Ripe fruits of pepper (Capsicum annuum) are resistant to the anthracnose fungus, Colletotrichum gloeosporioides, whereas unripe-mature fruits are susceptible. A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between the ripe fruit of pepper and C. gloeosporioides was previously cloned. Deduced amino acid sequence of PepEST cDNA showed homology to both esterases and lipases, and contained -HGGGF- and -GXSXG- motifs and a catalytic triad. Inhibition of PepEST activity by a specific inhibitor of serine hydrolase demonstrated that a serine residue is critical for the enzyme activity. Expression of PepEST gene was fruit-specific in response to C. gloeosporioides inoculation, and up-regulated by wounding or jasmonic acid treatment during ripening. PepEST mRNA and protein was differentially accumulated in ripe vs. unripe fruit from 24 h after inoculation when C. gloeosporioides is invading into fruits. Immunochemical examination revealed that PepEST accumulation was localized in epidermal and cortical cell layers in infected ripe fruit, but rarely even in epidermal cells in infected unripe one. Over-expression of PepEST in transgenic Arabidopsis plants caused restriction of Alternaria brassicicola colonization by inhibition of spore production, resulting in enhanced resistance against A.brassicicola. These results suggest that PepEST is involved in the resistance of ripe fruit against C.gloeosporioides infection.

A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between pepper (Capsicum annuum) and the anthracnose fungus Colletotrichum gloeosporioides has been previously cloned. Glutathione-S-transferase-tagged recombinant PepEST protein expressed in Escherichia coli showed substrate specificity for p-nitrophenyl esters. Inoculation of compatible unripe pepper fruits with C. gloeosporioides spores amended with the recombinant protein did not cause anthracnose symptoms on the fruit. The recombinant protein has no fungicidal activity, but it significantly inhibits appressorium formation of the anthracnose fungus in a dose-dependent manner. An esterase from porcine liver also inhibited appressorium formation, and the recombinant protein inhibited appressorium formation in the rice blast fungus, Magnaporthe grisea. Inhibition of appressorium formation in M. grisea by the recombinant protein was reversible by treatment with cyclic AMP (cAMP) or 1,16-hexadecanediol. The results suggest that the recombinant protein regulates appressorium formation by modulating the cAMP-dependent signaling pathway in this fungus. Taken together, the PepEST esterase activity can inhibit appressorium formation of C. gloeosporioides, which may result in protection of the unripe fruit against the fungus.

The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripe-mature-green fruit. Using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in the compatible interaction, and then remains elevated in the incompatible interaction. In the compatible interaction, the expression of PepCYP is transient. The induction of PepCYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process.

The aim of this study was to establish a stable transformation method for hot pepper using the hygromycin phosphotransferase (hpt)/hygromycin selection strategy. Explants from aseptic pepper seedlings were inoculated with Agrobacterium tumefaciens carrying pCAMBIA1301. A number of calli were developed on the medium containing hygromycin to discriminate the induction of “false-positive buds,” and then shoots were successfully regenerated from the hygromycin-resistant calli. Southern and Northern hybridization analysis indicated that the hpt gene was integrated and expressed in the transgenic pepper plants (T0) and transmitted to the progeny (T1) without genetic modification. Most T1 progenies derived from self-pollination revealed a 3:1 segregation ratio for hygromycin resistance, indicating that one copy of the T-DNA was integrated into the respective transgenic lines. Both uidA and hpt genes were stably expressed in the T1 generation and coinherited in the progenies. Finally, homozygous progenies were identified in the T1 generation of the transgenic peppers, and the homozygous state was maintained in all progenies tested (T2). The results show the reliability and stability of the hpt/hygromycin selection protocol for pepper transformation.

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