Enzyme activity and protein levels of squalene synthase in unripe and ripe pepper fruits after inoculation by <i>C. gloeosporioides</i>.

<p><b>A</b>, Enzyme activity assay of squalene synthase (SS). The SS enzyme and [³H]-FPP were incubated for 20 min at 30°C. Error bars indicate the standard deviation from three independent measurements. Means with different letters indicate significant difference at <i>P<0.05</i>, using LSD and DMRT. <b>B</b>, Immunoblot analysis showing the protein levels of squalene synthase. The time after fungal inoculation is indicated in hours on top of each lane. Microsomal proteins (10 µg/lane) were subjected to detect SS protein. β-tubulin and SDS-PAGE of Ponceau S staining were included as loading controls.</p

Developmentally Regulated Sesquiterpene Production Confers Resistance to <i>Colletotrichum gloeosporioides</i> in Ripe Pepper Fruits

<div><p>Sesquiterpenoid capsidiol, exhibiting antifungal activity against pathogenic fungus, is accumulated in infected ripe pepper fruits. In this study, we found a negative relation between the capsidiol level and lesion size in fruits infected with <i>Colletotrichum gloeosporioides</i>, depending on the stage of ripening. To understand the developmental regulation of capsidiol biosynthesis, fungal-induced gene expressions in the isoprenoid biosynthetic pathways were examined in unripe and ripe pepper fruits. The sterol biosynthetic pathway was almost shut down in healthy ripe fruits, showing very low expression of hydroxymethyl glutaryl CoA reductase (<i>HMGR</i>) and squalene synthase (<i>SS</i>) genes. In contrast, genes in the carotenoid pathway were highly expressed in ripe fruits. In the sesquiterpene pathway, 5-epi-aristolochene synthase (<i>EAS</i>), belonging to a sesquiterpene cyclase (<i>STC</i>) family, was significantly induced in the ripe fruits upon fungal infection. Immunoblot and enzyme activity analyses showed that the STCs were induced both in the infected unripe and ripe fruits, while capsidiol was synthesized discriminatively in the ripe fruits, implying diverse enzymatic specificity of multiple STCs. Thereby, to divert sterol biosynthesis into sesquiterpene production, infected fruits were pretreated with an SS inhibitor, zaragozic acid (ZA), resulting in increased levels of capsidiol by more than 2-fold in the ripe fruits, with concurrent reduction of phytosterols. Taken together, the present results suggest that the enhanced expression and activity of EAS in the ripe fruits play an important role in capsidiol production, contributing to the incompatibility between the anthracnose fungus and the ripe pepper fruits.</p></div

Inhibitory effect of capsidiol on the growth of <i>C. gleosporioides</i>.

<p><b>A</b>, Anti-fungal activity assay of capsidiol with filter paper discs. Discs were impregnated with 10 µL of 0, 0.1 and 1 mM of capsidiol and placed on the agar media 5 mm apart from the advancing edge of the mycelium. Discs impregnated with 10 µL of 1 mM squalene (Sq) or farnesol (F) were also included as controls. <b>B</b>, Evaluation of spore germination and appressorium formation by capsidiol treatment. Spores were amended on cover glasses with 10 µL of capsidiol dissolved in sterile water at concentration of 0, 0.02, 0.2 and 1 mM, then incubated for 24 h. Bar = 10 µm. <b>C</b>, Protective activity of capsidiol against anthracnose disease on pepper fruits. 10 µL of 0, 0.02, 0.2 and 1 mM capsidiol was amended with spores of <i>C. gloeosporioides</i> on the unripe fruits, and lesion development was observed 9 days after inoculation.</p

Fungal-induced expression of metabolic pathway genes for phytosterol, sesquiterpene and carotenoid biosyntheses in pepper fruits after inoculation by <i>C. gloeosporioides</i>.

<p><b>A</b>, Metabolic pathways for phytosterol, sesquiterpene and carotenoid biosyntheses. Genes encoding the enzymes used in the present study are indicated in italics. <i>HMGR</i>, 3-hydroxy-3-methyl glutaryl CoA reductase; <i>IPI</i>, isopentenyl diphosphate isomerase; <i>FPS</i>, farnesyl diphosphate synthase; <i>SS</i>, squalene synthase; <i>GGPS</i>, geranylgeranyl diphosphate synthase; <i>CCS</i>, capsanthin capsorubin synthase; <i>EAS</i>, 5-epi-aristolochene synthase (i.e. a sesquiterpene cyclase (<i>STC</i>)); <i>EAH</i>, 5-epi-aristolochene hydroxylase. ZA (zaragozic acid) is an inhibitor of squalene synthase. <b>B</b>, Relative expression of genes involved in phytosterol, sesquiterpene and carotenoid biosynthesis. Total RNA (10 µg/lane) was used for hybridization with ³²P-labelled cDNA of the indicated gene. The membranes were rehybridized with rRNA to ensure equal loading.</p

Development of anthracnose symptoms in infected pepper fruits and antifungal activity of capsidiol.

<p><b>A</b>, Representative anthracnose symptoms on unripe green and ripe red fruits 9 days after infection. <b>B</b>–<b>C</b>, Measurements of lesion diameters (B) and capsidiol levels (C) in the unripe and ripe fruits infected with <i>C. gloeosporioides</i>. Statistically significant changes between unripe and ripe fruits are indicated by ** and ***, indicating <i>P</i><0.01 and 0.001, respectively, using two-way ANOVA. At least 20 fruits were counted per replicate. Each value represents the mean ± SD of four replicates.</p

Effects of ZA pretreatment on fungal-induced accumulation of sterols and capsidiol in the unripe and ripe pepper fruits.

<p>Squalene (<b>A</b>), phytosterols (<b>B</b>) and capsidiol (<b>C</b>) levels in the infected unripe and ripe pepper fruits, with or without pretreatment of 10 µM ZA, were determined by GC analyses. Unripe/FI and Ripe/FI, unripe or ripe fruits with fungal infection; Unripe/FI+ZA and Ripe/FI+ZA, fungal-infected unripe or ripe fruits with ZA pretreatment. The data represent the mean values ± SD from three independent experiments. Significance test was performed by two-way ANOVA in comparison between ZA pretreated and non-treated infected fruits (*, **, and *** indicating <i>P</i><0.05, 0.01, and 0.001, respectively).</p

Fungal-induced activity and protein accumulation of 5-epi-aristolochene synthase in pepper fruits after inoculation by <i>C. gloeosporioides</i>.

<p><b>A</b>, Enzyme activity assay of 5-epi-aristolochene synthase. The EAS/STC activity was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109453#s2" target="_blank">Materials and Methods</a>. Error bars indicate the standard deviation from three independent measurements. Means with different letters indicate significant difference at <i>P<0.05</i>, using one way ANOVA and DMRT between control and fungal infected fruits. <b>B</b>, Immunoblot analysis showing the protein levels of EAS/STC. Soluble proteins (10 µg/lane) were subjected to detect EAS/STC proteins. β-tubulin and SDS-PAGE of Ponceau S staining were included as loading controls.</p