Graduate student University of Toledo Toledo, Ohio
Body of Abstract: Silicon (Si) mitigates biotic stress in plants. However, the molecular mechanism(s) by which it does so remain(s) unclear. Microarray analysis identified a Nicotiana tabacum Si-induced gene that closely resembled Defensins. Defensins are cysteine-rich polypeptides that play an important role in innate immunity for both animals and plants. Further analysis of the putative N. tabacum defensin showed it to be part of a small gene family consisting of 14 members. Members of this gene family shared >76% amino acid sequence identity. Furthermore, all members contained 6 or more histidines, indicating that these proteins are part of the Solanaceous histidine-rich defensins (HRDs) an important subgroup of the Defensin superfamily. These proteins were named NtHRD1s (the first histidine-rich defensin gene family from N. tabacum) and divided into groups based on sequence identity. Nearly all NtHRD1s showed Si-induction in whole roots although some were more highly induced in upper and others in lower roots. Most showed Si-induction in upper leaves. To elucidate the possible functions of the NtHRD1s, their intracellular localization was examined. NtHRD1s were tagged with fluorescent proteins, delivered into Nicotiana benthamiana leaf cells by agroinfiltration and visualized by confocal microscopy. NtHRD1s were localized to the cell surface. Two of the NtHRD1 proteins were also present within the nucleus. While the significance of nuclear localization is unclear, the cell surface is a useful place for NtHRD1s to repel pathogen invasion. To examine their possible antimicrobial effects, N. benthamiana leaves were agroinfiltrated with constructs expressing each individual NtHRD1 gene. Two days after infiltration, the leaves were individually inoculated with plant fungal pathogens: Sclerotinia sclerotiorum or Botrytis cinerea and disease progress was monitored. Four of the NtHRD1s provided protection against S. sclerotiorum and all six, against B. cinerea. Taken together, these data could possibly explain some of the antimicrobial activities attributed to Si.