Silverleaf Whitefly Induces Salicylic Acid Defenses and Suppresses Effectual Jasmonic Acid Defenses 1[W]

The basal defenses important in curtailing the development of the phloem-feeding silverleaf whitefly ( Bemisia tabaci type B, SLWF) on Arabidopsis thaliana were investigated. Sentinel defense gene RNAs were monitored in SLWF-infested and control plants. SA-responsive gene transcripts accumulated locally ( PR1, BGL2, PR5, SID2, EDS5, PAD4 ) and systemically ( PR1, BGL2, PR5 ) during SLWF nymph feeding. In contrast, JA- and ET-dependent RNAs ( PDF1.2, VSP, HEL, THI2.1, FAD3, ERS1, ERF1 ) were repressed or not modulated in SLWF-infested leaves. To test for a role of SA and JA pathways in basal defense, SLWF development on mutant and transgenic lines that constitutively activate or impair defense pathways was determined. By monitoring the percentage of SLWF nymphs in each instar, we show that mutants, which activate SA defenses ( cim10 ) or impair JA-defenses ( coi1 ), accelerated SLWF nymphal development. Reciprocally, mutants that activate JA defenses ( cev1 ) or impair SA defenses ( npr1, NahG) slowed SLWF nymphal development. Furthermore, when npr1 plants, which do not activate downstream SA defenses, were treated with MeJA, a dramatic delay in nymph development was observed. Collectively these results showed that SLWF-repressed, JA-regulated defenses were associated with basal defense to the SLWF. comparing RNA levels of well-characterized SA-, JA- and ET-defense genes. Here we show that SLWFs induced SA-signaling pathways and suppressed or did not alter expression of JA/ET-regulated genes. To test for the role of these defense pathways in basal resistance, five Arabidopsis SA and JA mutant/transgenic lines ( npr1, cim10, coi1, cev1, and NahG) and wild-type Columbia were utilized to monitor SLWF nymphal development and sentinel SA- and JA-defense gene RNAs. These experiments and infestation studies with MeJA-treated npr1 plants demonstrated that basal defenses suppressed by SLWF-feeding were critical for constraining nymphal development. The RNAs for genes important in events upstream of SA or for the synthesis of SA, such as SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) , ENHANCED DISEASE 5 EDS5) , and PHYTOALEXIN DEFICIENT 4 (PAD4) were also elevated after nymphal feeding These results indicated that, like biotrophic pathogens, the SA-defense pathway was activated. If similar to pathogen-plant interactions, this pathway could have a role in basal defense to SLWFs.

Unlike chewing insects, less is known about molecular responses to insects from other feeding guilds. Phloem-feeding insects are intriguing due to their "stealthy" feeding mechanisms that cause little damage to the plant tissue as they establish direct access to amino acids and carbohydrates through the vascular tissue. To date, most studies of phloem-feeding insects have examined aphid interactions including Myzus persicae (green peach aphid) with tomato or Arabidopsis, Myzus nicotianae (tobacco aphid) with tobacco, Macrosiphum euphorbiae (potato aphid) with tomato, and Schizaphis graminum (greenbug aphid) with sorghum (Fidantsef et al., 1999;Moran and Thompson, 2001;Ellis et al., 2002;Martinez de Ilarduya et al., 2003;Voelckel, 2004;Zhu-Salzman et al., 2004;De Vos et al., 2005;Li et al., 2006;Thompson and Goggin, 2006). By monitoring the RNA levels of sentinel defense genes after aphid feeding, studies in Arabidopsis show that SA-regulated transcripts increase (Moran and Thompson, 2001;Moran et al., 2002;De Vos et al., 2005). Wound-and JA/ET-regulated genes are induced transiently or at lower levels during M. persicae-Arabidopsis and M. euphorbiae-tomato aphid feeding (Moran and Thompson, 2001;Martinez de Ilarduya et al., 2003).
Transcriptome analysis after aphid feeding on Arabidopsis further confirmed the trends observed by Moran and Thompson (Moran et al., 2002;De Vos et al., 2005).
These changes in RNA levels suggest that responses to aphid feeding are more similar to "pathogen" defense responses than "chewing insect" defenses. While SA-regulated transcripts are induced, the role of SA in Arabidopsis' basal defense to aphids remains controversial (Moran and Thompson, 2001;Mewis et al., 2005;Pegadaraju et al., 2005).
In addition, recent experiments have shown mutations in PAD4, which is regulated by SA, increase susceptibility to M. persicae; pad4 susceptibility is correlated with a delayed aphid-induced senescence (Pegadaraju et al., 2005). In contrast to M. persicae-Arabidopsis interactions, basal SA defenses decrease M. euphorbiae longevity in tomato and SA is important in Mi1-mediated resistance to potato aphids (Li et al., 2006).
Like aphids, the silverleaf whitefly (SLWF; Bemisia tabaci type B; B. argentifolii) is an obligate phloem-feeding pest. Although these animals share membership in the same feeding guild, aphid and whitefly feeding are not synonymous. Unlike aphids, which probe extensively and are more mobile in their feeding habits, whitefly nymphs feed continuously from the same location throughout their 28 + -day nymphal development (Gill, 1990;Byrne and Bellows, 1991;Johnson, 1999;Freeman et al., 2001). The compared to aphids. In addition, whiteflies and aphids are likely to have different salivary components that may elicit different responses from their host (Walling, 2000).
The response of crop plants to SLWF feeding suggests that the JA/ET and novel defense pathways are induced (van de Ven et al., 2000;Walling, 2000). In tomato, JA/ET-regulated basic PR genes accumulate to higher levels than SA-regulated acidic PR gene transcripts (Puthoff et al., unpublished results). Genes identified through differential RNA display in response to SLWF feeding in tomato and squash, Whitefly Induced1 (Wfi1) and SILVERLEAF WHITEFLY INDUCED 1 (SLW1), respectively, have also been shown to be JA inducible (van de Ven et al., 2000;Walling, 2000). Novel pathways appear to contribute to SLWF defense in crop plants (van de Ven et al., 2000;Walling, 2000). For example, SLW3 transcripts do not accumulate in response to feeding by a closely related whitefly biotype (B. tabaci Type A) or after application of known defense-response chemicals (van de Ven et al., 2000). Although these studies demonstrate the complexity and dynamic interactions between crops and the SLWF, these plants lack the powerful genetic and genomic resources afforded by the plant model system Arabidopsis thaliana. Further studies that examine the role of both the JA and SA-defense pathways in Arabidopsis in response to SLWF are necessary to allow comparisons with aphid-induced responses.
The SLWF is a generalist and infests a wide variety of crop plants including members of the Brassicaceae. Infestations of Brassica oleracea in the field have been reported as high as 10 nymphs per cm 2 indicating that members of this family are natural hosts for this phloem-feeding pest (Liu, 2000). Therefore, it is timely to harness the genetic resources in the model plant Arabidopsis, a member of the Brassicaceae, to provide insights into the mechanisms that contribute to basal resistance to phloemfeeding whiteflies. Here, a foundation for Arabidopsis responses to SLWF feeding is provided by comparing RNA levels of well-characterized SA-, JA-and ET-defense genes. Here we show that SLWFs induced SA-signaling pathways and suppressed or did not alter expression of JA/ET-regulated genes. To test for the role of these defense pathways in basal resistance, five Arabidopsis SA and JA mutant/transgenic lines (npr1, cim10, coi1, cev1, and NahG) and wild-type Columbia were utilized to monitor SLWF nymphal development and sentinel SA-and JA-defense gene RNAs. These experiments and infestation studies with MeJA-treated npr1 plants demonstrated that basal defenses suppressed by SLWF-feeding were critical for constraining nymphal development.

Instar Feeding
To date, over 30 defense genes are aligned into complex SA-, JA-and ET-signaling cascades (Glazebrook, 2001;Devoto and Turner, 2003;Shah, 2003). Other defense genes have been identified, but their role or placement in defense signaling has yet to be determined. Transcriptome analysis of SLWF feeding in Arabidopsis thaliana ecotype Columbia has implicated that the SA-dependent pathway is induced, while the JAdependent pathway shows no change or is repressed (Kempema et al. 2006). These transcript profile studies suggest that Arabidopsis perceives and responds to SLWF more like a pathogen than a tissue-damaging herbivore. Figure 1 summarizes the trends in known defense gene expression gleaned from Kempema et al. (2006).
The microarray data indicated that increases in SA-regulated defense gene RNAs are detected by 28 days after SLWF feeding (Kempema et al., 2006). To assess the timing of defense gene activation in response to SLWF nymph feeding, the levels of two sentinel defense gene RNAs were assessed at 0, 7, 14, 21 and 28 days after SLWF infestation ( Fig. 2A). Transcripts for the SA-regulated PATHOGENESIS-RELATED PROTEIN 1 (PR1) gene were first detected at 14 days after infestation and increased to highest levels by 28 days. In contrast, the levels of the JA-regulated PLANT DEFENSIN PROTEIN 1.2 (PDF1.2) RNAs were not detected in control, non-infested or SLWFinfested leaves within the 28-day period.
To confirm the microarray data reported by Kempema et al (2006), RNAs control, non-infested and 21-day SLWF-infested plants from three independent biological experiments were used (Fig. 2B). Transcripts for the SA-regulated genes, PR1, PR5, and β-1,3-GLUCANASE 2 (BGL2; PR2) increased after 21 days of nymph feeding compared to non-infested control plants. The RNAs for genes important in events upstream of SA or for the synthesis of SA, such as SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), ENHANCED DISEASE SUSCEPTIBILITY 5 (EDS5), and PHYTOALEXIN DEFICIENT 4 (PAD4) were also elevated after nymphal feeding (Fig.   2B). These results indicated that, like biotrophic pathogens, the SA-defense pathway was activated. If similar to pathogen-plant interactions, this pathway could have a role in basal defense to SLWFs. RNA levels declined in SLWF-infested leaves relative to control leaves, while ETHYLENE RESPONSE FACTOR (ERF1) RNA levels were at similar levels in both infested and control leaves (Fig. 2B).

Local and Systemic Induction of Defense Genes in Response to SLWF Infestation
To evaluate if Arabidopsis mounts a systemic response to SLWF feeding, the change in SA and JA sentinel gene RNAs were examined both in local infested leaves and apical non-infested leaves (systemic) after a 21-day infestation. RT-PCR with genespecific primers showed that, unlike responses to aphids, the trends identified in SLWFinfested Arabidopsis leaves were also observed in apical, non-infested leaves. SAregulated gene transcripts (PR1, PR5, and BGL2) accumulated both locally and systemically after nymph feeding (Fig. 3). JA-responsive RNAs (VSP1 and PDF1.2) were not present or were at lower levels in both local and systemic leaves. Collectively, the whole-plant response to SLWF infestation was distinctive from what has been observed with other phloem-feeders in Arabidopsis.

Repression of JA Responses Enhances SLWF Development
To assess the role of SA-and JA-signaling pathways in defense against SLWFs, lines with impaired SA (npr1 and NahG) and JA (coi1) signaling were examined (Cao et al., 1994;Feys et al., 1994;Lawton et al., 1995). This was complemented with lines that constitutively activated SA (cim10) and JA (cev1) defenses (Ellis and Turner, 2001;Maleck et al., 2002). Although cim10 is less well-characterized than some mutants that constitutively express SA defenses, it was chosen for these studies because it does not display a dwarf phenotype, nor does it produce the spontaneous lesions that are commonly observed in SA over-expression lines (Maleck et al., 2002 Mutant and wild-type plants were infested with SLWFs (> 100 nymphs/plant) to assess impacts on nymphal development using a no-choice bioassay (Fig. 4). SLWF development was assayed by scoring the total number of insects at each developmental stage (1 st , 2 nd , 3 rd or 4 th instars) on each of the 8 replicate plants. The percent of insects that had reached advanced stages of development (4 th instars) by day 24 was calculated and compared between all six lines using a Tukey Multiple Comparison Test ( Fig. 4; Supplemental Fig. 1). In addition, to assess defense pathway activation during SLWF feeding, the changes in levels of marker genes PR1, BGL2, PDF1.2, and VSP1 RNAs were monitored in all lines. Most SA-and JA-defense mutants have not been utilized in long-term infestation or infection studies. Therefore, the examination of defense gene transcripts in these defense mutants provided further characterization of both SA-and JA-dependent gene expression at later times in plant development. Some defense genes are expressed at higher basal levels in older plants (Kus et al., 2002). Therefore it was important that the defense gene transcripts were monitored in the mutant noninfested plants and after challenge with SLWFs in order to interpret bioassay results. npr1 and NahG plants, respectively, when compared to WT plants (Fig. 4). In accordance with this finding, the percentage of SLWFs in their 2 nd and 3 rd instars rose. In these mutants, the SA-regulated RNAs PR1 and BGL2 accumulated to lower levels than in wild-type in both non-infested and infested leaves and, in a reciprocal fashion, JAdependent PDF1.2 and VSP1 transcripts increased compared to WT (Fig. 5). These data indicated that by abolishing SA defenses and/or enhancing JA defenses in npr1 and NahG plants enhanced defenses active against SLWF nymphs, as reflected in significant delays in nymphal development, were displayed.
Similarly, on the JA-pathway over-expression mutant cev1 significantly fewer nymphs reached the 4 th instar (13%) than on wild-type plants (Fig. 4). Consistent with the constitutive activation of JA defenses in cev1 plants (Ellis and Turner, 2001), the JAdependent transcripts PDF1.2 and VSP1 accumulated to high levels in uninfested cev1 than WT leaves (Fig. 5). Despite elevated JA defenses, SLWF nymph feeding caused SA transcripts (PR1, BGL2) to accumulate in infested cev1 leaves; in fact, PR1 and BGL2 RNAs accumulated to similar levels in RNA blot analysis in the cev1 mutant and wild type plants (data not shown). PDF1.2 transcripts increased, while VSP1 transcripts decreased after SLWF infestation. Collectively, these data indicated that the SLWF nymphs provided signals that allowed for strong expression of SA-regulated genes and repression of VSP1 in the cev1 mutant. The fact that nymphs feeding on cev1 plants exhibited delayed development relative to WT plants and the SA-pathway gene RNAs accumulated in both cev1 and WT plants suggested that enhanced JA responses, and not SA defenses, were responsible for delaying the development of SLWF nymphs.
As cross-talk between JA and SA defense pathways is commonly associated with responses to biotic threats and displayed in defense mutant studies, it was important to further dissect the relative importance of the suppressed JA and induced SA defenses in SLWF basal resistance. npr1 plants uncouple the cross-talk between SA and JA signaling. For example, during Pseudomonas syringae pv. tomato infection, npr1 plants have reduced levels of SA and PR1 RNAs but JA signaling is preserved (Spoel et al., 2003). Therefore, comparisons of untreated and MeJA-treated npr1 plants should allow the role of JA-regulated defenses to be assessed. npr1 plants were treated with MeJA or served as controls. Unlike previous experiments (Fig. 4), these infestations were performed at 24 o C, which significantly accelerated nymphal development (Fig. 6) nymphs feeding on control npr1 plants were in their 4 th instar. Smaller numbers of 1 st , 2 nd and 3 rd instars were also noted in control plants (Fig. 6). In contrast, the MeJA-treated npr1 plants had no 4 th -instar nymphs. Nymphs were primarily in their 1 st (33%) and 2 nd (65%) instars (Fig. 6). MeJA had a dramatic effect on SLWF nymphal development on npr1 plants clearly demonstrating the importance of JA-regulated defenses in basal resistance and curtailing SLWF nymphal development.

DISCUSSION
The SA and/or JA/ET-regulated defense pathways are important in basal and genefor-gene resistance to pathogens and herbivores. After perception of a biotic threat, plants fine-tune the balance of defense pathways to orchestrate the "best" defense response to its intruder (Reymond and Farmer, 1998;Walling, 2000;Kunkel and Brooks, 2002). The cross-talk between the SA and JA pathways is thought to minimize expression of costly and ineffective defenses that divert C-and N-resources from plant vegetative growth, thereby avoiding compromises to plant vitality and reproduction. This view is supported by the facts that SA-induced defenses are important in the induced basal and gene-for-gene defenses against biotrophic pathogens (Glazebrook, 2005).
Similarly, JA-induced defenses confer resistance to necrotrophic pathogens and insects.
Pests and pathogens have leveraged this molecular communication mechanism to enhance their success on host plants (Mudgett, 2005;Chisholm et al., 2006). While some pathogens evade host defenses by actively catabolizing antimicrobial compounds (Bouarab et al., 2002), there is a growing evidence that plant pathogens produce effectors that antagonize defense signaling networks (Hammond-Kosack and Parker, 2003;Kamoun, 2006). The complexity of the arms race between host and attacker is exemplified by Pseudomonas syringae, which uses an array of effectors to suppress expression of defense genes and secondary metabolites, suppress programmed cell death, avoid R gene-mediated resistance, suppress cell wall remodeling, and potentially alter gene expression programs and turnover of defense regulatory proteins (He et al., 2004;Cui et al., 2005;Mudgett, 2005;Chisholm et al., 2006;Janjusevic et al., 2006). By simultaneously evaluating SLWF nymph development on mutants from both SA and JA defense pathways and after exogenous MeJA-treatments, it appears that SLWFs should be added to the set of pathogens and pests that manipulate host-plant defense responses to their own advantage. SLWF nymphs have an intimate and long-term interaction with their host plants. With the exception of the crawler, which emerges from the egg, SLWF nymphs are immobile and feed almost continuously for approximately 28 days under optimal Arabidopsis conditions. SLWF nymphs provided strong and reproducible signals that were perceived by Arabidopsis resulting in increases in SA-regulated defenses and suppression of JAregulated defenses (Fig. 2). The accumulation of PR gene RNAs after SLWF feeding in Arabidopsis was SA-and NPR1-dependent, as transcripts did not accumulate to wildtype levels in NahG and npr1 plants (Fig. 5). SA-dependent defense gene RNAs accumulated both in local, infested leaves and systemically in non-infested apical leaves (Fig. 3). Previous studies in squash and tomato also show local and systemic induction of defense genes after SLWF feeding (van de Ven et al., 2000;Walling, 2000). In contrast, systemic activation of defenses was not observed in compatible M. persicae-Arabidopsis and M. euphorbiae-tomato interactions (Moran and Thompson, 2001;Martinez de Ilarduya et al., 2003). This suggests that SLWFs may provide more potent signals, more mobile signals, or larger quantities of signals (due to its prolonged feeding habits) to its host plant.
By using mutant and transgenic lines that alter SA and JA defenses, the branch of Arabidopsis defense signaling that antagonizes SLWF nymph development was identified. There was a strong correlation of SLWF success (as measured by the rate of nymphal development) with the absence of JA defenses and presence of SA defenses (Fig. 4). For example, SLWF nymph development was more rapid on cim10 and coi1 than WT plants (Fig. 4); coi1 and cim10 plants accumulated the SA-regulated PR1 and BGL2 RNAs and displayed reduced JA defenses (PDF1.2 and VSP1 RNAs) (Fig. 5).
Reciprocally, cev1, NahG and npr1 mutants had an enhanced basal resistance to SLWFs; the delayed SLWF nymph development was correlated with enhanced JAregulated defenses in these lines (Fig. 5). The fact that SA-dependent RNAs were abundant in cev1, cim10, and WT plants, but only cev1 displayed an increased basal resistance suggested that JA-dependent defenses, and not SA defenses, were responsible for the delays in nymph development observed on cev1 and cim10 plants ( Figs. 4 and 5).
The importance of JA-regulated defenses in basal resistance to SLWFs was also supported by comparing SLWF development on untreated and MeJA-treated npr1 plants. npr1 mutants lack the ability to activate SA defenses (Spoel et al., 2003) and MeJA treatments accentuated the npr1 delay in SLWF nymph development relative to the untreated npr1 plants (Fig. 6). Collectively, these data and those reported above indicated that the suppressed JA-regulated defenses were important in slowing SLWF nymphal development. Furthermore, the highly induced SA defenses did not appear to significantly contribute to the basal resistance to SLWFs in Arabidopsis; although SAdependent defenses may have a role in other aspects of the SLWF-Arabidopsis interaction, such as host choice, fecundity or longevity. These data contrasted to the preferential induction of SA defenses observed in biotrophic pathogen-plant interaction and the importance of SA defenses in both basal and R gene-mediated resistance (Glazebrook, 2005).
The data presented here support the idea that SLWFs enhance their success on Arabidopsis plants by failing to activate or suppressing the effectual JA-regulated defenses. It is possible that SLWFs evade activation of the JA pathway, since SLWFs cause little tissue damage (intracellular punctures) until they establish feeding sites at a minor veins of the phloem (Cohen et al., 1996;Walling, 2000). SLWFs could also prevent the activation of JA defenses by introducing inhibitors that directly or indirectly antagonize JA signaling pathway activation or action. Finally, SLWFs strongly activated SA defenses, even in cev1 plants. Therefore, it is possible that SLWFs down-regulated the effectual JA defenses via SA cross-talk in WT plants. The SLWF effector(s) that induce SA defenses and/or suppress JA defenses are unknown, but are presumed to be salivary components synthesized by the whitefly or one of its endosymbionts (Walling, 2000). While whitefly saliva is not well characterized biochemically (Funk, 2001), the watery and sheath salivas of other hemipterans, such as aphids, are rich in potential defense signaling molecules including pectinases, complex carbohydrates, proteins, peroxidases, phospholipids, amylases, lipases and/or phosphatases (Miles, 1999;Walling, 2000).
Additional evidence for herbivore manipulation of plant defenses (the "decoy" hypothesis) to enhance insect performance is accumulating from studies with both tissue-damaging herbivores and phloem-feeding aphids (Zhu-Salzman, 2005;Thompson and Goggin, 2006). For example, Helicoverpa zea larvae egest saliva containing glucose oxidase (GOX) into their feeding sites to suppress the JA-regulated defenses that deter larval growth (Musser et al., 2002). While GOX uses glucose to produce hydrogen peroxide to activate SA defenses, such as PR1 protein accumulation, an SAindependent mechanism is responsible for suppression the effectual JA-mediated defenses of tobacco, such as nicotine production (Musser et al., 2002; Several studies from the molecular plant-aphid interaction literature also support the "decoy" hypothesis. It should be noted that changes in JA-or SA-defense gene RNA levels and aphid population dynamics on defense mutants have varied, presumably due to the differences in aphid-infestation experimental design (Moran and Thompson, 2001;Ellis et al., 2002;Moran et al., 2002;De Vos et al., 2005;Mewis et al., 2005;Pegadaraju et al., 2005). In general, rises in PR RNAs have been noted and, like SLWFs, JAdefense gene RNAs are often suppressed or not highly induced after aphid feeding on Arabidopsis (Moran and Thompson, 2001;Ellis et al., 2002;Moran et al., 2002;De Vos et al., 2005). More variation is observed in defense mutant studies. The clear reciprocal phenotypes of SA-and JA-defense mutants, as was seen for SLWFs, have not been documented in the Arabidopsis-aphid literature. While several studies have shown that M. persicae population growth is slowed in cev1, npr1 and NahG lines or after MeJA treatments (Moran and Thompson, 2001;Zhu-Salzman et al., 2004;Mewis et al., 2005), other studies showed neither NahG, npr1, nor coi1 changed M. persicae population dynamics relative to WT plants (Moran and Thompson, 2001;Ellis et al., 2002;Mewis et al., 2005;Pegadaraju et al., 2005).
Given the variability in the aphid-plant interactions studies to date, the simultaneous analyses of five defense mutants were crucial in providing a comprehensive and reproducible picture establishing the importance of JA-regulated defenses in deterring SLWF nymphal development. While the specific JA-dependent genes important in SLWF defense have yet to be identified, basal defense towards SLWF in Arabidopsis appeared to be antibiotic. Preliminary no-choice egg-deposition and choice bioassays show that SLWF exhibits no preference for any of the mutants altered in constitutive defenses including cell wall composition, secondary metabolites and trichome density (data not shown). Both generalist (M. persicae) and specialist (Brevicoryne brassicae) aphid interactions with Arabidopsis suggest that JA-dependent defenses have antibiotic effects on aphids (Mewis et al., 2005). However, in tomato, Mi1.2-mediated resistance towards SLWF feeding is antixenotic in that it acts to deter the whitefly from establishing a feeding site (Nombela et al., 2003) If viewed in the broadest terms, the SLWF-Arabidopsis interactions bear a semblance to Arabidopsis interactions with fungal biotrophs like Erysiphe spp (Reuber et al., 1998, Kempema et al., 2006; both sets of organisms induce SA-dependent defenses. However, when basal resistance mechanisms are investigated, the SLWF and fungal biotrophs are distinct. While SA defenses are essential for the basal and induced resistance mechanisms for control of fungal biotrophs, SA-induced defenses did not appear to contribute to the mechanisms that dictate basal resistance to SLWFs. Interestingly, Arabidopsis appeared to mount a completely ineffectual response to SLWF feeding as effective JA-dependent defenses were not induced in WT plants. Further experiments that examine the role of crosstalk, SLWF salivary components and downstream responses will allow identification of elicitor(s) and mechanism(s) for retarding nymphal development, which contributes to the basal resistance in Arabidopsis. The JA-mediated delays in SLWF nymph development could be used in the future to engineer resistance to SLWFs. Delays in insect development are considered important resistance mechanisms impacting insect population dynamics and providing a longer period of time for natural enemies, such as parasitoid wasps, to attack the insects (Pechan et al., 2000;Dicke and Hilker, 2003).

Plant Growth and Insect Maintenance
Arabidopsis thaliana ecotype Columbia (wild-type, WT)plants used in the local and systemic defense gene transcript studies (Figs. 1-3) were grown and infested as described in Kempema et al. (2006). Plants used in the bioassays WT, coronatine The rosette diameter and number of leaves on three-week-old cev1 plants was approximately the same as two-week-old plants from the other lines. coi1 plants were identified from a F 2 seed pool on ½ Murashige and Skoog medium (10 g L -1 Suc and 0.8% [w/v] agar content) containing 30 µM methyl jasmonate (MeJA)/0.01% ethanol (EtOH) (Bedoukian Research, Danbury, CT). At seven days, homozygous coi1 seedlings were identified by elongated roots and normal above-ground organ morphology (Feys et al., 1994). The coi1 plants were transferred to pots containing soil.
cim10 mutants have WT stature, do not display necrotic lesions and constitutively over express SA and SA-regulated defense genes (Maleck et al., 2002). These features distinguish cim10 relative to other constitutive immunity mutant and made cim10 an excellent choice for SLWF infestations. The levels of PR1 RNAs in cim10 and WT plants were determined using RT-PCR and gene-specific primers in non-infested 2-week-old and 3-week-old plants to confirm the cim10 constitutive immunity phenotype prior to the time of SLWF infestation ( Supplementary Fig. 2).

Whitefly Infestations
Adult male and female whiteflies (totaling 30 to 100 depending on the experiment) were collected from SLWF-infested B. napus leaves by aspiration into 15-ml falcon tubes. A tube containing male and female SLWFs was placed upright in each pot. This number of whiteflies per plant resulted infestation levels similar to infestation levels experienced by Brassica plants in the field (Liu, 2000). Arabidopsis plants typically had > 100 feeding nymphs per plant. Nylon bags (5 by 10 inch) were placed around each pot and secured with a rubber band. Whiteflies were released by unscrewing the falcon tube. Control pots were bagged but not infested. After seven days, all adult flies were Therefore after 17 days, plants were scored for number of nymphs at each developmental stage as described for the developmental bioassays above. The experiment was replicated twice.

Data Analysis
Defense genes induced or repressed 1.5-fold by microarray analysis were identified by Kempema et al. (2006). Briefly, microarray data was background adjusted using Robust Multiarray Analysis (RMA) and differential analysis performed using Significant Analysis of Microarray (SAM). Data from the no-choice bioassay was analyzed using a one-way unstacked ANOVA and Tukey Multiple Comparison Test with Minitab software (Minitab, State College, PA). Data for the npr1 MeJA treatment experiment was analyzed using Student's T-Test.

Reverse Transcription and Polymerase Chain Reactions (PCR)
Total RNA was extracted from rosette leaves using TRIzol reagent (Invitrogen, Carlsbad, CA). The quality of the RNA was checked on a 1% agarose denaturing gel (0.5% MOPS, 0.8% formaldehyde). Before the reverse transcriptase reaction, 1 µg of RNA was treated with TURBO DNase as indicated in the manufacturer's instructions (Ambion, Austin, TX).