Arabidopsis vegetative storage protein is an anti-insect acid phosphatase

Abstract

Indirect evidence previously suggested that Arabidopsis (Arabidopsis thaliana) vegetative storage protein (VSP) could play a role in defense against herbivorous insects. To test this hypothesis, other AtVSP-like sequences in Arabidopsis were identified through a Basic Local Alignment Search Tool search, and their transcriptional profiles were investigated. In response to methyl jasmonate application or phosphate starvation, AtVSP and AtVSP-like genes exhibited differential expression patterns, suggesting distinct roles played by each member. Arabidopsis VSP2 (AtVSP2), a gene induced by wounding, methyl jasmonate, insect feeding, and phosphate deprivation, was selected for bacterial expression and functional characterization. The recombinant protein exhibited a divalent cation-dependent phosphatase activity in the acid pH range. When incorporated into the diets of three coleopteran and dipteran insects that have acidic gut lumen, recombinant AtVSP2 significantly delayed development of the insects and increased their mortality. To further determine the biochemical basis of the anti-insect activity of the protein, the nucleophilic aspartic acid-119 residue at the conserved DXDXT signature motif was substituted by glutamic acid via site-directed mutagenesis. This single-amino acid alteration did not compromise the protein's secondary or tertiary structure, but resulted in complete loss of its acid phosphatase activity as well as its anti-insect activity. Collectively, we conclude that AtVSP2 is an anti-insect protein and that its defense function is correlated with its acid phosphatase activity.

Figure 1.

Differential mRNA expression of AtVSP-like genes. Total RNA was extracted from untreated Arabidopsis seedlings, as well as seedlings treated with MeJA and phosphate starvation, respectively. Gene-specific primers were used in real-time RT-PCR. At4g25150 was excluded from the graph as its primers failed to result in PCR product. Relative transcript abundance was calculated according to Salzman et al. (2005). Transcript levels in control seedlings were arbitrarily set at 1. Error bars indicate sd of the mean induction fold (n = 2).

Figure 2.

Production of recombinant proteins in E. coli. A, Constructs for expression of AtVSP2, Nus-AtVSP2, and Nus-AtVSP2(D119E). While AtVSP2 expressed via pET28a vector was soluble, mutated AtVSP2(D119E) could only be detected in the insoluble fraction. Thus, both cDNAs were cloned into the pET44a vector and proteins were expressed as fusion protein with Nus. B, SDS-PAGE analysis of expression and purification of recombinant proteins. The supernatant of bacterial cell extract and Ni²⁺ chelate affinity-purified recombinant proteins were subjected to SDS-PAGE and stained with Coomassie Blue. C, Site-specific mutation did not disrupt AtVSP2 structure. Shown are far-UV circular dichroism spectra of Nus-AtVSP2, Nus-AtVSP2(D119E), and Nus.

Figure 3.

AtVSP2 is a pH- and divalent cation-dependent acid phosphatase. A, Reactions were buffered by 50 mm citric acetate (pH 2.5–4.0), sodium acetate (pH 3.5–5.5), and Tris-acetate buffers (pH 5.5–7.5). B, Divalent cations Mg²⁺, Co²⁺, Zn²⁺, Ca²⁺, or Mn²⁺ at levels specified were evaluated for effects on phosphatase activity of AtVSP2 in 50 mm sodium acetate (pH 4.5). pNPP at 10 mm was used as substrate and incubated with purified recombinant proteins for 30 min at 37°C. Enzymatic activity was measured by release of p-nitrophenol according to a standard curve.

Figure 4.

AtVSP2 is an anti-insect protein. A, Artificial seeds containing various doses of AtVSPs were infested with viable cowpea bruchid eggs. Within-seed developmental time was recorded and analyzed by one-way ANOVA. Fisher's protected lsd test (P = 0.05) was used for mean separation. Error bars indicate standard error. Means followed by the same letter are not significantly different at P = 0.05. ∞ indicates 100% mortality under a specified dose. B, Neonate Drosophila melanogaster were reared on AtVSP2 and AtVSP2-free diets. The development time from neonate larvae to pupae were recorded. The effect of AtVSP was analyzed by one-way ANOVA and Bonferroni multiple means comparison tests. Error bars indicated standard error. Means followed by the same letter were not significantly different at P = 0.05. ∞ indicates 100% mortality under a specified dose. C, Newly hatched southern corn rootworm larvae were reared in a diet containing AtVSP2 at doses indicated. Survival data were recorded as insects developed and were log₁₀ transformed. The regression analysis was used to calculate regression equations and for each treatment; control, y = −0.01541x + 2.01737 (P < 0.0001); 0.05% AtVSP2, y = −0.02963x + 2.04037 (P < 0.0001); 0.1% AtVSP2, y = −0.03128x + 2.02984 (P < 0.0001); 0.2% AtVSP2, y = −0.01877x + 1.99290 (P < 0.0001); 0.3% AtVSP2, y = −0.06101x + 1.98955 (P < 0.0001); 0.4% AtVSP2, y = −0.06072x + 1.84539 (P < 0.0001). Linear regression R² values were indicated in the graph. Comparative analysis was performed between each treatment. A probability of 0.05 was used to determine statistical significance. s, Significant; n.s., nonsignificant.

Figure 5.

Anti-insect activity of AtVSP2 is correlated with its acid phosphatase activity. The neonate Drosophila larvae were reared on a diet containing Nus-AtVSP2, Nus-AtVSP2(D119E), and Nus recombinant proteins. Surviving larvae were recorded daily until pupation, and the development time from neonate to pupae was calculated. The effect of Nus-AtVSP2, Nus-AtVSP2(D119E), and Nus on the survival (A) and development time (B) was analyzed by one-way ANOVA and Bonferroni multiple means comparison tests. Error bars indicate standard error. Means followed by the same letter are not significantly different at P = 0.05. CK, Control.