Kunitz Trypsin Inhibitor: An Antagonist of Cell Death Triggered by Phytopathogens and Fumonisin B1 in Arabidopsis

Molecular Plant

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Volume 1

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Number 3

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Pages 482–495

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May 2008

RESEARCH ARTICLE

Kunitz Trypsin Inhibitor: An Antagonist of Cell Death Triggered by Phytopathogens and Fumonisin B1 in Arabidopsis Jing Lia,b, Gu¨nter Bradera and E. Tapio Palvaa,1 a Viikki Biocenter, Department of Biological and Environmental Sciences, Division of Genetics, University of Helsinki, POB 56, FIN-00014, Helsinki, Finland b Current address: Sainsbury Laboratory, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom

INTRODUCTION Plants possess a large arsenal of protease inhibitor (PI) genes that have been proposed to function as storage proteins, regulators of endogenous proteinases, and most notably in defense against herbivores with potent applications in transgenic crops (Jofuku and Goldberg, 1989; Schuler et al., 1998). PIs are often part of an inducible, jasmonic acid (JA)associated defense pathway and accumulate upon wounding, pathogen, and herbivore damage in leaves (Farmer et al., 2003). Soybean (Glycine max) contains different types of PIs including Bowman-Birk trypsin inhibitors and Kunitz trypsin inhibitors (KTI). Both of these inhibitor types are specific for serine proteases, and KTIs have specific inhibitory activity solely against trypsin proteases that cleave polypeptides after Lys and Arg. At least 10 KTI genes with different expression patterns and functionalities exist in soybean (Gotor et al., 1995; Jofuku and Goldberg, 1989). KTIs inhibit the proteolytic enzymes found within herbivore guts, resulting in an inhibition of insect growth, such as in the larvae of the moth Spodoptera littoralis (Leo et al., 1998; Marchetti et al., 2000; Schuler et al., 1998). Over the past two decades, much effort has been directed to address potential roles of specific KTIs in protection of plants against insect pests. However, expression of KTI genes in plants does not always effectively reduce damage or confer protection against insects. For instance, expres-

sion of the soybean gene SKTI3 in transgenic poplars, tobacco and sugarcane did not lead to enhanced field resistance against various lepidopterous species, albeit in-vitro inhibition of the midgut-proteolytic enzymes by KTIs does occur (Confaloneri et al., 1998; Falco et al., 2003; Nandi et al., 1999). Programmed cell death (PCD) or apoptosis—an active and controlled cell suicide essential for the development, homeostasis and host-pathogen interactions of multi-cellular organisms—involves controlled induction and activation of cellular mechanisms and genetic programs. Control of the PCD is essential for its containment to specific tissues and hence proteolysis by cysteinyl Asp-specific proteases (caspases) and serine proteases are tightly regulated and specific protease inhibitors play crucial roles in the cellular regulation of proteases during the PCD process (Shi, 2002; Woltering et al., 2002). In mammalian cells, eight inhibitors of apoptosis proteins (IAP) have been identified. They directly bind via a baculovirus IAP repeat zinc-binding (BIR) domain and confer protection from death-

1 To whom correspondence should be addressed. E-mail [email protected], fax +358-9–191–59076, tel. +358-9-191-59600.

ª The Author 2008. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssn013, Advance Access publication 29 April 2008

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ABSTRACT Programmed cell death (PCD) is a central regulatory process in both plant development and in plant responses to pathogens. PCD requires a coordinate activation of pro-apoptotic factors such as proteases and suppressors inhibiting and modulating these processes. In plants, various caspase-like cysteine proteases as well as serine proteases have been implicated in PCD. Here, we show that a serine protease (Kunitz trypsin) inhibitor (KTI1) of Arabidopsis acts as a functional KTI when produced in bacteria and in planta. Expression of AtKTI1 is induced late in response to bacterial and fungal elicitors and to salicylic acid. RNAi silencing of the AtKTI1 gene results in enhanced lesion development after infiltration of leaf tissue with the PCD-eliciting fungal toxin fumonisin B1 (FB1) or the avirulent bacterial pathogen Pseudomonas syringae pv tomato DC3000 carrying avrB (Pst avrB). Overexpression of AtKTI1 results in reduced lesion development after Pst avrB and FB1 infiltration. Interestingly, RNAi silencing of AtKTI1 leads to enhanced resistance to the virulent pathogen Erwinia carotovora subsp. carotovora SCC1, while overexpression of AtKTI1 leads to higher susceptibility towards this pathogen. Together, these data indicate that AtKTI1 is involved in modulating PCD in plant–pathogen interactions.

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Here, we isolated and characterized an Ecc elicitor-induced Kunitz trypsin inhibitor gene (AtKTI1) encoding a functional KTI protein in Arabidopsis. Using both gain of function and RNAi silencing approaches, we demonstrate that the protein plays a regulatory role in PCD antagonizing pathogen and FB1-induced cell death.

RESULTS A Putative Trypsin Inhibitor Gene (AtKTI) Induced in Response to Ecc Elicitors We have employed suppressive subtractive hybridization (SSH) to identify Arabidopsis genes upregulated by Ecc elicitors (culture filtrates, CF) and hence potentially involved in pathogen recognition, defense signaling or production of antimicrobial compounds (Brader et al., 2001). One of the isolated clones, containing a 326-bp fragment, showed a 99% match with the coding region of a putative Kunitz trypsin inhibitor (KTI) and has an ‘A’ instead of a ‘G’ in the third position of the codon for Leu14 compared to its database entry At1g73260. We obtained a full-length cDNA of this gene named AtKTI1 from a library for CF-treated Arabidopsis plants containing the same synonymous substitution. The deduced amino acid sequence of AtKTI1 contains a predicted 28aa signal peptide at the N-terminal region (Figure 1). AtKTI1 shows substantial similarity to a number of putative proteins from other plant species, including a KTI precursor from Brassica oleracea (BOU18995), a root nematode-induced miraculin homologue (LEU70076) from Lycopersicon esculentum, and, interestingly, an HR-related gene product (NTU66263) triggered by viruses in tobacco (Karrer et al., 1998). In Arabidopsis, the AtKTI1 protein is a member of a small family of seven proteins with predicted KTI function (Figure 1). The closest homologues to At1g73260 AtKTI1 are a miraculin homologue (At1g17860), a protein encoded by the droughtrepressed Dr4 gene (At1g73330; Gosti et al., 1995), and a Dr4-related protein (At1g73325). AtKTI1 does not share significant nucleotide homology with any other gene in Arabidopsis. However, none of the above proteins annotated as TIs has demonstrated trypsin inhibitory activity. The predicted AtKTI1 product shares less than 30% amino acid identity (Figure 1) across its entire structure to the KTI3 of Glycine max (AF233296) with confirmed inhibitor activity (Jofuku and Goldberg, 1989). The aa Arg87 and Ile88 of KTI3 are crucial for activity, and are replaced by His and Ala in the two other, non-functional KTI1 and KTI2 of soybean (Jofuku and Goldberg, 1989). The alignment in Figure 1 shows a substitution of Arg87 and Ile88 with the similar aa Lys and Val, respectively, indicating that AtKTI1 might have retained its inhibitor functionality.

Defense-Related Signals Trigger AtKTI1 Expression The induction of the AtKTI1 gene by Ecc CF suggested an involvement of AtKTI1 in plant responses to Ecc, a pathogen that

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inducing stimuli by inhibiting active caspases (Salvesen and Duckett, 2002). In turn, antagonists of IAP are also involved in the regulation of PCD. HtrA2 is such an IAP antagonist and encodes a serine protease, which is released from the mitochondria to the cytosol upon apoptosis stimulation and inhibits the function ofXIAP(a3chromosome-linkedIAP) bydirect binding resulting in induction of cell death (Jin et al., 2003; Yang et al., 2003). Over the past few years, caspase-mediated PCD has been extensively addressed in plant cells and, although the PCD machinery might be somewhat conserved between animal and plant kingdoms, cell death executors and regulators defined in animal PCD are largely missing from the Arabidopsis genome (Chichkova et al., 2004; Della Mea et al., 2007; Estelle, 2001; Lam et al., 2001; Lam, 2004). Different groups of caspase-like proteases participating in PCD of plants have been identified (for a review, see Piszczek and Gutman, 2007), including metacaspases (Suarez et al., 2004; Uren et al., 2000; Vercammen et al., 2004; Watanabe and Lam, 2004), vacuolar processing enzymes (VPEs, legumains; Hatsugai et al., 2004, 2006; Kuroyanagi et al., 2005), and serinyl Asp-specific proteases (saspases; Coffeen and Wolpert, 2004). A number of biochemical and pharmacological studies indicates the involvement of saspases and other types of serine proteases in plant PCD (Antao and Malcata, 2005; Coffeen and Wolpert, 2004; Groover and Jones, 1999; Sasabe et al., 2000; Yano et al., 1999). Specific inhibitorsof PCD in plant cellsinclude cystein proteaseinhibitors (Solomon et al., 1999) and the Bax inhibitor 1 (BI-1), presumably interacting with VPEs (Watanabe and Lam, 2004, 2006). Genes encoding KTIs or homologues have also been suggested to be involved in the regulation of the PCD (Karrer et al., 1998; Park et al., 2001). In contrast to the role of KTIs in insect resistance, however, little is known about physiological functions of KTIs in plant signaling. Park et al. (2001) suggested that SKTI3 might act as a regulator in cellular defense responses and that it is associated with the hypersensitive response (HR). However, so far, no genetic evidence demonstrates that trypsin inhibitors are components of the complex machinery of PCD. HR is one classical form of PCD in plant pathogen interactions, where host detection of pathogen-encoded effectors or other components leads to rapid cell collapse around the site of infection and restriction of pathogen spreading (Dangl and Jones, 2001). In Arabidopsis ecotype Col-0, HR is triggered by Pseudomonas syringae pv maculicola and pv tomato (Pst) strains carrying one of the avirulence genes avrB, avrRpt2, avrRpm1, avrPphB, or avrRps4 (Weigel and Glazebrook, 2002). The elicitor HrpN of Erwinia carotovora subsp. carotovora SCC1 (Ecc SCC1) can also trigger lesion formation in different plant species including Arabidopsis (Kariola et al., 2003). Fumonisin B1 (FB1) is a fungal toxin, which acts as a competitive inhibitor of ceramide synthase and disrupts sphingolipid metabolism (Desai et al., 2002). FB1 is produced by the necrotrophic fungal pathogen Fusarium moniliforme and triggers PCD in a similar manner as avirulent bacterial pathogens in different plant species including Arabidopsis (Asai et al., 2000; Desai et al., 2002; Stone et al., 2000).

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Figure 1. Kunitz Trypsin Inhibitors. Alignment of the At1g73260 KTI protein with homologues found in other plants and with the six other members of the KTI family of Arabidopsis. Alignment was created using MCOFFEE (Moretti et al., 2007). The closest homologue of At1g73260 in Arabidopsis is At1g17860, with 36% identities and 53% positives. Accession numbers for the KTIs of other plant species are BOU18995 (AAB68964) from Brassica oleracea (51% identities/63% positives compared with At1g73260), LEU70076 ( = AAC63057) from tomato (40%/56%), NTU66263 ( = AAC49969) from tobacco (38%/61%) and AF233296 from soybean (Kunitz TI 3; 26%/50%). The arrow is indicating the end of the N-terminal signal domain. The aa critical for trypsin inhibitor activity in soybean KTI 3 (Jofuku and Goldberg, 1989) and their aligned aa are framed with a rectangle.

can trigger both salicylic acid (SA)- and jasmonate/ethylene (JA/ET)-dependent defense gene expression (Li et al., 2004; Kariola et al., 2005). To elucidate the potential involvement of AtKTI1 in plant defense, we characterized expression of the gene in plants exposed to CF, SA, methyl jasmonate (MeJA),

and 1-aminocyclopropane-1-carboxylic acid (ACC, a natural precursor of ET), H2O2, and mechanical wounding. Expression of the AtKTI1 gene was induced in response to CF, SA, H2O2, and wounding treatments (Figure 2). AtKTI1 transcript levels were weakly induced in the CF and H2O2-treated leaves 5 or

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sis. The purified full-length AtKTI1 protein was slightly bigger (;23 kDa) and was non-functional as a trypsin inhibitor in our assay (data not shown) indicating that only the cleaved mature variant acts as functional trypsin inhibitor and that expression of the full-length protein in E. coli does not result in correctly cleaved AtKTI1.

AtKTI1 Does Not Inhibit Protease Activity of Ecc Previous studies have demonstrated that extracellular proteases are necessary for normal progression of infection and generation of disease symptoms by Ecc (Marits et al., 1999). Consequently, AtKTI1 protein might theoretically inhibit the activity of extracellular proteases of Ecc and hence help to prevent or contain the spreading maceration caused by Ecc infection. To address this possibility, we tested the effects of the active recombinant protein on CF containing multiple extracellular proteases. Robust protease activity could be detected when Ecc SCC1 was grown in LB medium for 30 h at 28
C.

Functional Characterization of AtKTI1 To elucidate the functional identity of the AtKTI1 gene, we produced an N-terminally His-tagged protein with and without the predicted signal peptide (predicted by SignalP, Bendtsen et al., 2004) in E. coli and determined its biological activity. As shown in Figure 3A, the purified recombinant AtKTI1 protein produced without the signal peptide had the expected size of about 21.5 kDa and exhibited similar inhibitory effect on bovine trypsin activity as soybean TI (Figure 3B), strongly suggesting that AtKTI1 encodes a functional KTI in Arabidop-

Figure 3. Production of Recombinant AtKTI1 Protein and In-Vitro Assay of Trypsin Inhibitory Activity.

Figure 2. Induction of AtKTI1 by Exogenous Application of Signal Molecules and Wounding Treatment in Arabidopsis. Four-week-old soil-grown Arabidopsis wild-type (Col-0) plants were treated with Ecc culture filtrates (CF), 5 mM SA, 100 lM JA, 100 lM ACC, 100 lM H2O2, and environment stimuli wounding (W), respectively. Leaf samples were collected at the indicated time-points. 20 lg of total RNA was used for RNA-gel analysis. RNA blots were hybridized with a DIG-labeled AtKTI1 cDNA probe. Experiments were repeated three times, with similar results.

(A) Coomassie-stained SDS-PAGE gel showed bacterial overexpression and purification of His-tagged AtKTI1 protein without signal peptide. Molecular weight markers are shown in the left lane and the purified AtKTI1 is indicated by an arrow in the right lane. (B) In-vitro bovine trypsin (10 lg) inhibition by the recombinant AtKTI1 protein. Soybean trypsin inhibitor was assayed in parallel as a positive control. Trypsin activity was measured with the substrate Na-benzoyl-L-arginine ethyl ester (BAEE), as described in the Methods. Reaction mixtures contained 50 BAEE units bovine pancreas trypsin, resulting in a DA253 of 0.04 per min without addition of an inhibitor. Values indicate the mean of three samples (6 SD). Experiments were repeated three times, with similar results.

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8 h, respectively, after treatment and continued to increase until 24 h. SA treatment caused a robust expression at 24 h whereas only a weak accumulation of AtKTI1 transcripts was observed following wounding of mature leaves at this timepoint. In contrast, MeJA or ACC treatments did not lead to any detectable increase in AtKTI1 mRNA in leaves (Figure 2). It is interesting to note that publicly available microarray data accessed via Genevestigator (Zimmermann et al., 2004) show a clear increase of AtKTI1 transcript 24 h after Pst DC3000 treatment. This increase is more pronounced in strains carrying the avrRpm1 avirulence gene and develops here already 6 h after treatment. Interestingly, microarray data analysis performed with Genevestigator shows that the expression profile of AtKTI1 differs from most of the other Arabidopsis KTI genes listed in Figure 1. In addition to AtKTI1, only At3g04320 is induced by Pst DC3000 with or without avrB, while the closest homologues At1g17860 and At1g73330 as well as At1g72290 are not induced. The expression pattern might indicate specific roles for the individual KTIs of Arabidopsis.

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The protease activity of Ecc SCC1 was strongly inhibited by the metalloprotease inhibitor EDTA (Table 1). In contrast, the recombinant AtKTI1 had no significant effect on the extracellular protease activity even at a ratio of 2:1 (recombinant AtKTI1:CF-induced total proteins, Table 1). Thus, we conclude that the Arabidopsis trypsin inhibitor AtKTI1 does not inhibit the major Ecc-derived extracellular proteases.

Overexpression of AtKTI1 Does Not Affect Resistance of Arabidopsis to S. littoralis

Overexpression of AtKTI1 Enhances Susceptibility of Arabidopsis to Ecc Since recombinant AtKTI1 did not exhibit inhibitory effect on Ecc-produced extracellular proteases and overexpression did not show enhanced resistance to insects, we considered the possibility that the CF and SA-inducible expression of AtKTI1 might be involved in modulating Arabidopsis defense response to Ecc through an as yet unknown mechanism. Surprisingly, after Ecc SCC1 infection, most plants of the overexpression line S13 displayed significantly accelerated maceration, not only in inoculated, but also in systemic leaves (Figure 4B). In contrast, the other two lines (S8 and S16) tested did not exhibit this enhanced maceration phenotype but were

RNAi Silencing Causes Enhanced Disease Resistance of Arabidoposis to Ecc SCC1 As overexpression of AtKTI1 resulted in enhanced susceptibility to E. carotovora, silencing of the gene should have the opposite effect. To achieve this, we employed an RNAi silencing approach (Smith et al., 2000) to down-regulate AtKTI1. Interestingly, some primary soil-grown transformants developed lesions at an early developmental stage and could not survive or showed dwarf phenotypes (Figure 5A). Two stable independent and homozygous lines (RNAi-16 and RNAi-68) were used for the subsequent studies. The efficacy of RNAi silencing was verified by RNA-gel blots (Figure 5B). Both lines exhibited morphological traits different from controls such as serrated leaves and reduced size in up to 3-week-old plants (Figure 5B). Line RNAi-16 appeared to show a more severe phenotype and was smaller in size than the other line. When challenged with Ecc SCC1 at concentrations causing strong maceration in wild-type and vector controls, transgenic AtKTI1-silenced plants showed only limited maceration that was restricted to the inoculated leaf (Figure 5C). In contrast, vector control plants developed more severe disease symptoms and maceration was spreading to systemic leaves (Figure 5C) leading to a higher percentage of dead plants than in the AtKTI1-silenced lines RNAi-16 and RNAi-68 (Figure 5D). Taken together, these results strongly suggest that silencing of the AtKTI1 gene reduces sensitivity of Arabidopsis to Ecc SCC1.

AtKTI1 Controls Induced Cell Death in Arabidopsis Table 1. Effects of Inhibitors on Extracellular Protease Activity in Culture Supernatant Fluids of Ecc SCC1.

Inhibitors

Inhibitor concentration

Percentage inhibition

EDTA

10 mM

96.6 6 6.8

Recombinant AtKTI1

15 lgm1
1*

4.9 6 5.5

*Total protein concentration of CF was adjusted to 7.5 lg ml
1. Results are the means of two experiments, each with two replicates (6 SD).

The enhanced lesion formation and the altered leaf morphology in the RNAi-silenced lines suggested that AtKTI1 might be involved in containing PCD and also be involved in modulating pathogen-triggered cell death. Avirulent pathogen-triggered hypersensitive response (HR) is one of the best characterized examples of PCD in plants. To elucidate the involvement of AtKTI1 in this type of PCD, we employed a strain of Pst DC3000 carrying avrB, which is avirulent on Arabidopsis Col-0. We characterized the HR triggered by infiltration of Pst DC3000 avrB (5 3 106 cfu mL
1) into Arabidopsis leaves of

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To assess the in-planta role of AtKTI1 in defense, we constructed several independent, homozygous lines overexpressing AtKTI1. A total of 22 primary transformants in Arabidopsis Col-0 were obtained and the transgene expression was verified by RNA-gel blot analysis (Figure 4A). The homozygous progenies (T4 generation) of three independent lines S8, S13, and S16 each with a single insertion of the transgene were used for subsequent experiments. Overexpression of AtKTI1 had no obvious effect on growth, morphology, or fertility of Arabidopsis. Since PIs are very common defense proteins against insects (Schuler et al., 1998) and soybean KTI3, when overexpressed in tobacco and potato, reduces the survival and the growth of S. littoralis (Marchetti et al., 2000), we assessed the effect of AtKTI1 overexpression on Arabidopsis resistance against S. littoralis. Neither leaf consumption nor larval growth and mortality displayed significant differences between AtKTI1 overexpressor and control plants (data not shown), indicating that overexpression of AtKTI1 does not contribute to resistance of Arabidopsis against S. littoralis.

similar to vector control (data not shown). In agreement with this observation, no substantial differences in the bacterial growth were observed between vector control and lines S8 or S16, whilst line S13 showed clearly increased colonization by Ecc leading to the death of the majority of the tested plants (Figure 4C). To determine whether there was a correlation between endogenous levels of AtKTI1 and the exaggerated disease symptoms, we assayed AtKTI1 levels in leaf extracts from all three overexpression lines and the vector control. As shown in Figure 4D, TI activity was highly increased in line S13, while line S8 only exhibited some activity. No TI activity was detectable in line S16 and the control line. Thus, the compromised resistance in Ecc-infected line S13 is probably caused by the high cellular level of AtKTI1 and suggests that the virulent pathogen Ecc can benefit from increased AtKTI1 production.

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Figure 4. Overexpressing AtKTI1 in Arabidopsis and Responses of Overexpression Lines to Ecc SCC1. (A) AtKTI1 overexpression transformants were confirmed by RNAgel analysis. Non-treated mature leaves from overexpression and vector control pCP60 (VC) lines were collected. Each lane contained 20 lg total RNA. The RNA blot was hybridized with the DIG-labeled AtKTI1 cDNA probe. (B) Development of soft-rot symptoms on overexpression plants after Ecc infection. Fifteen 4-week-old plants from each line (T3 generation) were inoculated with Ecc SCC1 by pipetting 10 lL of 105 cfu mL
1 on leaves. Representative leaves were detached and photographed 12 h after inoculation and whole plants were documented 48 h after inoculation. Red arrows indicate the inoculated leaves and yellow arrows indicate systemically macerated leaves. Left panels, vector control pCP60 (VC); right panels, AtKTI1 overexpressing Arabidopsis line S13.

(C) The percentage of dead plants was calculated from three independent experiments performed with 28 plants for AtKTI1 overexpressors S8, S13 and S16 and vector control pCP60 (VC), as in (B) 7 d after inoculation with Ecc SCC1. Bars represent SD. Plants were considered dead if maceration spreads to buds and stems resulting in detachment of the roots. (D) Transgenic plants express functional, detectable proteins. Trypsin inhibitory activity of leaf crude extracts from AtKTI1 overexpressors S8, S13 and S16 and vector control pCP60 (VC) line was determined with bovine trypsin and Na-benzoyl-L-arginine ethyl ester (BAEE) as a substrate, as described in the Methods. Reaction mixtures contained 50 BAEE units bovine pancreas trypsin, resulting in a DA253 of 0.013 per min without addition of an inhibitor. ND, not detectable. Value indicates a mean of three samples (6 SD). Experiments were repeated twice, with similar results.

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vector control, AtKTI1 RNAi, and overexpressor lines; 48–72 h after the infiltration, overexpression of AtKTI1 resulted in clearly reduced damage when compared with control plants under high (100% RH) humidity (Figure 6A). No difference between overexpressor lines and vector controls can be seen at lower humidity or a smaller inoculum size (data not shown). In contrast, RNAi lines showed clearly more severe Pst DC3000 avrB-induced damage compared to vector controls (Figure 6B). These results argue that AtKTI1 modulates pathogen-related damage control in Arabidopsis. Fumonisin B1 (FB1)—a PCD-eliciting fungal toxin—is a sphinganine analogue that has been shown to trigger dosagedependent cell death in Arabidopsis that shares many features with avirulent pathogen-induced HR (Stone et al., 2000; Asai et al., 2000). Because AtKTI1 overexpression or RNAi silencing caused opposite effects on Pst DC3000 avrB-induced cell death in transgenic plants, we speculated that AtKTI1 might function as a negative regulator of pathogen-triggered cell death. To test this hypothesis, we first examined FB1 induction of the AtKTI1 gene. RNA-gel blot analysis revealed that expression of AtKTI1 was induced after 24 h of treatment with 10 lM FB1, whilst the highest level of AtKTI1 transcripts was detected at 48 h followed by a decline at later time-points (Figure 7A). The transient expression of AtKTI1 suggested a potential involvement of AtKTI1 in formation of FB1-induced lesions. To test this, we characterized FB1-induced lesion formation in RNAi-silenced and overexpression plants using 1, 2.5, 5, and 10 lM FB1. Two AtKTI1 overexpression lines (S8 and S13) showed reduced lesion formation at high FB1 concentrations (10 lM), resulting in clear lesion formation in vector control (Figure 7B) and wild-type (Stone et al., 2000). RNAi silencing of AtKTI1 was shown to significantly enhance the formation of FB1-induced lesions. The accelerated lesion formation was dose-dependent and evident at concentrations of <2.5 lM FB1 in RNAi-16 and RNAi-68. Infiltration of a 1 or 2.5 lM FB1 solution into mature leaves of plants of RNAisilenced line 16 resulted in formation of confluent lesions on the infiltrated leaves within 1 week (Figure 7C) or 4 d (Figure 7D), respectively. However, only scattered visible lesions formed on FB1-infiltrated leaves of control plants.

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There was no substantial difference at a higher concentration (>5 lM) between RNAi-16 and control plants (data not shown). Similar results were obtained in RNAi-68 (data not shown). These data demonstrate that the AtKTI1 gene is likely to be involved in control of FB1-induced cell death.

DISCUSSION

Figure 5. Phenotype of RNAi Lines and their Response to Ecc SCC1. (A) Some primary transformants (RNAi-AtKTI1-T1, 3-week-old) developed lesions in early developmental stage and exhibited aberrant leaf shape and smaller size in comparison to the vector control (pCP60) shown in the right lower corner. (B) Growth phenotypes of two independent and stable RNAi lines. T4 generation of 3-week-old RNAi-16, RNAi-68, and pCP60 vector control plants were photographed. Arrows indicate serration of leaves. RNA-gel analysis shown below each transgenic line indicates reduced AtKTI1 expression in both RNAi lines corresponding to the growth phenotype. Mature leaves from RNAi lines and control plants were treated with CF and collected after 24 h. Each lane contained 8 lg total RNA. The RNA blot was hybridized with the DIGlabeled AtKTI1 RNA probe. (C) Fifteen 4-week-old plants from each line were inoculated with Ecc SCC1 by pipetting 10 lL of 106 cfu mL
1 on leaves. Representative plants of RNAi-16 and pCP60 vector control plants were photographed 4 d after inoculation. The experiment was performed with T3 and T4 generation three times, with similar result. The red arrow indicates the inoculated leaf and yellow arrows point to spreading infection and maceration. (D) The percentage of dead RNAi-16, RNAi-68, and pCP60 vector control plants was calculated from three independent experiments as in (C) 7 d after inoculation with Ecc SCC1. Bars represent SD. Plants were considered dead if maceration spreads to buds and stems resulting in detachment of the roots.

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In this work, we show that the AtKTI1 gene triggered by pathogen-derived elicitors encodes a functional serine protease inhibitor and antagonizes pathogen-associated cell death in Arabidopsis. This is indicated by increased lesion formation in AtKTI1 RNAi-silenced lines after fumonisin B1 (FB1) treatment and after infection with avirulent Pst avrB (Figures 6B and 7). Moreover, AtKTI1 overexpression leads to reduction of lesion formation after FB1 and Pst avrB treatment (Figures 6A and 7B). Two lines of evidence indicate that AtKTI1 encodes a functional Kunitz trypsin inhibitor: (1) Purified His-tagged AtKTI1 protein produced in E. coli inhibits proteolytic activity of commercial bovine trypsin as efficiently as soybean TI and (2) crude leaf extracts of plants overexpressing the AtKTI1 transgene exhibit strongly enhanced trypsin inhibitory activity. Based on its activity, we can estimate that cellular levels of KTI in the overexpression line S13 reaches up to ;0.5% of total soluble protein. In spite of the high levels, we could not detect any deleterious effect on the growth and development of the pest insect S. littoralis when feeding on AtKTI1 overexpressing lines. Previous studies have shown that the soybean KTI3 can inhibit S. littoralis only at very high (.1% of total soluble proteins) leaf concentrations (Leo et al., 1998; Marchetti et al.,

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(A) One leaf of each plant was infiltrated with approximately 2 ll of avirulent Pst DC3000 avrB suspension at a concentration of 5 3 106 (cfu mL
1). Thirty 4-week-old plants were treated from AtKTI1 overexpression line S13 and the vector control line (pCP60), respectively. Treated plants were covered by transparent lid to keep 100% RH. Images of representative plants were taken and scored 3 d after inoculation. The infiltrated leaf is indicated with an arrow. Definition of symbols: +, scattered lesions covering less than 25% of the infiltrated area; ++, visible scattered lesions with occasional dry necrotic areas; +++, confluent necrosis that covers more than 50% of the infiltrated area. These experiments are representative of two independent replicates. (B) Fully expanded leaves from two independent RNAi lines and control plants (three leaves per plant) were infiltrated with approximately 2 lL of Pst DC3000 avrB suspension at a concentration of 2–3 3 105 (cfu mL
1). Three-week-old plants were treated and incubated under 70% RH and representative leaves were scored and excised three days after infiltration for photographing. All experiments were performed with T3 and T4 generation, with similar results.

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2000). Consequently, based on our results, it seems unlikely that AtKTI1 would be an efficient inhibitor of insect growth in wild-type Arabidopsis Col-0 under normal circumstances. Furthermore, recombinant AtKTI1 does not appear to have a direct role in defense against the virulent pathogen Ecc. The purified AtKTI1 protein does not inhibit the activity of extracellular proteases secreted by Ecc grown in LB medium (Table 1). Ecc produces mainly metalloproteases in these growth conditions and we cannot rule out the possibility that Ecc might secrete other types of extracellular proteases or that certain types of proteases are modulated in their activity by cofactors present in planta conditions. However, since plants overexpressing AtKTI1 show enhanced susceptibility to Ecc infection (Figure 4) and RNAi plants with reduced AtKTI1 levels are more resistant to Ecc (Figure 5), a direct inhibitory effect of AtKTI1 on Ecc proteases seems unlikely. The insect and pathogen assays together point to a possible a role of AtKTI1 rather in cellular signaling than as acting as an inhibitory protein directed against proteins of invading organisms. Our data show RNAi silencing of AtKTI1 increases lesion formation both after treatment with the fungal toxin FB1 and after infection with avirulent Pst avrB, while overexpression suppresses lesion formation after infection with the avirulent pathogen and after FB1 treatment (Figures 6 and 7). Moreover, RNAi-mediated silencing of AtKTI1 disturbs normal developmental processes, so that some of RNAi-silenced lines develop spontaneous lesions at the early developmental stage. The homozygous lines RNAi-16 and RNAi-68 have been characterized in more detail and have an altered, serrated leaf shape and are smaller than vector control (Figure 5A and 5B) and wild-type plants. These observations indicate that AtKTI1 may be involved in modulating both developmental and pathogenrelated PCD. PCD is executed in plant cells by cysteine proteases (metacaspases and VPEs) and specific serine proteases termed saspases (Coffeen and Wolpert, 2004; Piszczek and Gutman, 2007). These characterized proteases are not likely candidates to be inhibited by AtKTI1. Saspases cleave their substrates after Asp (Coffeen and Wolpert, 2004) and KTIs specifically inhibit serine proteases cleaving after Lys and Arg (Jofuku and Goldberg, 1989). Metacaspases can also cleave at the C-terminal end of Lys or Arg (Vercammen et al., 2004; Watanabe and Lam, 2005) but are, however, cysteine proteases; therefore, inhibition by AtKTI1 seems unlikely. Previous pharmacological studies, however, indicate also the involvement of other serine proteases and especially TIs in various forms of plant PCD. For instance, serine proteases were proposed to be responsible for the activation of signal transduction pathways

Definition of symbols: +, scattered lesions covering less than 25% of the infiltrated area; ++, visible scattered lesions with occasional dry necrotic areas; +++, confluent necrosis that covers more than 50% of the infiltrated area. These experiments are representative of two independent replicates.

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Figure 6. Responses of AtKTI1 Transgenic Lines to Pst DC 3000 Carrying an avrB Gene.

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Figure 7. FB1 Induces Altered Lesion Formation in Arabidopsis AtKTI1 Overexpression and RNAi Plants. (A) Four-week-old soil-grown Arabidopsis wild-type (Col-0) plants were treated with 10 lM FB1 (FB) or 0.14% methanol in water (C). Leaf samples were collected at the indicated time-points. 20 lg of total RNA was used for RNA-gel analysis. RNA blots were hybridized with a DIG-labelled AtKTI1 cDNA probe. (B) Visible symptoms on representative plants photographed 1 week after 10 lM FB1 infiltration. Four-week-old soil-grown AtKTI1-overexpressing lines S8 and S13 and vector control (pCP60) plants were treated with 10 lM FB1 and symptoms were photographed and scored 72 h after infiltration. Definition of symbols: +, visible scattered lesions; ++, visible scattered lesions with occasional dry necrotic areas; +++, confluent necrosis that covers large parts of the infiltrated area. These experiments are representative of two independent replicates. (C) Visible symptoms on representative plants photographed 1 week after 1 lM FB1 infiltration. FB1 lesions predominantly occur on older leaves. Four-week-old soil-grown Arabidopsis AtKTI1 RNAi line 16 and vector control (pCP60) plants were treated with 1 lM FB1 and symptoms were photographed 1 week after infiltration. (D) Visible symptoms on representative leaves after 2.5 lM FB1 infiltration. Four-week-old soil-grown Arabidopsis AtKTI1 RNAi line 16 and vector control (pCP60) plants were treated with 2.5 lM FB1 and symptoms on treated leaves were assessed and scored 4 d after infiltration. Definition of symbols: +, visible scattered lesions; ++, visible scattered lesions with occasional dry necrotic areas; +++, confluent necrosis that covers partially the infiltrated area. These experiments have been repeated twice with similar results. AtKTI1 RNAi line 16 and vector control (pCP60) plants treated with 2.5 lM FB1 have been trypan-blue stained 4 d after infiltration to show damaged cells and representative leaves are shown.

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leading to cell death in tobacco (Sasabe et al., 2000; Yano et al., 1999). Also, PCD triggered by chemicals such as FB1 and camptothecin in tomato suspension cells are markedly inhibited by specific serine protease inhibitors (De Jong et al., 2000). A 40-kDa serine protease is implicated in tracheary element (TE) cell death and a soybean trypsin inhibitor can inhibit trypsin-triggered cell death mimicking PCD of TEs (Groover and Jones, 1999). Also, SKTI3 was found identical to the previously so-called soybean elicitation competency factor associated with wounding or HR (Park et al., 2001). These studies, together with our results, indicate a possible role for AtKT1 in modulating plant pathogen-related PCD. How AtKTI1 protein antagonizes pathogen-related host cell death and which proteinases are inhibited remain to be determined. Apart from a possible inhibition of so far uncharacterized caspase-like proteases, a more indirect role of AtKTI1 is possible. In mammalian cells, certain serine proteases are involved in the removal of IAP caspase inhibitors (Salvesen and Duckett, 2002; Jin et al., 2003; Yang et al., 2003). For example, the mitochondrial located serine protease HtrA2 is thought to play pro-apoptotic function via activating a caspase-dependent pathway (Li et al., 2002), but probably induces cell death also through extra-mitochondrial caspaseindependent pathways (Suzuki et al., 2001). The suggested

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METHODS Plant Growth and Pathogen Infection All Arabidopsis thaliana plants in this study are in Col-0 background and were grown in soil in a controlled-environment room at 22
C and 70% relative humidity, on a 12-h light/12-h dark cycle, with 50 lmol m2 sec
1 cool white fluorescent illumi-

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nation. Plants were 4 weeks old at the time of infection. Erwinia carotovora subsp. carotovora (Ecc) SCC1 (wild-type strain described in Pirhonen and Palva, 1988) was grown overnight in LB medium, cells were collected by centrifugation, washed, re-suspended in 0.9% NaCl and used to infect plants at a concentration of 105–106 cfu mL
1, as indicated in the figures. For Pseudomonas syringae pv. tomato (Pst) DC3000 avrB inoculations, bacteria were grown at 28
C for 18–24 h in King B liquid medium supplemented with 100 lg ml
1 rifampicin and 25 lg ml
1 kanamycin, re-suspended into 10 mM MgCl2 at a concentration of 2 3 105–5 3 106 (cfu mL
1), as indicated in the figures. Infiltrations were performed with a needle-less 1-ml syringe.

Insect Assays Larvae of Spodoptera liitoralis were from a laboratory colony reared on a bean-based diet, as described (Srivastava and Proksch, 1991). Freshly hatched larvae were grown on lettuce before the experiment. In a no-choice assay, three 2nd instar larvae per plant (15 plants in a closed mini greenhouse) were placed on 4-week-old Arabidopsis plants and their weight gain and survival rate were documented after 6 d. In a choice assay, four 3rd instar larvae were put in between two control and two AtKTI1 overexpression (line 8, 13, or 16) plants in a closed mini greenhouse and leaf consumption was estimated after 1–3 d.

Chemical Application, Wounding and CF Treatment All chemicals were purchased from Sigma-Aldrich. Ecc culture filtrate (CF) was prepared as described previously (Vidal et al., 1997). 1-aminocyclopropane-1-carboxylic acid (ACC) (100 lM), methyl jasmonate (MeJa; 100 lM in 0.1% (v/v) ethanol), methyl viologen (25 lM) and salicylic acid (SA; 5 mM) and CF were applied as 5 3 5 lL droplets on four fully expanded leaves for each plant, as described (Li et al., 2004). The stock solution of Fumonisin B1 (7 mM in methanol) was diluted with water to the indicated concentrations and infiltrations were performed with a needle-less 1-ml syringe. For wounding treatment, each leaf was pressed eight times with forceps. Trypan blue staining was performed as described by Bowling et al. (1997).

Bacterial Over-Expression, Purification and Assay of Recombinant AtKTI1 The full-length AtKTI1 gene was amplified by polymerase chain reaction (PCR) from a cDNA library of Arabidopsis plants treated with CF by using the forward primer (5#-CCATTGGCTATAATTATGACAAAAACTACC-3’) and the reverse primer (5#-AGTTTTCAAGAAAACAGAGAGCGGGTGTT-3’) and subsequently cloned into pCR2.1 to result in pS4C4. After sequence confirmation, the coding regions with and without the putative N-terminal signal sequence were sub-cloned into the BamHI and HindIII restriction sites of the pQE30 vector (Qiagen) with the following primer pairs: 5#-CGGGATCCACAAAAACTACCAAAACCATG-3’ and 5#-CCCAAGCTTGTCCTCTCACATAGTCTTG-3’ or 5#-AAGGATCCGCGGTTGTAGACATCGA3’ and 5#-CCCAAGCTTGTCCTCTCACATAGTCTTG-3#, respectively,

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location or association of AtKTI1 with mitochondria (Heazlewood et al., 2004) and its role in modulating FB-1 and avrBinduced cell death (Figures 6 and 7) indicates that AtKTI1 might be involved in regulating cell death, possibly due to inhibition of an HtrA2-like enzyme in plants. Future research should provide information on the interacting proteins of AtKTI1 in planta and how AtKTI1 is mechanistically modulating plant PCD. Surprisingly, overexpression of AtKTI1 increases susceptibility and RNAi silencing increases resistance to the virulent pathogen Ecc despite the growth impairments of silenced plants (Figures 4 and 5). This raises two questions: (1) what is the reason for the altered resistance? and (2) why is the gene induced by pathogens and by the defense hormone SA, if overexpression impairs the resistance of the plants? Treatment of Arabidopsis with the elicitor harpin of Ecc SCC1 leads to formation of HR-like lesions in Arabidopsis leaves, followed by enhanced resistance of the plants against subsequent infection with this pathogen (Kariola et al., 2003). HR in general is accompanied by an oxidative stress response and both reactive oxygen species (ROS) and HR, as such, have been implied as signals leading to enhanced resistance in subsequent infections (Heath, 2000). Also, lesion mimic mutants are often more resistant to various pathogens (Lorrain et al., 2003). In this respect, it seems reasonable to assume that the enhanced capacity of RNAi-AtKTI1silenced plants to develop PCD leads to elevated resistance. In accordance, reduction of PCD formation in AtKTI1 overexpression lines leads to increased susceptibility. The second intriguing question is why AtKTI1 is induced by pathogens or elicitors if reduced expression results in increased resistance. One possible explanation is that Ecc modulates the plant defense pathways by activating plant gene expression for its own benefit, resulting in reduced resistance to Ecc, as suggested earlier (Kariola et al., 2005). However, another important point to be considered is the late induction of AtKTI1 after pathogen treatment. AtKTI1 is not only induced late by both FB1 and Ecc elicitors, but also publicly available microarray data accessed via Genevestigator (Zimmermann et al., 2004) show a clear increase of AtKTI1 transcript only 24 h after Pst DC3000 treatment. This increase is earlier (after 6 h) and more pronounced when strains carrying the avrRpm1 avirulence gene were used for infection, which suggests a role in local containment of cell death after an initial HR. HR and PCD are fine-tuned processes in plants and clearly require also negative regulators to contain lesion development (Lam et al., 2001). After an initial HR and HR signaling, later containment of the HR might be crucial for optimizing the plant defense response and to minimize damage after pathogen attack.

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Ai = ½ðDA253c
DA253i Þ=DA253c  3 100%; where Ai represented inhibitory activity, DA253c the absorbance increase of control, and DA253i the absorbance increase in presence of recombinant AtKTI1 protein. As a positive control, soybean trypsin inhibitor (Sigma-Aldrich, catalog number: T9003) was used.

RNA Gel Blot Analysis Total RNA from plants was prepared by SDS/phenol/chloroform extraction and LiCl precipitation (Kingston, 1997). 10 lg RNA was denatured in formamide, separated by electrophoresis through formaldehyde agarose gels, and blotted onto a positively charged nylon membrane (Roche) via capillary transfer with 20 SSC (Sambrook et al., 1989). A plasmid containing a 326-bp cDNA fragment obtained from a subtracted cDNA library (Brader et al., 2001) and the plasmid pS4C4 containing a full-length cDNA fragment of AtKTI1 were used to synthesize DIG-labeled DNA or DIG-labeled RNA probes with T7 RNA polymerase (Promega) according to manufacture’s instruction (Roche).

Construction of AtKTI1 Overexpression and RNAi Vector and Plant Transformation To generate the AtKTI1 overexpression construct pCP60-S4C4, the PstI-HindIII fragment from pS4C4 was inserted in pBluescript II (SK+) to create pS4C4-1. The fragment obtained by XbaI-KpnI restriction digestion of pS4C4-1 was cloned to the pCP60 binary plasmid (Li et al., 2004) downstream of the cauliflower mosaic virus (CaMV) 35S promoter. For construction of the RNAi vector, the fragment obtained by BamHI-HindIII

restriction digestion of pR4C4-I was cloned into the pBluescript II (SK+) to create pSA4C4-1. An antisense-arm containing the restriction sites HindIII and KpnI obtained by PCR with primers 5#-GGGGTACCTATGACAAAAACTACC-3’ and 5#-CCCAAGCTTGAAGTACTGCTTCCTC-3’ was inserted in pSA4C4-I to give pSA4C4-II. The BamHI-KpnI fragment from pSA4C4-II was moved to pCP60 to result in the RNAi construct pCP60-SA4C4. The empty pCP60 vector and the generated constructs pCP60-S4C4 and pCP60-SA4C4 were transformed to Agrobacterium tumefaciens GV2260 and used for transformation of Arabidopsis Col-0 plants by floral dip, as described by Clough and Bent (1998). Transformants were selected according to their kanamycin resistance. Homozygous lines were selected by segregation ratio analysis and RNA-gel analysis of total leaf RNA using DIG-labeled RNA probe. For each construct, at least two representative lines carrying one single insert were used for subsequent studies.

Determination of Endogenous AtKTI1 Levels in Transgenic Arabidopsis Plants Mature leaves from 4-week-old overexpression lines and a vector control line were collected. Each sample contains 18 leaves from six individual plants. For each line, protein extracts from three samples were independently prepared as described by Leo et al. (1998) and protein concentrations were determined according to Bradford and adjusted to 1.5 lg ll
1 with the extraction buffer. The crude protein extracts were directly used to detect their trypsin inhibitory activity in the abovementioned BAEE reaction system, with the following modifications. Reaction mixtures contained 10 ll bovine pancreas trypsin solution, 60 ll phosphate buffer and 230 ll protein extracts or an equal volume of the extraction buffer instead of crude protein extracts as control. 100 ll incubated mixtures were added to cuvette containing 900 ll BAEE solution and incubation time was increased to 15 min.

Assays for Activity of Ecc Extracellular Proteases in CF and Inhibition of Proteolysis Ecc SCC1 was grown at 28
C in LB medium for 18 h and protein concentrations of CFs were estimated using the method of Bradford (1976). Azocasein tests were used to quantify the level of inhibition of bovine pancreas trypsin. 1-mL reaction mixtures containing 2% azocasein (Sigma), 750 ll CF, 200 ll 1 M Tris HCl buffer (pH 8.0), and either 50 ll of water or 10 mM EDTA (final concentration) or 15 lg His-tagged AtKTI1 were incubated at 30
C. After 0 or 180 min, the reaction was stopped. Protease activity is presented as the increase in A436 over 3 h, as described by Braun and Schmitz (1980).

FUNDING This study was supported by the Helsinki Graduate School in Biotechnology and Molecular Biology (J.L.), the Academy of Finland (Finnish Centre of Excellence Programme) and the Biocentrum Helsinki.

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resulting in pR4C4-I and pR4C4-II. pR4C4-I and pR4C4-II with confirmed sequences were individually transformed into E. coli strain M15 (pREP4). Recombinant AtKTI1 proteins were affinitypurified under native conditions as described in the manufacturer’s protocol (Qiagen). The size of fusion proteins with six His residues at the N-terminus of the recombinant AtKTI1 proteins was determined by 12% SDS-PAGE. Protein concentrations were determined as in Bradford (1976). Recombinant AtKTI1 protein activity was determined by measuring the change in A253 due to cleavage of the trypsin substrate Na-benzoyl-L-arginine ethyl ester (BAEE, Sigma-Aldrich), as previously described (Worthington Enzyme Manual, 1993), with some modifications. Briefly, reaction mixtures containing 10 ll bovine pancreas trypsin solution (0.5 lg ll
1, Sigma), 20 ll sodium phosphate buffer (0.5 M, pH 6.5) and 5 lg recombinant AtKTI1 in elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0) or an equal volume of elution buffer as control were adjusted to 100 ll with H2O and were incubated at room temperature for 30 min. 35-ll incubation mixtures were added to a cuvette containing 965 ll BAEE substrate (0.25 mM BAEE, 67 mM phosphate buffer, pH 7.0). Absorbance increase in A253 for 4 min was recorded. The inhibitory activity of recombinant AtKTI1 was calculated according to:

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ACKNOWLEDGMENTS We thank L. Miettinen and H. Mikkonen for excellent technical assistance and M. Montesano for discussion. We thank H. Greger and B. Brem of the University of Vienna (Austria) for providing us kindly with Spodoptera littoralis. No conflict of interest declared.

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