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ATP hydrolyzing salivary enzymes of caterpillars suppress plant defenses - PubMed

ATP hydrolyzing salivary enzymes of caterpillars suppress plant defenses

Shuang Wu et al. PLoS One. 2012.

Abstract

The oral secretions of herbivores are important recognition cues that can be used by plants to mediate induced defenses. In this study, a degradation of adenosine-5'-triphosphate (ATP) in tomato leaves was detected after treatment with Helicoverpa zea saliva. Correspondingly, a high level of ATPase activity in saliva was detected and three ATP hydrolyzing enzymes: apyrase, ATP synthase and ATPase 13A1 were identified in salivary glands. To determine the functions of these proteins in mediating defenses, they were cloned from H. zea and expressed in Escherichia coli. By applying the purified expressed apyrase, ATP synthase or ATPase 13A1 to wounded tomato leaves, it was determined that these ATP hydrolyzing enzymes suppressed the defensive genes regulated by the jasmonic acid and ethylene pathways in tomato plant. Suppression of glandular trichome production was also observed after treatment. Blood-feeding arthropods employ 5'-nucleotidase family of apyrases to circumvent host responses and the H. zea apyrase, is also a member of this family. The comparatively high degree of sequence similarity of the H. zea salivary apyrase with mosquito apyrases suggests a broader evolutionary role for salivary apyrases than previously envisioned.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Activities and expressions of ATP hydrolyzing enzymes in H. zea tissues.

A, in-gel ATP hydrolysis activity test among different tissues of H. zea. Lane 1–5, total crude homogenates from labial glands, fat body, hemolymph, Malpighian tubules and midgut, respectively. B, the relative expression levels of three target genes among different tissues of H. zea. Actin was used as the internal reference. Values are expressed as mean ± SE (n = 3). Significance was tested for each gene separately. Different letters above each bar indicate statistical difference determined by ANOVA analysis followed by the Duncan's Multiple Range Test (P<0.05).

Figure 2
Figure 2. The molecular evolutionary trees of target proteins with amino acid sequences from other insect species.

A, B, C represent the evolutionary trees for H. zea apyrase, ATP synthase and ATPase 13A1, respectively. The corresponding GeneBank accession numbers for H. zea apyrase, ATP synthase and ATPase 13A1 are ADK90114, ADJ95799 and ADN88179, respectively.

Figure 3
Figure 3. SDS-PAGE analyses of protein expression and purification.

A, expression time-courses of the fusion proteins in E. coli. Lane a, control 1, total cellular proteins from E. coli Rosetta 2 (DE3) after being induced by 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 2.5 h. Lane b, control 2, expression of empty pET-43.1b(+) vector protein after being induced by 1.0 mM IPTG for 2.5 h. Lane 1–5, 6–10 and 11–15 show the expressions of fusion apyrase, ATP synthase and ATPase 13A1 after being induced by 1.0 mM IPTG for 2, 3, 4, 5 and 6 h, respectively. B, SDS-PAGE analysis of purified apyrase (lane 1), ATP synthase (lane 2) and ATPase 13A1 (lane 3) from E. coli. Lane M, protein molecular weight markers. C, native PAGE analysis of purified apyrase (lane 1), ATP synthase (lane 2) and ATPase 13A1 (lane 3). Arrows represent the locations of target proteins.

Figure 4
Figure 4. The relative expression levels of defense genes among tomato leaves after different treatments.

Total RNAs were extracted from tomato leaves after 24 h of different treatments. Different quantities of proteins with the same total ATP hydrolysis activity of 74.3 pmol·L−1·min−1 were indicated. Ubiquitin was used as the internal reference. The expression level of each gene was normalized to the level in treatment with purified pET-43.1b(+) vector protein, which contain the impure protein only. Values are expressed as mean ± SE (n = 3). Significance was tested for each gene separately. Different letters above each bar indicate statistical difference determined by ANOVA analysis followed by the Duncan's Multiple Range Test (P<0.05).

Figure 5
Figure 5. Trichome productions in tomato leaves after 10 days of different treatments.

Values are expressed as mean ± SE (n = 5). Asterisks indicate statistical difference determined by ANOVA analysis followed by the Duncan's Multiple Range Test (P<0.05).

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