Browsing by Author "Sathoff, Andrew E"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Antimicrobial Activity of Brassica rapa Nectar Lipid Transfer Protein(2017) Sathoff, Andrew E; Samac, Deborah A; Holl, Catherine; Schmidt, Tony; Carter, ClayAntimicrobial peptides (AMPs) provide an ancient, innate immunity conserved in all multicellular organisms. In plants, there are several large families of AMPs defined by sequence similarity. The nonspecific lipid transfer protein (LTP) family is defined by a conserved signature of eight cysteines and has a compact structure with a lipid-binding hydrophobic cavity. The antimicrobial activity of LTPs varies greatly among plant species. An LTP from Brassica rapa (variety R-o-18) nectar was tested for antimicrobial activity. In a 96-well microplate, each well contained half strength potato dextrose broth, approximately 2000 spores, and concentrations of LTP peptide up to 300 μg/mL in a total volume of 100 μL. After 48 hours of incubation at 25 C in the dark, absorbance of the wells was measured at 595 nm on a microplate reader to quantify the inhibition of fungal growth. The amount of LTP needed to inhibit growth of pathogen strains by 50% (IC50) was calculated. This Brassica LTP was most effective against Trichoderma and Bipolaris oryzae with IC50 values of 0.78 μM and 1.71 μM, respectively. Additionally, both Colletotrichum trifolii and Alternaria solani had IC50 values of less than 4.0 μM. The activity of this Brassica LTP at such low biological values indicates that it is a potent defense protein. These results suggest that transgenic expression of antimicrobial LTPs has the potential to lead to improved broad-spectrum disease resistanceItem Plant defensins inhibit growth of pathogens in the alfalfa crown rot disease complex(2016) Sathoff, Andrew E; Samac, Deborah AAlfalfa crown rot is a disease complex that severely limits alfalfa stand density and productivity in all alfalfa-producing areas. Currently, there are no viable methods of control. Plant defensins are small cationic antimicrobial peptides with a conserved signature of cysteines. Defensins have a γ-core motif, a cluster of positively charged residues, which is essential for antimicrobial activity. The γ-core motifs of five synthetic defensins were tested for antimicrobial activity against the pathogens in the alfalfa crown rot disease complex. In a 96-well microplate, each well contained half strength potato dextrose broth, approximately 2000 spores, and concentrations of defensin peptide up to 30 μg/mL in a total volume of 100 μL. After 48 hours of incubation at 25 C in the dark, absorbance of the wells was measured at 595 nm on a microplate reader to quantify the inhibition of fungal growth. The amount of defensin needed to inhibit growth of pathogen strains by 50% (EC50) was calculated. The γ-core motif of MtDef4 was shown to be the most effective peptide with EC50 values of 5.3 μM against Phoma medicaginis and 6.9 μM against Fusarium oxysporum f.sp. medicaginis. In addition, MtDef4 had activity against Pseudomonas syringae pv. syringae and Xanthomonas alfalfae subsp. alfalfae but not the oomycete Aphanomyces euteiches in in vitro assays. These results indicate that transgenic expression of plant defensins in alfalfa has the potential to lead to improved crown rot resistance.Item Transgenic expression of Medicago truncatula PR10 and PR5 promoters in alfalfa shows pathogen induced up-regulation of transgene expression(2016) Sathoff, Andrew E; Samac, Deborah AGenetic modification of alfalfa for introduction of novel traits requires promoters for controlling gene expression. Promoters that are constitutively activated for expression of genes that enhance disease resistance pose a great energy load on the plant and exert a strong selective pressure on the pathogens. Promoters that are induced upon pathogen invasion are needed for engineering plants with disease resistance. Medicago truncatula promoter regions of pathogenesis-related (PR) genes, PR5 and PR10, were identified as being highly up-regulated during the initial stages of infection by root and foliar pathogens. These promoters were PCR amplified and cloned into plant transformation vectors ahead of the β-glucuronidase (gus) gene. Agrobacterium mediated transformation was used to create transgenic lines of alfalfa (cultivar Regen SY27x). The transgenic plants were stained for GUS activity. In uninoculated plants, GUS activity was primarily seen in the root vascular tissues. No activity was observed in uninoculated leaves. With fungal pathogen infection, staining was greatly enhanced and allowed for stain visualized in the leaves. Quantitative PCR assays were done to quantify pathogen-induced GUS expression, as well as expression of PR5 and PR10 in infected leaves. RNA was extracted from symptomatic infected leaves after inoculation and converted to cDNA. Using specific primers, transcript accumulation was compared between cDNA from mock inoculated and inoculated plant tissue. In plants with the PR10:GUS or PR5:GUS constructs, GUS transcripts accumulated 41- to 378-fold over the mock inoculated plants at 7 days after inoculation with Phoma medicaginis, depending on the plant line. GUS transcripts were also strongly up-regulated in response to Colletotrichum trifolii and Pseudomonas syringae pv. syringae. Consistently, the PR10 promoter had greater fold amplifications and greater activity than the PR5 promoter. In response to P. medicaginis, transcripts of the PR10 gene were up-regulated 31- to 221-fold at 7 days after inoculation and transcripts of the PR5 gene were up-regulated 44- to 60-fold. These experiments show that the M. truncatula PR10 promoter is functional in alfalfa for expression of transgenes and up-regulates genes after infection by a range of alfalfa pathogens.