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. 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. 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.

The SNAKIN/GASA family comprises antimicrobial peptides with proven activity against bacteria and fungi, making them promising candidates for improving disease resistance in citrus rootstocks. In sixty-seven new GASA variants from a citrus germplasm collection, the presence of the characteristic 12-cysteine domain was confirmed and were classified into three subfamilies. The absolute expression levels of ten representative genes were analyzed in floral tissues, young leaves, and mature leaves from five citrus accessions with contrasting susceptibility to Xanthomonas citri. Expression profiling revealed tissue-specific patterns, with higher transcript abundance in juvenile and floral tissues of tolerant accessions. Meta-analysis of HLB-related RNA-seq datasets revealed the upregulation of specific GASA genes. Three genes from Poncirus trifoliata—PtGASA6, PtGASA8, and PtGASA10—were then selected for functional validation in Nicotiana benthamiana. Transient overexpression of PtGASA6 and PtGASA10 significantly reduced disease symptoms caused by Pseudomonas syringae and heightened the hypersensitive response to X. citri, whereas PtGASA8 showed no detectable effect. Notably, PtGASA6 enhanced the hypersensitive response by 30% more than PtGASA10, while PtGASA10 delayed necrosis by 40% more than PtGASA6, indicating distinct antimicrobial mechanisms. Together, these results identify PtGASA6 and PtGASA10 as strong candidates for breeding and biotechnological strategies aimed at improving broad-spectrum bacterial disease resistance in citrus.

Potato virus Y (PVY) and potato leafroll virus (PLRV) are the most damaging viruses for potato production worldwide. Mixed infections not only result in additive detrimental effects on plant growth and tuber yield but also complicate the development of durable and broad-spectrum viral resistance. Heterologous protection against PVY can be achieved through the expression of the coat protein (CP) of lettuce mosaic virus (LMV) (CPLMV), conferring resistance via a capsid protein-mediated mechanism. On the other hand, we have previously demonstrated that transgenic lines expressing the PLRV ORF2 (RepPLRV) exhibit resistance to different PLRV isolates. In this study, potato transgenic lines of cv. Kennebec expressing CPLMV and RepPLRV were developed to confer dual virus resistance. Transgenic and non-transgenic control plants were molecularly and phenotypically characterized in greenhouse and field conditions. Across multiple growing seasons, two selected transgenic lines consistently displayed robust resistance to both major viruses, without exhibiting yield penalties or noticeable phenotypic alterations. These results constitute a significant advancement, demonstrating that dual resistance to PVY and PLRV can be achieved while preserving the original agronomic performance of the cultivar. This breakthrough not only contributes to long-term crop productivity but also provides a more sustainable strategy for managing viral diseases in potato production.

The SNAKIN/GASA family comprises antimicrobial peptides with proven activity against phytopathogens that may play a significant role in citrus rootstock breeding. We identified 67 new curated GASA variants present in a germplasm collection. They were checked for the diagnostic 12-cysteine-domain and classified into three GASA subfamilies. Absolute expression of ten representative GASA genes was analyzed in floral, young leaf and mature leaf tissues from five accessions with contrasting levels of disease tolerance against Xanthomonas citri. Expression profiling revealed tissue-specific patterns, with higher transcript abundance in juvenile and floral tissues of tolerant accessions. Meta‑analysis of HLB‑related RNA‑seq datasets revealed the upregulation of specific GASA genes. Three candidate genes (PtGASA6, PtGASA8, and PtGASA10) derived from Poncirus trifoliata (a rootstock cultivar conferring disease tolerance), were selected and functionally assessed via transient overexpression in Nicotiana benthamiana. Overexpression of PtGASA6 and PtGASA10 significantly reduced disease symptoms caused by Pseudomonas syringae and enhanced hypersensitive response to Xanthomonas citri, whereas PtGASA8 showed no effect. Specific time-course dynamics and structural predictions suggest distinct antimicrobial mechanisms among these 2 genes. These findings provide new genetic targets for breeding and biotechnological strategies aimed at improving broad-spectrum bacterial disease resistance in citrus.

Potato virus Y (PVY) and potato leafroll virus (PLRV) are the most damaging viruses for potato production worldwide. Mixed infections not only result in additive detrimental ef-fects on plant growth and tuber yield but also complicate the development of durable and broad-spectrum viral resistance. Heterologous protection against PVY can be achieved through the expression of the CP protein of lettuce mosaic virus (CPLMV) conferring re-sistance via a capsid protein-mediated mechanism. On the other hand, we have previ-ously demonstrated that transgenic lines expressing the PLRV ORF2 (RepPLRV) exhibit resistance to different PLRV isolates. In this study, potato transgenic lines of cv. Kennebec expressing CPLMV and RepPLRV were developed to confer dual virus resistance. Trans-genic and non-transgenic control plants were molecularly and phenotypically character-ized in greenhouse and field conditions. Across multiple growing seasons, two selected transgenic lines consistently display robust resistance to both mayor viruses, without ex-hibiting yield penalties or noticeable phenotypic alterations. These results constitute a significant advancement, demonstrating that dual resistance to PVY and PLRV can be achieved while preserving the original agronomic performance of the cultivar. This breakthrough not only contributes to long-term crop productivity but also provides a more sustainable strategy for managing viral diseases in potato production.