloem-feeding activity was repressed in the different organs of PP2OETAt1. Inversely, the phloem phase of aphid feeding from the PP2OETAt1 flower stalk lasted as longer as feeding from the same organ of the transgenic control plant (Figure 6d), suggesting that aphids did not have a preference between both plants in feeding from flower stalks.
Expression of AtPP2-A1 promoter-GUSis organ-unspecific
Analysis of major activities of aphid feeding from uidAETAt (uidA-expressing transgenic Arabidopsis thalian a) and transgenic control plants
Control plant (SD)
Student's t-test (n = 20)
Total duration of nonpuncturing, min
Duration of pathway phase, min
Total duration of phloem phase, min
Although harpin proteins and their functions as proteinaceous elicitors in eliciting plant defense responses have been found for decades [1–3], many aspects of the mechanisms that underlie harpin-induced defenses remain unclear. Important questions include, for example, how a harpin protein as an exogenous signal is perceived by plants and how the signal perception is connected to a transducer to trigger a cellular pathway. Great attentions have been paid to plant signal transduction in harpin-induced resistance to pathogens [3, 6, 30, 31, 37, 44] and insect herbivores [2, 4, 8, 34]. We have used HrpNEa, the first-characterized  and well-studied harpin [2–7], as a model of proteinaceous elicitors to characterize induced resistance against insect herbivores [2, 4, 8, 34], particularly the green peach aphid M. persicae, a generalist phloem-feeding insect . It has been shown that the HrpNEa treatment and M. persicae infestation have some overlapping effects on the induction of plant responses [4, 25, 30–34], especially the PBD mechanism that is suggested to involve the lectin-type phloem protein PP2 [23, 24] as a component [14–16, 25, 26]. Although 30 members of the PP2 multigene family have been identified in Arabidopsis  and AtPP2-modified Arabidopsis mutants generated , little is known about biological effects, especially on resistance to insects, of the genes and mutants http://www.arabidopsis.org. The purpose of this study is to elucidate the function of AtPP2-A1 in resistance to M. persicae in Arabidopsis plants when treated with HrpNEa and under the condition of AtPP2-A1 overexpression.
We show that the treatment of Arabidopsis with HrpNEa induces a repression in M. persicae feeding from the plant phloem (Figure 1; Table 1) and colonization of plants by the insect (Figure 2). Based on the EPG patterns, applying HrpNEa to WT Arabidopsis impedes aphids in stylet puncturing of the plant cell, en route to the vascular tissue while looking for the phloem, and, especially, in the phloem-feeding activity. So the HrpNEa treatment is likely to induce changes in cell wall properties unfavorable to aphid feeding, but this notion remains to be examined. HrpNEa-induced deterrent effect on the phloem-feeding activity has been found in the EPG data analyzed either by hour or based on the four-hour record as a whole (Table 1). The phloem-feeding activity could be reflected in the EPG by duration of the phloem phase composed of E1 and E2 salivations (Figure 1), which are essential for ingestion of the phloem sap [13, 16, 17, 35]. Shortened duration of the phloem phase, in both E1 and E2 salivations (Table 1), suggests that the insect's effort in ingestion of the phloem sap is repressed under the HrpNEa treatment condition compared with control. It is also pertinent to propose that the HrpNEa treatment impacts the insect-plant interaction. In terms of the insect, E1 and E2 saliva are believed to prevent protein clogging inside the sieve element and prevent phloem proteins from clogging inside the capillary food canal [13, 16], respectively. In the plant side, phloem protein plugging of the sieve element presumably serves as a physical barrier to aphid feeding from the phloem . The lectin-type phloem protein PP2 [23, 24] is supposed to play a role in plant response to the feeding stress [21, 25, 26].
Molecular and genetic evidence supports a role of Arabidopsis PP2 gene AtPP2-A1 in HrpNEa-induced repression of M. persicae feeding from the plant phloem. RT-PCR analyses (Figure 3) suggest that AtPP2-A1 is the most HrpNEa-responsive gene of 30 members of the PP2 multigene family . PLACE Web Signal Scan  provides a clue to molecular basis of HrpNEa response and the AtPP2-A1 induction as well. For example, the gene promoter contains three copies of the consensus GT-1 binding box GA/GA/TAAA/T (#S000508) . This element is involved in the regulation of salicylic acid signaling [45, 46], which otherwise can be activated by HrpNEa treatment in Arabidopsis . Moreover, previous studies have shown that HrpNEa-induced resistance to M. persicae is regulated by the ethylene signaling pathway , which essentially involves perception of the ethylene signal by the receptor ETR1, the signal transduction to the integral membrane protein EIN2 , and the regulation of ethylene responsive factors (ERFs) [47, 48]. The W-box TGACC/T (#S000457) present in the AtPP2-A1 promoter has been shown as required for wounding-induced activation of the ERF3 gene . The ERF3 protein is a regulator of ethylene signaling , which otherwise is activated to regulate induced resistance to M. persicae in Arabidopsis plants responding to HrpNEa treatment . Thus, AtPP2-A1 is pertinently thought a part of the signaling pathway that is required for HrpNEa response, at least during induction of the plant resistance to M. persicae . A role of AtPP2-A1 in the induced resistance has been elucidated by evidence obtained from investigating ten mutants (Table 2) in comparison with the WT plant. The investigation demonstrates that deterrent effect of the HrpNEa treatment on aphid feeding from the phloem requires a functional AtPP2-A1 gene in the plant (Figure 4). This notion is especially supported by the absence of HrpNEa-induced repression of the phloem-feeding activity in the atpp2-a1/E/142 mutant (Figure 4; Table 3). This result offers a novel angle to further understanding on the PBD mechanism. Previously, this defensive mechanism was known as a result of plant responses to attacks by phloem-feeding insects [14, 15, 20–22] and other stresses, such as wounding [16, 21, 22, 26]. Now, the PBD mechanism is known to occur as a result of plant response to HrpNEa, a proteinaceous elicitor of plant defenses [1–7, 49]. This notion, however, remains to be examined in regard to how AtPP2-A1 contributes to PBD in response to the HrpNEa treatment.
The function of AtPP2-A1 in conferring repression of the phloem-feeding activity is further supported by evidence obtained from investigating PP2OETAt (AtPP2-A1-overexpression transgenic A. thaliana) plants (Figure 5). Levels of AtPP2-A1 expression are significantly greater in the 10 tested PP2OETAt lines than in the transgenic control plant, conforming to the experimental design for the gene overexpression. In the different PP2OETAt lines, durations of the phloem phase are decreased with increases in levels of AtPP2-A1 expression, suggesting that AtPP2-A1 overexpression confers a repression in the phloem-feeding activity of M. persicae. These observations also identify the PP2OETAt1 line as the greatest expresser of AtPP2-A1 and the greatest repressor of colonization and feeding by the insect. Reminiscently of cell-to-cell PP2 RNA movement in cucurbits  and distant phloem transport flowering signals , little amount of the AtPP2-A1 transcript in flower stalks (Figure 6) may result from organ-to-organ trafficking to fulfill the requirement for flower development. In the other organs, however, AtPP2-A1 expression is consistent with the repression of aphid feeding from the organs (Figure 6). The organ-unspecific feature of AtPP2-A1 expression and function is also suggested indirectly by investigating the transgenic plant uidAETAt1. In the plant, uidA expression under direction of the AtPP2-A1 promoter is found in various organs (Figure 7), but these organs do not have a repressive effect on aphid feeding (Table 4). This result indirectly supported that AtPP2-A1 plays a role, only when expressed itself, in repression of the phloem-feeding activity. Consistent to our observations on uidAETAt1, a previous study detected GUS activity in different organs of transgenic plants that expressed uidA under direction by the AtPP2-A promoter . In the present study, both uidA transcript and GUS activity were detected in different organs of uidAETAt1 (Figure 7). Due to our failure in obtaining transgenic plants that had been designed to express AtPP2-A1-uidA under control by the AtPP2-A1 promoter, now we can not provide more convening evidence for coincident organ localization in AtPP2-A1 expression and aphid feeding repression. However, repression of the phloem-feeding activity seems a consistent attribute of the different PP2OETAt lines (Figure 5) and a consistent attribute of the different organs of PP2OETAt1 (Figure 6) as well, owing to AtPP2-A1 overexpression in both cases. In the case of PP2OETAt1, whenever the level of AtPP2-A1 expression is greater in an organ than in the others, aphid feeing from the organ incurs a stronger repression (Figure 6). These observations offer a convincing support for the function of AtPP2-A1 in conferring the plant resistance shown as a repression in phloem-feeding activity of the insect. The results also indicate a defensive significance of ubiquitous organ-unspecific expression of PP2 genes in plants demonstrated previously  and observed in this study (Figure 7).
The contribution of lectin-type phloem proteins, such as PP2, to the PBD mechanism is believed owing to their functions as a physical barrier that prevents insects from phloem feeding . A preceding event is the formation of the PP1-PP2 complex, which, however, has been demonstrated only in cucurbits, whereas, other plant families do not have any PP1-like protein . The role of PP1-PP2 aggregation in the clogging of sieve plates has been the matter of long standing debates that have not yet been solved, and still remains a hypothesis that is beyond elucidating scopes of the present study. Lectin-type phloem proteins take only a small proportion of phloem sap proteins that have potential of defensive significance in plants under attacks by phloem-feeding insects . Thus, lectin-type phloem proteins are only one of different PBD components and are not likely to play an entire role in plant resistance against attacks by the insects [14–21]. Subtle differences in aphid population, the insect escape from leaf colonies, for example, between HrpNEa-treated plants and control plants (Figure 2), between atpp2-a1/E/142 and WT (Figure 4), and between PP2OETAt and transgenic control plants (Figure 5), also imply components alternative to AtPP2-A1 in impacting aphid behaviors while colonizing the plants. Alternative defense components are further indicated by HrpNEa-induced impediments to aphid feeding activities observed in the first-hour EPG monitoring (Table 1). However, we do not have evidence yet to show a proportion of AtPP2-A1's contribution to resistance against M. persicae in Arabidopsis plants either when treated with HrpNEa or under the condition of AtPP2-A1 overexpression.
Moreover, AtPP2-A1 is a member of the PP2 multigene family [23, 27] and atpp2-a1/E/142 is one of AtPP2 mutation alleles in Arabidopsis . The other AtPP2 genes and AtPP2-modified mutants seem not involved in HrpNEa-induced repression of aphid feeding from the phloem (Figure 4). This result suggests that different members of the PP2 multigene family may have different functions in the plant. So far, AtPP2-A1 is the only phloem protein demonstrated as a lectin with the ability to bind N-acetylglucosamine oligomers, and recombinant AtPP2-A1 has been shown to affect weight gain in M. persicae nymphs in an artificial diet . The induction of AtPP2-A1 may be an indirect effect of the HrpNEa treatment, which is multifunctional, inducing plant growth enhancement , resistance to pathogens , insects  and drought stress , and resistance-associated cell death [1, 6]. These multiple effects have been determined separately; and so whether they are simultaneous is unclear. It is also unclear if other PP2 genes affect plant defenses rather than resistance. In several species of angiosperms, including Arabidopsis, different PP2 genes are expressed in various organs during plant growth and development . The ubiquitous organ-unspecific PP2 expression suggests that different PP2 genes may fulfill distinct functions at a special stage of plant growth and development. It is possible that a particular PP2 gene may have different functions depending on plant growth and development processes or depending on an immediate requirement for encountering with distinct challenges, such as attacks by insects and infection by pathogens. Studies to test this hypothesis represent an interesting avenue for further research.
The HrpNEa treatment has a deterrent effect on the phloem-feeding activity of M. persicae and the deterrent effect occurs in WT Arabidopsis rather than the atpp2-a1/E/142 mutant. The phloem-feeding activity can be also repressed as a result of AtPP2-A1 overexpression. Both sets of evidence support the conclusion that AtPP2-A1 plays a role in Arabidopsis resistance to the insect, particularly at the phloem-feeding stage. The accompanied change of aphid population in leaf colonies suggests that the function of AtPP2-A1 is related to colonization of the plant and may have a broader importance for the plant-insect interaction.
Plant growth and treatment
Arabidopsis genotypes used in this study included the ecotype Col-0, transgenic plants created in this study, and AtPP2-A sequence-indexed T-DNA insertion lines generated previously (Table 2). Both types of modified plants were created under the background of Col-0 and characterized as homozygous at the transgene and T-DNA insertion loci, respectively, before use in the experiments. Plants were grown in 9-cm pots, 1 plant/pot for the EPG monitoring and 5 plants/pot for other experiments, under 22°C and 250 μE/m2/s illumination . A long day (16-h light/8-h dark) photoperiod was applied to plants for transformation and AtPP2-A1 expression in different organs, and short day (with 12-h light/12-h dark) was used in other experiments. Plants grown in short day were used at different stages of growth and development depending on experimental purposes. Transgenic plants were used in different experiments since the 35th day after planting. Thirty-day-old plants of the WT and mutants were treated with EVP and HrpNEa, respectively. EVP and HrpNEa were prepared [1, 6] as 10 μg/ml aqueous solutions and were applied in the presence of surfactant Silwet-77 (0.02%) by spraying plant tops with a low-pressure atomizer. Treated plants were used at 5 dpt in monitoring of aphid behaviors, and were used at 0 and 24 hpt in determination of gene expression.
A single isolate of M. persicae was collected from the field-grown radish (Raphanus sativus L.) near Nanjing in China. A clone of apterous agamic females was obtained by acclimatization in WT Arabidopsis grown in the chamber (22°C; 250 μE/m2/s; short day). The colony was maintained in nursery WT Arabidopsis seedlings and was transferred to fresh plants every two weeks. Uniform ten-day-old aphids were used in this study and were transferred to experimental plants with a fine paintbrush.
Aphid feeding behavior
Aphid feeding activities were observed by the EPG technique using the Giga Amplifier system (Laboratory of Entomology, Wageningen Agricultural University, Wageningen, The Netherlands; http://www.epgsystems.eu/systems.htm). Uniform ten-day-old aphids were placed on upper side of the top first expanded leaves of plants. For each genotype of the plant or each combination of a genotype and treatment (with EVP or HrpNEa), 20 aphids placed on 20 plants were monitored in five repetitions of experiments. Immediately after aphids were placed on leaves, a 20-mm diameter gold wire was attached to the dorsal surface of each aphid's abdomen using silver conductive paint. The other end of the wire was connected to a four-channel Giga-4 direct current amplifier with four channels and 109-Ω input resistance in an electrical circuit that is also connected to the plant via an electrode placed in the soil. The behavior of individual aphids was monitored for 4 hours. Voltage waveforms were digitized at 100 Hz with an A/D converter USB device. Waveform patterns were identified according to previously described categories . Waveform recordings were dissected each 5 second with the EPG analysis software STYLET 2.5 installed in a computer connected to Giga-4 direct current amplifier.
Uniform ten-day-old aphids were placed on the lower sides of the top two expanded leaves of plants; 10 aphids per leaf. A total of 1,200 aphids were monitored in four repetitions of the experiments for each single recombination of a treatment and a plant genotype. In each experimental repetition, 300 aphids were placed on 30 leaves of 15 plants treated specifically. Aphid movement from leaf colonies was monitored for five days, and the number of aphids in a leaf colony was scored at intervals in 24 hours. Aphid reproduction was surveyed twice a day, and in each survey, newborn nymphs were counted. Reproduction rate was quantified as the ratio between total numbers of nymphs produced in five days and total numbers of aphid adults that stayed in leaf colonies during the same period. Nymphs produced in five days were also monitored; the number of nymphs that run away from leaf colonies was accounted.
Determination of gene expression in plants
Information on genes analyzed by reverse transcriptase-polymerase chain reaction in this study
Product size (bp)
Information on sequence-indexed T-DNA insertion Arabidopsis mutants tested in this study (Table 2) was from The Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org) seed stock database. Mutant seeds were provided as either homologous (atpp2-a1/P/-210, atpp2-a10/P/-157, and atpp2-a3/I/1650) or heterozygous (the other seven mutants) at the insertion loci (Table 2). Homozygous progenies of heterozygous mutants were obtained by a PCR-based screening protocol according to information shown in Table 2. Provided mutant seeds were used to grow progeny plant lines and new seeds were harvested separately from five lines of each mutant. In the next generation, five lines of a mutant were grown for use to analyze the T-DNA insert and identify homozygous plants. Genomic DNA was isolated separately from ten plant individuals of each line and subjected to PCR analyses with Kan r -specific primers (Table 5). Seeds from the line that had Kan r in all the ten plant individuals were regarded as homozygous at the insertion locus.
Promoter sequences of the AtPP2-A genes (Figure 3b) were predicted with the AtcisDB program http://arabidopsis.med.ohio-state.edu/. Presence and locations of plant cis-acting regulatory DNA elements in the promoter sequences were determined by analyses with the PLACE Web Signal Scan program http://www.dna.affrc.go.jp/PLACE/signalup.html. The cis-acting regulatory DNA elements were correlated with genes and processes by browsing linked web information and publications.
Generation and characterization of transgenic plants
The binary vector pBI121 (EMD Bioscience Inc., Gibbstown, NJ, USA), which contains the NPT II gene encoding kanamycin resistance, 35S and uidA, was used to construct transformation units. Full length cDNA of the AtPP2 gene used in construction of pBI121::35S::AtPP2-A1 was obtained by RT-PCR conducted with RNA isolate from leaves of HrpNEa-treated plants and AtPP2-A1-specific primers (5'-CGG GATCCATGAGCAAGAAACATTGCTCAG-3' and 5'-CG AGCTCTTACTGTTTGGGACGAATTGCAACAC-3'; underline indicates protection bases; italics indicate BamH I and Sac I restriction bases). The gene was inserted into the pBI121 vector at the BamH I and Sac I restriction sites to replace uidA (Figure 5a). The AtPP2-A1 promoter was obtained by PCR using the genomic DNA from WT plant and the specific primers (5'-CC CAAGCTTGATAATTTTTCAAGACCC-3' and 5'-CGG GATCCAAACCAGTATGATGTATT-3'; underline indicates protection bases; italics indicate Hind III and BamH I restriction bases). The promoter sequence was inserted pBI121 at the Hind III and BamH I restriction sites to replace 35S (Figure 7a), creating pBI121::PP2P::uidA.
Recombinant vector was transferred into cells of Agrobacterium tumefaciems strain EHA105. A suspension of EHA105 cells containing the empty pBI121 vector (without AtPP2-A1 and uidA inserts) or the recombinant vector were used to transform WT Arabidopsis by blossom infiltration. Transformation with pBI121::GUS::AtPP2-A1 and pBI121::PP2P::uidA generated PP2OETAt and uidAETAt plants, respectively. Both types of transgenic plants were screened, multiplied and characterized by a previously described protocol . The phenotype of kanamycin resistance was used in screening of PP2OETAt candidates and the transgenic control plant candidates, respectively; the phenotype of both kanamycin resistance and GUS activity was used in screening of uidAETAt candidates. GUS activity was determined using the histochemical techniques described previously . Screened transgenic plants were allowed to self-pollinate and selected to the T3 generation . T3 homozygous progenies were used in this study. The genomic integration of the transgene in PP2OETAt was detected by Southern blot analysis . For Southern blots, Arabidopsis genomic DNA was digested with BamH I and Sac I, and transferred to a nylon membrane, followed by hybridization to a digoxigenin-labeled AtPP2-A1 probe prepared using the DIG Nucleic Acid Detection Kit [Roche Diagnostics (Shanghai) Trading Co., LTD].
Experiments were done three or four times with similar results. The student's t-test was used to compare data obtained from HrpNEa-treated plants with those obtained from EVP-treated plants, and to compare data obtained from the transgenic control plant with those obtained from each line of PP2OETAt. Quantitative data were also analyzed by the ANOVA test to compare differences among the transgenic control plant and different lines of PP2OETAt, and among different organs of transgenic plants.
We thank the two anonymous reviewers for comments on the manuscript and scrutiny on characters of the mutants, our colleagues Miss Qiuxia Wang and Dr. Shuwen Wu for assistance in the EPG technique and insect nursery maintenance, and all the students in the lab for assistance in insect survey. This study was supported by grants from China National Novel Transgenic Organisms Breeding Project (2009ZX08002-004B and 2008ZX08002-001) and National Science Foundation of China (30771441).
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