ABSTRACT
Snakin-1 is a member of the Solanum tuberosum Snakin/GASA family. We previously demonstrated that Snakin-1 is involved in plant defense to pathogens as well as in plant growth and development, but its mechanism of action has not been completely elucidated yet. Here, we showed that leaves of Snakin-1 silenced potato transgenic plants exhibited increased levels of reactive oxygen species and significantly reduced content of ascorbic acid. Furthermore, Snakin-1 silencing enhanced salicylic acid content in accordance with an increased expression of SA-inducible PRs genes. Interestingly, gibberellic acid levels were also enhanced and transcriptome analysis revealed that a large number of genes related to sterol biosynthesis were downregulated in these silenced lines. Moreover, we demonstrated that Snakin-1 directly interacts with StDIM/DWF1, an enzyme involved in plant sterols biosynthesis. Additionally, the analysis of the expression pattern of PStSN1::GUS in potato showed that Snakin-1 is present mainly in young tissues associated with active growth and cell division zones. Our comprehensive analysis of Snakin-1 silenced lines demonstrated for the first time in potato that Snakin-1 plays a role in redox balance and participates in a complex crosstalk among different hormones.
Subject(s)
Plant Growth Regulators , Plant Leaves , Plant Proteins , Plants, Genetically Modified , Solanum tuberosum , Phytosterols/biosynthesis , Phytosterols/genetics , Plant Growth Regulators/genetics , Plant Growth Regulators/metabolism , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/biosynthesis , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Solanum tuberosum/genetics , Solanum tuberosum/metabolismABSTRACT
BACKGROUND: Snakin-1 (StSN1) is a broad-spectrum antimicrobial cysteine-rich peptide isolated from Solanum tuberosum. Its biotechnological potential has been already recognized since it exhibits in vivo antifungal and antibacterial activity. Most attempts to produce StSN1, or homologous peptides, in a soluble native state using bacterial, yeast or synthetic expression systems have presented production bottlenecks such as insolubility, misfolding or low yields. RESULTS: In this work, we successfully expressed a recombinant StSN1 (rSN1) in Spodoptera frugiperda (Sf9) insect cells by optimizing several of the parameters for its expression in the baculovirus expression system. The recombinant peptide lacking its putative signal peptide was soluble and was present in the nuclear fraction of infected Sf9 cells. An optimized purification procedure allowed the production of rSN1 that was used for immunization of mice, which gave rise to polyclonal antibodies that detect the native protein in tissue extracts of both agroinfiltrated plants and stable transgenic lines. Our results demonstrated that this system circumvents all the difficulties associated with recombinant antimicrobial peptides expression in other heterologous systems. CONCLUSIONS: The present study is the first report of a successful protocol to produce a soluble Snakin/GASA peptide in baculovirus-infected insect cells. Our work demonstrates that the nuclear localization of rSN1 in insect cells can be exploited for its large-scale production and subsequent generation of specific anti-rSN1 antibodies. We suggest the use of the baculovirus system for high-level expression of Snakin/GASA peptides, for biological assays, structural and functional analysis and antibody production, as an important step to both elucidate their accurate physiological role and to deepen the study of their biotechnological uses.
