Abstract
Functional characterization of a defensin, J1-1, was conducted to evaluate its biotechnological potentiality in transgenic pepper plants against the causal agent of anthracnose disease, Colletotrichum gloeosporioides. To determine antifungal activity, J1-1 recombinant protein was generated and tested for the activity against C. gloeosporioides, resulting in 50% inhibition of fungal growth at a protein concentration of 0.1 mg·mL−1. To develop transgenic pepper plants resistant to anthracnose disease, J1-1 cDNA under the control of 35S promoter was introduced into pepper via Agrobacterium-mediated genetic transformation method. Southern and Northern blot analyses confirmed that a single copy of the transgene in selected transgenic plants was normally expressed and also stably transmitted to subsequent generations. The insertion of T-DNA was further analyzed in three independent homozygous lines using inverse PCR, and confirmed the integration of transgene in non-coding region of genomic DNA. Immunoblot results showed that the level of J1-1 proteins, which was not normally accumulated in unripe fruits, accumulated high in transgenic plants but appeared to differ among transgenic lines. Moreover, the expression of jasmonic acid-biosynthetic genes and pathogenesis-related genes were up-regulated in the transgenic lines, which is co-related with the resistance of J1-1 transgenic plants to anthracnose disease. Consequently, the constitutive expression of J1-1 in transgenic pepper plants provided strong resistance to the anthracnose fungus that was associated with highly reduced lesion formation and fungal colonization. These results implied the significance of the antifungal protein, J1-1, as a useful agronomic trait to control fungal disease.
Highlights
Higher plants have innate defense systems to protect themselves against biotic stresses [1,2,3]
The induction of the J1-1 was monitored in the pepper fruits infected with the fruitspecific fungal pathogen, C. gloeosporioides
The level of the J1-1 protein was increased at 3 hours after infection (HAI) in the ripe fruit and was maintained during the period of observation
Summary
Higher plants have innate defense systems to protect themselves against biotic stresses [1,2,3]. A range of protective molecules, including antimicrobial proteins, are synthesized in the tissues invaded by pathogens or accumulated during normal growth [4,5,6]. Defensins that belong to antimicrobial peptide superfamily are a large class of small peptides occurring in various living organisms, ranging from microorganisms to plants and mammals [7,8]. On the basis of structural and functional similarity with insect defensin, plant antimicrobial peptide called c–thionin in wheat and barley grains was renamed as defensin [9]. Plant defensins are composed of three anti-parallel b-strands and one ahelix with a characteristic three-dimensional folding stabilized by four disulfide bonds [10]. The cysteine-stabilized a-helix/b-sheet (CSab) motif confers great stability on the peptide to maintain the functional activity [11]
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