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First published online March 30, 2007; 10.1105/tpc.106.049353 The Plant Cell 19:1096-1122 (2007) © 2007 American Society of Plant Biologists Herbivory Rapidly Activates MAPK Signaling in Attacked and Unattacked Leaf Regions but Not between Leaves of Nicotiana attenuata[W]Department of Molecular Ecology, Max-Planck-Institute for Chemical Ecology, D-07745 Jena, Germany 1 To whom correspondence should be addressed. E-mail baldwin{at}ice.mpg.de; fax 49-3641-571102.
Mitogen-activated protein kinase (MAPK) signaling plays a central role in transducing extracellular stimuli into intracellular responses, but its role in mediating plant responses to herbivore attack remains largely unexplored. When Manduca sexta larvae attack their host plant, Nicotiana attenuata, the plant's wound response is reconfigured at transcriptional, phytohormonal, and defensive levels due to the introduction of oral secretions (OS) into wounds during feeding. We show that OS dramatically amplify wound-induced MAPK activity and that fatty acid–amino acid conjugates in M. sexta OS are the elicitors. Virus-induced gene silencing of salicylic acid–induced protein kinase (SIPK) and wound-induced protein kinase revealed their importance in mediating wound and OS-elicited hormonal responses and transcriptional regulation of defense-related genes. We found that after applying OS to wounds created in one portion of a leaf, SIPK is activated in both wounded and specific unwounded regions of the leaf but not in phylotactically connected adjacent leaves. We propose that M. sexta attack elicits a mobile signal that travels to nonwounded regions of the attacked leaf where it activates MAPK signaling and, thus, downstream responses; subsequently, a different signal is transported by the vascular system to systemic leaves to initiate defense responses without activating MAPKs in systemic leaves.
Over time, plants have acquired sophisticated defense mechanisms to cope with herbivory. Plants react to herbivore attack with finely tuned transcriptional changes (Hui et al., 2003  ; Reymond et al., 2004), elevated phytohormone biosyntheses (Reymond and Farmer, 1998), and, finally, the production of direct and indirect defense compounds (Kessler and Baldwin, 2001; Zavala et al., 2004). In Nicotiana attenuata, the application of Manduca sexta's oral secretions (OS) and regurgitants to mechanical wounds elicits (1) jasmonic acid (JA) and ethylene bursts that are greater than those elicited by mechanical wounding (Kahl et al., 2000); (2) high levels of trypsin proteinase inhibitor (TPI), an important direct defense compound (Halitschke and Baldwin, 2003; Zavala et al., 2004); and (3) the release of volatile organic compounds (VOCs), which function as indirect defenses by attracting predators to feeding herbivores (Kessler and Baldwin, 2001).
Several lines of evidence point to the importance of fatty acid–amino acid conjugates (FACs) in herbivore OS in eliciting herbivory-specific responses (Alborn et al., 1997
The mitogen-activated protein kinase (MAPK) cascade is a conserved pathway involved in modulating a large number of cellular responses in all eukaryotes (Herskowitz, 1995
Salicylic acid–induced protein kinase (SIPK) and wound-induced protein kinase (WIPK), two tobacco (Nicotiana tabacum) MAPKs and their homologs in other plant species, are rapidly activated after various challenges, such as ozone (Samuel et al., 2000 Here, we demonstrate that in N. attenuata, applying M. sexta OS to puncture wounds in leaves dramatically amplifies the wound-induced increase in SIPK activity and WIPK transcripts and that FACs in M. sexta OS are the responsible elicitors. We show that both SIPK and WIPK are upstream signaling components regulating wound- and OS-elicited JA, salicylic acid (SA), JA-Ile/JA-Leu conjugate, and ethylene biosynthesis. Transcriptional analyses revealed that SIPK and WIPK mediate the wounding- and OS-elicited accumulation of many defense-related genes, including three MAPKs and four calcium-dependent protein kinases (CDPKs); moreover, they even regulate each other's transcript accumulation, highlighting the complicated transcriptional crosstalk that occurs among protein kinases. After OS elicitation, a mobile signal quickly moves to particular undamaged regions of the elicited leaf and activates MAPK signaling and downstream responses. In contrast with the signaling observed within attacked leaves, a distinct signal travels to systemic distal leaves and activates defense-related responses without activating MAPKs.
Herbivory Activates MAPKs in N. attenuata MAPKs are rapidly activated after mechanical wounding (Bogre et al., 1997 ; Zhang and Klessig, 1998a). To determine if MAPKs are involved in activating defense responses to herbivore attack, N. attenuata leaves were wounded with a pattern wheel; immediately thereafter puncture wounds were treated with water (W+W) or fivefold-diluted M. sexta OS (W+OS), and MAPK activity was analyzed by an in-gel kinase assay using myelin basic protein as a substrate (Figure 1A
, top panel). After mechanical wounding, a 48-kD protein kinase was activated that reached a maximum at 10 min and rapidly declined to a basal level within 1 h; in comparison, leaves wounded with W+OS dramatically increased the activity of 48-kD kinase within 10 min and sustained elevated levels for 2 h. The in-gel activity assay also showed that a 44-kD kinase, although characterized by significantly less activity than the 48-kD protein kinase, was also activated, but this activity was not enhanced by the W+OS treatment. An unidentified 40-kD protein kinase having rather low levels of in-gel kinase activity was also activated 10 min after both W+W and W+OS treatments. Notably, W+W-treated plants showed higher activity levels of 40-kD protein kinase for as long as 2 h than did W+OS-treated plants. Schittko et al. (2000) demonstrated that applying highly diluted M. sexta OS (up to 1000-fold) to N. attenuata elicits the same level of JA burst as using undiluted OS. We also tested the ability of diluted OS to activate MAPKs (Figure 1A, bottom panel). Consistent with the JA analysis (Schittko et al., 2000), the in-gel kinase activity assay showed that even 1000-fold–diluted OS significantly increased the levels of SIPK activity above those of the W+W treatment.
Since the SIPK and WIPK from cultivated tobacco (Seo et al., 1995 ; Zhang and Klessig, 1998a) are also 48- and 44-kD kinases, we silenced transcript levels of N. attenuata's SIPK and WIPK by virus-induced gene silencing (VIGS) to confirm their identities (Ratcliff et al., 2001; Saedler and Baldwin, 2004). Agrobacterium tumefaciens was transformed with pTV-SIPK and pTV-WIPK constructs carrying cDNA fragments of N. attenuata's SIPK and WIPK. Although these two fragments share a certain homology, the longest perfect match is 16 nucleotides long. Given that a 22- or 23-nucleotide perfect match is the minimum size capable of initiating RNA interference (RNAi) (Thomas et al., 2001; Xu et al., 2006), neither construct had a cosilencing effect on the other's transcripts. Young N. attenuata leaves were inoculated with these construct-transformed Agrobacterium, generating SIPK-silenced (SIPK-VIGS) and WIPK-silenced (WIPK-VIGS) plants, respectively; plants inoculated with Agrobacterium, which carried empty vector (EV) pTV00, were used for comparison. No morphological differences associated with SIPK or WIPK silencing were observed. Eighteen days after inoculation, transcript levels of SIPK and WIPK were reduced by  90 and 80%, respectively (Figure 1B). An in-gel kinase assay was performed to examine activity levels in various kinase-silenced plants (Figure 1C). Compared with EV plants, SIPK-VIGS plants had greatly reduced 48-kD protein kinase levels; the 44-kD kinase bands apparent in EV plants were not detected in WIPK-VIGS plants, indicating that the 48- and 44-kD protein kinases are SIPK and WIPK, respectively. Repeated kinase assays showed the same results (data not shown). Notably, levels of WIPK activity were higher in SIPK-silenced plants than in EV plants. This increase was likely a result of decreased competition between SIPK and WIPK for their common upstream kinase, MEK2 (Yang et al., 2001), which was also demonstrated in cultivated tobacco plants: after ozone exposure, SIPK-silenced plants had higher levels of WIPK activity than did wild-type plants, a difference that could be attributed to decreased competition for the binding to MEK2 (Samuel and Ellis, 2002).
A recent study of the cultivated tobacco Ntf4 gene, a MAPK gene having high homology to SIPK, indicated that Ntf4 is also involved in stress responses (Ren et al., 2006
Wounding is known to elevate WIPK transcript levels in cultivated tobacco (Seo et al., 1995
SIPK and WIPK Regulate Transcript Levels of Three WRKY Transcription Factors
SIPK and WIPK Mediate Transcript Accumulation of Three MAPKs and Four CDPKs Liu et al. (2003) have elegantly demonstrated that SIPK mediates pathogen-elicited increases in WIPK transcript levels in cultivated tobacco. To determine if OS-elicited WIPK transcript levels are also mediated by SIPK, we measured transcript levels of WIPK in SIPK-silenced plants (Figure 3A
). Untreated SIPK-VIGS plants had similar WIPK transcript levels compared with EV plants, and transcript levels in EV plants increased almost 20 and 80 times 1.5 h after W+W and W+OS treatments, respectively. In SIPK-VIGS plants, WIPK transcript levels were only 50 and 25% of those in EV plants after W+W and W+OS treatments. We conclude that OS-elicited activation of SIPK enhances levels of WIPK transcript in N. attenuata. SIPK transcript levels in WIPK-VIGS plants were significantly reduced compared with EV plants, even when untreated; both treatments slightly increased SIPK transcript levels in EV plants but not in WIPK-VIGS plants (Figure 3B).
To determine if other MAPKs are also transcriptionally regulated by SIPK and WIPK, transcript levels of N. attenuata MPK4, a homolog of Arabidopsis thaliana MPK4, were measured with q-PCR (Figure 3C). Levels of MPK4 transcripts were about three times as high in untreated EV plants as in untreated SIPK- and WIPK-VIGS plants. Both W+W and W+OS treatments increased MPK4 transcript levels in EV plants but not in SIPK- and WIPK-VIGS plants, except that 3 h after OS elicitation, transcript levels in SIPK-VIGS rebounded to those in EV plants. Two N. attenuata MAPKs, Naf3 and Naf6, which are orthologous to Ntf3 and Ntf6 of cultivated tobacco, were cloned and their transcript levels measured (Figures 3D and 3E). In EV plants, Naf3 levels increased significantly 0.5 h after W+W treatment (P = 0.010, unpaired t test) and then quickly declined before rebounding by 1.5 h. OS treatment dramatically amplified the wound-induced 0.5 h peak in Naf3 transcript levels, which rapidly declined. When untreated, EV and SIPK-VIGS plants showed similar levels of Naf3 transcript, while levels in WIPK-VIGS plants were significantly reduced. After W+W treatment, lower levels of Naf3 transcripts were detected in SIPK-VIGS and WIPK-VIGS plants compared with EV plants, except after 1 h when Naf3 levels in EV plants transiently declined. W+OS-treated WIPK-VIGS plants had lower levels of Naf3 transcripts than did EV plants, except after 3 h; levels in SIPK-VIGS plants were lower than in EV plants only at 0.5 h and significantly higher at 1.5 h. In contrast with the response in Naf3 transcripts, Naf6 transcripts were not elevated. W+W and W+OS treatments gradually decreased Naf6 transcript levels in EV plants. When untreated, SIPK-VIGS plants had significantly lower levels of Naf6 transcripts, but WIPK-VIGS and EV plants had the same. Following W+W treatment, SIPK-VIGS and WIPK-VIGS plants had lower levels of Naf6 transcripts than did EV plants before 3 h, when SIPK-VIGS plants still had low levels, but levels in WIPK-VIGS plants returned to those found in EV plants. W+OS treatment significantly reduced levels in both SIPK-VIGS and WIPK-VIGS plants for up to 1 h.
In addition to MAPKs, CDPKs are also known to be involved in plant development and defense reactions (Romeis et al., 2000 While CDPK2 transcript levels were strongly suppressed in SIPK-VIGS plants (to 40% of those in EV plants), levels in WIPK-VIGS plants did not differ significantly from those in EV controls (Figure 4A ). After W+W treatment, CDPK2 transcripts in SIPK-VIGS plants remained low throughout the 3-h time course, but in OS-treated plants, levels returned to those found in EV plants by 3 h.
In EV plants, OS elicitation increased levels of CDPK4 transcripts; these reached maximum levels after 1.5 h, whereas in W+W-treated plants, levels of CDPK4 transcripts peaked as early as 0.5 h and after 1.5 h attained similar levels to those in OS-elicited plants (Figure 4B). Compared with EV plants, untreated SIPK-VIGS and WIPK-VIGS plants showed significantly lower levels of CDPK4 transcripts. CDPK4 transcript levels were lower in SIPK-VIGS plants than in EV plants for up to 3 h after both treatments. WIPK-VIGS plants, by contrast, showed a different profile: following W+W treatment, their levels of CDPK4 transcripts were lower than those of EV plants before 1.5 h, but no difference was observed thereafter; however, in W+OS-treated WIPK-VIGS and in EV plants, levels of CDPK4 transcripts were similar as early as 0.5 h. We also measured the transcript accumulation profiles of CDPK5 (Figure 4C). After both treatments, EV plants dramatically increased the levels of CDPK5 transcripts; W+OS treatment in particular led to an almost 20-fold increase after 1.5 h, while W+W treatment resulted in an almost 10-fold increase after 1 h. When untreated, both SIPK-VIGS and WIPK-VIGS plants showed similar levels of CDPK5 transcripts compared with those in EV plants. However, after either elicitation, levels of CDPK5 transcripts in both SIPK-VIGS and WIPK-VIGS plants were significantly lower than in EV plants for up to 3 h. After this time, CDPK5 transcript levels were similar in all plants, except that W+W-treated SIPK-VIGS plants still showed slightly but significantly lower levels of CDPK5 transcripts. Similarly, substantially lower levels of CDPK8 transcripts before and after either treatment were detected in SIPK-VIGS and WIPK-VIGS plants, compared with transcript levels in EV plants (Figure 4D). Following W+W treatment, EV plants had increased CDPK8 transcript levels after 0.5 and 1.5 h, whereas after W+OS treatment, levels of transcripts significantly increased only after 0.5 h (P = 0.0092, unpaired t test) and then quickly declined, as was seen in Naf3 transcripts following the same treatment (Figure 3D). CDPK8 transcript levels in SIPK-VIGS and WIPK-VIGS plants were significantly suppressed throughout the time course, except that 3 h after W+OS treatment, CDPK8 transcript levels in SIPK-VIGS plants rebounded to those in EV plants. In summary, both SIPK and WIPK regulate the transcript levels of MPK4, Naf3, Naf6, CDPK4, CDPK5, and CDPK8. The transcript level of CDPK2 is mediated by SIPK but not WIPK. Furthermore, SIPK and WIPK regulate each other's transcript levels.
Silencing SIPK and WIPK Attenuates OS-Elicited JA, SA, JA-Ile/JA-Leu, and Ethylene Biosynthesis
W+OS treatment elicited higher JA levels than did W+W, and these were remarkably reduced in SIPK-VIGS and WIPK-VIGS plants compared with EV plants. After either treatment in SIPK-VIGS plants, JA levels in particular were reduced by almost 80%; by contrast, JA levels in WIPK-VIGS plants were reduced by only
Seo et al. (1999) demonstrated that WIPK of cultivated tobacco is involved in wound-induced SA accumulation. To determine if this is also the case in N. attenuata, we measured SA levels in SIPK-VIGS, WIPK-VIGS, and EV plants. When untreated, SIPK-VIGS plants showed significantly lower levels of SA than did EV plants, whereas WIPK-VIGS plants and EV plants had similar levels (Figure 6A
). One hour after W+W treatment, EV plants had slightly increased SA levels, but these quickly decreased to basal levels. Both SIPK- and WIPK-VIGS plants had lower levels of SA than did EV plants up to 1 h; after that, levels of SA in both plants gradually reached the same as those in EV plants. W+OS elicitation dramatically increased SA levels by the 3 h harvest in EV plants, but this increase was suppressed in SIPK- and WIPK-VIGS plants. Notably, W+W and W+OS treatments didn't elicit significantly different SA levels in EV plants for up to 1.5 h (P > 0.12, unpaired t test). Isochorismate synthase (ICS) is one of the key enzymes involved in SA biosynthesis. Following either treatment, levels of ICS transcripts were lower in SIPK- and WIPK-VIGS plants compared with those of EV plants, except at the 3 h harvest, when levels of ICS transcripts in SIPK-VIGS plants rebounded to those in EV plants (Figure 6B).
JA-Ile is emerging as an important signal compound activating defense responses to herbivores (Staswick and Tiryaki, 2004 ; Kang et al., 2006). The elicited dynamics in JA-Ile/JA-Leu levels (Figure 7A
) closely followed those of JA (Figure 6A), with the exception that JA-Ile/JA-Leu levels were not as reduced as the JA levels in WIPK-VIGS plants. In N. attenuata, JASMONATE RESISTANT4 (JAR4) and JAR6 are enzymes conjugating JA, Ile, and Leu to form JA-Ile and JA-Leu (Kang et al., 2006; Wang et al., 2007). q-PCR analyses revealed that transcripts of both genes were strongly elicited by either treatment in EV plants; SIPK-VIGS and WIPK-VIGS plants had significantly lower levels of JAR4 and JAR6 transcripts than did EV plants (Figure 7B). Although higher levels of JA-Ile/JA-Leu were found in WIPK-VIGS plants than in SIPK-VIGS plants after either elicitation, both had the same levels of JAR4 and JAR6 transcripts. A THREONINE DEAMINASE (TD) gene in N. attenuata also plays important roles in JA-Ile biosynthesis, converting Thr to Ile at the wound site and thus supplying the amino acid for this conjugation reaction (Kang et al., 2006). After either treatment, TD transcripts were dramatically increased in EV plants, and these increases were suppressed in SIPK-VIGS and WIPK-VIGS plants (Figure 7C). These data suggest that both SIPK and WIPK regulate the levels of JAR4, JAR6, and TD transcripts.
M. sexta herbivory and OS elicitation, but not wounding, increase ethylene biosynthesis and emissions in N. attenuata (Kahl et al., 2000 ). To determine if OS-elicited ethylene production correlates with the activation of MAPKs, we analyzed ethylene production in SIPK-VIGS, WIPK-VIGS, and EV plants. Leaves cut from petioles and subsequently treated with W+W or W+OS were sealed in 250-mL flasks for 5 h; the accumulated ethylene was measured with a photoacoustic laser spectrometer (Figure 8A
). No detectable amount of ethylene was observed in any plant treated with W+W. After W+OS treatment, both EV and WIPK-VIGS plants accumulated similar levels of ethylene, but SIPK-VIGS plants only accumulated about half as much, indicating that SIPK, but not WIPK, plays an important role in regulating ethylene biosynthesis after herbivory. In cultivated tobacco, elegant gain-of-function analyses have demonstrated that SIPK/WIPK activation mediates transcript accumulations of ACC SYNTHASE (ACS) and ACC Oxidase (ACO) genes (Kim et al., 2003). In EV plants, both treatments strongly increased levels of ACS3 to a similar degree; although OS treatment amplified the wound response for ACO1 transcripts, ACO3 transcripts did not respond (Figures 8B to 8D). When untreated, both WIPK-VIGS and SIPK-VIGS plants had lower levels of ACS3 transcripts (Figure 8B), and SIPK-VIGS plants had lower ACO3 transcripts (Figure 8C) than did EV plants. After both treatments, SIPK-VIGS plants had significantly suppressed transcript levels of both ACO genes for the duration of the time course, and for the ACS3 transcript, suppressed levels were found at all harvests with the exception of the 3 h harvest, when levels returned to those in EV plants. By contrast, WIPK-VIGS plants had only transiently lower ACS3 transcript levels after OS elicitation (Figure 8B); all other ethylene biosynthetic transcripts were at levels comparable to (for W+OS elicitations) or higher than (for W+W elicitations) those found in EV plants. For example, ACO1 transcript levels in WIPK-VIGS plants were 1.8 times higher 3 h after wounding than those in EV plants (P = 0.024, unpaired t test), but OS elicitation abolished the difference between EV and WIPK-VIGS plants (Figure 8D). These data indicate that both SIPK and WIPK positively regulate OS-elicited JA, SA, and JA-Ile; moreover, SIPK positively regulates ethylene biosynthesis.
JA and Ethylene Signaling Do Not Mediate OS-Elicited MAPK Activity Research with cell suspension cultures and in-gel kinase assays has shown that treatment with methyl jasmonate (MeJA) or ethylene activates kinases (Kumar and Klessig, 2000 ; Ouaked et al., 2003). To determine whether this is also the case with intact plants, we analyzed kinase activity and WIPK transcript accumulation in N. attenuata plants that had been treated with MeJA and ethephon; these readily diffuse into plants to release JA and ethylene, respectively. Up to 1 h after treatment, slightly increased levels of WIPK transcripts and SIPK activity were detected in MeJA- and ethephon-treated plants; however, these levels were not higher than those in lanolin- and MES-treated plants, their respective controls. These increased levels are likely to have resulted from the plants being touched when these chemicals were applied. Even after 6 h, MeJA and ethephon treatments didn't elevate SIPK or WIPK activity or WIPK transcript levels more than their respective controls (see Supplemental Figure 1A online), although applying MeJA dramatically elicited the JA-responsive gene TPI, and ethephon effectively released a large quantity of ethylene as measured by a photoacoustic spectrometer (see Supplemental Figures 1A and 2 online). MAPK activity was also analyzed in transgenic plants impaired in their JA and ethylene signaling. We used an antisense-LOX3 line (asLOX3) that elicited only 50% of the JA produced by W+OS elicitation in wild-type N. attenuata plants (Halitschke and Baldwin, 2003), and a COI1-silenced line (irCOI1), whose ability to perceive JA was remarkably reduced (Paschold et al., 2007). To study the impact of ethylene production and perception on MAPK activation, we used an ACO-silenced line (irACO) of N. attenuata, which produced substantially less ethylene after OS elicitation than wild-type plants did, and a line overexpressing a mutant Arabidopsis ETR1 gene, whose ethylene perception was highly attenuated (ETR1) (von Dahl et al., 2007). After these transgenic plants were challenged with W+OS, in-gel kinase activity assays revealed that their levels of MAPK activity were the same as those of wild-type plants (see Supplemental Figure 1B online). All these data suggest that JA and ethylene signaling do not mediate OS-elicited MAPK activity levels in N. attenuata plants.
Silencing SIPK and WIPK Leads to Decreased Levels of TPI Activity and Phe Ammonia-Lyase mRNA
Phe ammonia-lyase (PAL) is another gene important for plant defense (Bennett and Wallsgrove, 1994 ). We investigated PAL transcript levels in EV, SIPK-VIGS, and WIPK-VIGS plants (Figure 9B) and found that they were suppressed in SIPK-VIGS plants but not in WIPK-VIGS plants. In SIPK-VIGS plants, suppression lasted for the duration of the experiment, except in OS-elicited plants, and levels of PAL transcripts returned to those of EV plants by 3 h. WIPK-VIGS plants had significantly higher levels of PAL transcripts than did EV plants 3 h after either elicitation, suggesting that WIPK negatively regulates PAL transcript levels.
FACs in M. sexta OS Are Elicitors That Activate Herbivory-Specific MAPK Signaling
Consistent with the activity assay, RNA gel blotting analysis revealed that levels of WIPK transcript were highly amplified when different FACs were applied to puncture wounds compared with when both 10% DMSO, the solvent for FACs, and the pure water treatments were used (Figure 10B). Similar levels of WIPK transcripts were found when leaves were treated with FAC-free OS and with W+W. RNA gel blotting also demonstrated that FAC-treated and W+OS-treated samples accumulated comparable levels of WIPK transcripts. Furthermore, we measured the JA bursts elicited by different FAC treatments. One hour after elicitation, all plants treated with OS or with different FACs had accumulated similarly elevated levels of JA (P = 0.69, ANOVA), levels that were 1.5 times higher than those in water-, 10% DMSO-, and FAC-free-OS–treated plants (Figure 10C). These data strongly support the idea that FACs are important elicitors in M. sexta OS; FACs are recognized by N. attenuata and in turn trigger downstream responses by activating MAPKs.
OS Elicitation of MAPKs at the Wound Site and in Systemic Tissues within a Leaf
Remarkably, W+OS application in regions 0 and 1 elicited SIPK activity in distinct regions of the leaf (Figure 11B). When region 0 was elicited, high levels of SIPK activity were observed in treated region 0; slightly lower levels were detected in region 1; SIPKs in region 3 were also activated, although levels were much lower than those in regions 0 and 1; in region 2, no elevation of SIPK activity was observed. Over time, SIPK activity levels in all regions waned, and region 2 remained unelicited throughout. Eliciting region 1 produced a distinct distribution of kinase activity: SIPK activity levels increased greatly in region 1 but only slightly in region 0. However, SIPK activity levels in region 3 also highly increased, reaching those of region 1. Similarly, region 2 did not increase SIPK activity levels when region 1 was treated with OS. These findings suggest that certain mobile signals move to various regions of the herbivore-attacked leaf and elicit SIPK activity. The duration of the response was also influenced by the region that was elicited. Elicited kinase levels lasted longer when leaves were treated in region 1. Even in leaves that had been elicited 1.5 h earlier in region 1, SIPK activity levels were higher than in leaves elicited in region 0 (Figure 11B, kinase activity panels). These results suggest that there might be less negative SIPK regulator activity at the tip than at the base of a leaf. The pattern of WIPK transcript accumulation followed the pattern of SIPK activity: regions 0 and 1 showed greatly elevated WIPK transcript levels when leaves were elicited in 0, and regions 1 and 3 showed highly elevated WIPK transcript levels when region 1 was elicited (Figure 11B), confirming the idea that WIPK transcript accumulation is regulated by SIPK. Moreover, in undamaged regions of the herbivore-attacked leaf, WIPK is also likely involved in herbivore resistance. To examine how fast the mobile signals move across the leaf, we analyzed the spatial distribution of SIPK activity shortly after elicitation in S0 leaves (Figure 11C). When leaves were treated in region 0, region 1 showed elevated SIPK activity levels as soon as 5 min afterwards. Similarly, when leaves were treated in region 1, region 3 also showed increased levels of SIPK activity after only 5 min. After 10 min, SIPK activity levels in leaves treated in either region 0 or 1 were similar to those harvested after 30 min. We compared SIPK activity levels in leaves at different developmental stages (Figure 11D). When OS were applied to region 0, after 30 min both younger (S3) and older (S0) leaves had elevated SIPK activity levels in both regions 0 and 1. Notably, eliciting region 1 in older leaves rapidly activated SIPK activity levels in 1 and 3. Region 3 of younger leaves showed only slightly enhanced SIPK activity levels after 30 min, suggesting that the maturation of the vascular system that comes with age allows the OS-elicited responses to be propagated more vigorously within an elicited leaf.
JA, SA, and ethylene are known to regulate the transcript levels of defense genes in many plant species (Dong, 1998
To investigate if the spatial pattern of JA levels also matched the distribution of LOX3 accumulation, JA levels produced in different regions were examined 1 h after treating either region 0 or 1 with W+OS, which is when wild-type plants accumulate the most JA (Figure 11E). Leaves in which region 0 had been elicited showed elevated JA levels in regions 0 and 1; remarkably, region 0 showed almost 7 times more JA than adjacent region 1, though such a dramatic difference did not characterize levels of SIPK activity and LOX3 transcripts in these two regions. When leaves were treated in region 1, JA was detected only in regions 1 and 3, which was also consistent with the spatial distribution of SIPK activity and LOX3 transcript levels. Similarly, region 3 had
TD and TPI are two genes involved in plants' direct defenses against herbivores (Zavala et al., 2004
Systemic TPI Elicitation Doesn't Require SIPK Activation in Systemic Leaves
JA and ethylene are known to play a central role in mediating a plant's responses to herbivore attack, which involves reconfiguring a plant's wound response (reviewed in Reymond and Farmer, 1998 ; Kessler and Baldwin, 2002a). How plants perceive herbivory and along which signaling pathways the messages are transformed into cellular responses are poorly understood. Here, we demonstrate that applying M. sexta OS to wounds in N. attenuata leaves rapidly and markedly elicits MAPKs more than wounding does. By removing FACs from OS and applying synthetic FACs, we demonstrate that FACs elicit these responses. Silencing SIPK and WIPK by VIGS revealed that both kinases regulate the transcript levels not only of WRKY transcription factors, MAPKs, and CDPKs but also of JA and ethylene, which highlights their central role in the OS-elicited, hormonally mediated, signal transduction network. By applying JA and ethylene and using transgenic plants impaired in the production and perception of JA and ethylene, we show that neither OS-elicited JA nor ethylene production contribute to OS-elicited MAPK activation. OS elicitation activates MAPKs and other defense responses not only in the damaged regions of a leaf but also in particular nondamaged regions. The spatial distribution of these responses depends on the developmental stages of the leaves and on the locations of herbivore damage, both of which implicate vascular maturation in the signaling. In addition to these responses in the attacked leaf, enhanced levels of TPI transcripts were found in unelicited leaves on the same plant. However, this between-leaf systemic signaling of defense responses occurs without MAPK activation in systemic leaves and suggests the existence of a mobile signal other than the one activating responses within attacked leaves.
Herbivore-Specific Elicitors, FACs, and Plant Perception of Herbivory
Although no FAC-specific receptors have yet been identified, some evidence suggests that ligand–receptor interactions play important roles in plant resistance to pathogens. In wild-type Arabidopsis protoplast, flagellin binds to receptor FLS2 and initiates downstream responses through MEKK1, MKK4/MKK5, and MPK3/MPK6 signaling cascades. In the fls2 mutant, these responses are absent, indicating the critical role of flagellin perception by receptor FLS2 (Asai et al., 2002
SIPK and WIPK Regulate Transcript Levels of Defense-Related Genes in N. attenuata |
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ABSTRACT
INTRODUCTION
-bergamotene emission, JA bursts, and extensive OS-specific transcript accumulation). Moreover, adding synthetic FACs back to FAC-free OS restored all of the OS-elicited responses, and treating wounds with aqueous FAC solutions mimicked the effects of OS (Alborn et al., 1997
