Plant Pathology Faculty and Staff Research

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Genome editing in alfalfa (Medicago sativa) to hyper-accumulate phosphate
(2019) Samac, Deborah A; Miller, Susan S; Dornbusch, Melinda R; Curtin, Shaun J
Rock phosphate, the main source of phosphate (P) for crop fertilizers, is a finite resource that is predicted to be depleted in 50-100 years. P is a critical nutrient in agriculture and its application can dramatically improve plant productivity. However, many soils have excess amounts of P from application of animal manures and runoff of phosphate from agricultural lands is the major source of nonpoint water pollution in the Midwestern US. The goal of this project is to create mutations by gene editing in the ubiquitin E2 conjugating enzyme PHO2, involved in P signaling and P homeostasis in alfalfa so that plants hyper-accumulate phosphate. Such plants could be used to reduce soil P levels and reclaim P for use as a fertilizer. From a draft diploid Medicago sativa genome scaffold sequence and the alfalfa transcriptome database (AGED), three PHO2 genes were identified. The genes, two of which are >99% homologous (a/b), each have seven exons interspersed by six introns. The open reading frames are 912 amino acids except when an alternate splice site is used in a/b gene transcript resulting in a 902 amino acid sequence. Alfalfa plants grown under P limiting conditions expressed low levels of the a/b transcripts with higher levels seen for PHO2c, while application of higher P induced increased expression mainly of the a/b transcripts. Under high P conditions, roots and shoots accumulated 4.1x and 2.5x more P than in low P conditions, respectively. An initial CRISPR/Cas9/Cys4 reagent targeting all three genes was generated and used to transform alfalfa cv. RegenSY. A total of 67 verified transgenic plants were screened by acrylamide gel shift assays, cloning, and sequencing to identify plants with mutations. Mutations ranging from a 1 bp insertion to a 25 bp deletion were identified in a total of 10 plants and some plants had multiple targets hit. Recently, a second attempt at CRISPR/Cas9 mutation utilized a cassette vector system with either the tRNA or Cys4 splicing system and exonuclease components. Initial screening results indicate that the tRNA splicing system may have yielded greater numbers of mutations. TaqMan probes were designed to identify plants with changes in the target sites and were verified by restriction digestions, cloning, and sequencing. Data on inheritance of mutations and phosphate accumulation in edited plants will be presented. The results of these experiments demonstrate that editing of multiple targets can be accomplished in alfalfa, although the tetraploid inheritance of genes complicates analysis.
Lignin reduction in alfalfa (Medicago sativa) does not affect foliar disease resistance
(2018) Samac, Deborah A; Ao, Samadangla; Dornbusch, Melinda R; Grev, Amanda M; Wells, M Scott; Martinson, Krishona; Sheaffer, Craig C
Disruptions in the lignin biosynthetic pathway have been shown to reduce disease resistance in a number of crops. Recently, genetically modified alfalfa (Medicago sativa) varieties have been marketed with reduced lignin and improved forage quality traits, including increased digestibility by ruminants at later stages of plant maturity. The objective of this study was to compare foliar disease resistance in three reference alfalfa varieties, 54R02, DKA43-22RR, WL355.RR, and the reduced lignin variety, 54HVX41, to evaluate the effect of the reduced lignin trait on foliar disease resistance. Alfalfa plants in research plots at three locations in Minnesota were evaluated for percent defoliation caused by foliar pathogens at four maturity stages; early bud, bud, early flower, and flowering; with natural inoculum. Spring black stem and leaf spot, Leptosphaerulina leaf spot, and common leaf spot were observed from June through September in all locations on all varieties. Summer black stem and leaf spot was most prevalent in August on all varieties at one location. The amount of defoliation increased with maturity stage for all varieties. When harvest was delayed until the flowering stage, moderate to severe (32 to 64%) leaf loss occurred, depending on location. Alfalfa varieties did not differ in percent defoliation at any maturity stage indicating that the reduced lignin trait did not affect foliar disease resistance.
Alfalfa leaf protein concentrate: A sustainable protein source for aquaculture feeds
(2019) Samac, Deborah A; Coburn, Jessica E M; Phelps, Nicholas; Wells, M Scott
Interest in local, sustainable aquaculture is continuing to expand across the United States. To ensure profitability and high consumer acceptance, a plant-based non-GMO source of protein is needed for replacing fishmeal in aquaculture diets. Most replacements in current use are derived from seeds, and often contain undesirable antinutritional components. Alfalfa (Medicago sativa) is a high biomass perennial legume that is grown across the U.S. for use in animal feeds. We evaluated the yield and composition of protein concentrates derived from fresh alfalfa foliage. Furthermore, a commercial alfalfa protein concentrate (APC) was used to replace fishmeal in diets for yellow perch (Perca flavescens) and rainbow trout (Oncorhynchus mykiss). Weight gains, growth rate, and feed conversion ratios were measured.
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 A
Genetic 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.
Antimicrobial Activity of Brassica rapa Nectar Lipid Transfer Protein
(2017) Sathoff, Andrew E; Samac, Deborah A; Holl, Catherine; Schmidt, Tony; Carter, Clay
Antimicrobial 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 resistance
Plant defensins inhibit growth of pathogens in the alfalfa crown rot disease complex
(2016) Sathoff, Andrew E; Samac, Deborah A
Alfalfa 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.
Antibacterial Activity of Plant Defensins Against Alfalfa Crown Rot Pathogens
(2017) Sathoff, Andrew; Velivelli, Siva; Shah, Dilip; Samac, Deborah A
Alfalfa (Medicago sativa) is the fourth most widely grown crop in the United States. Alfalfa crown rot is a disease complex that severely decreases alfalfa stand density and productivity in all alfalfa-producing areas. Currently, there are no viable methods of disease control. Plant defensins are small cationic antimicrobial peptides with a conserved signature of cysteines. The in vitro and in planta antifungal activity of plant defensins has been extensively studied. However, their antibacterial activity has been less well characterized. Defensins have a γ-core motif, a cluster of cationic and hydrophobic residues, which is essential for antimicrobial activity. The γ-core motifs of five synthetic defensins were tested for antibacterial activity against the bacterial pathogens in the alfalfa crown rot disease complex. Full length defensins, expressed using a Pichia pastoris expression system, were tested to compare antibacterial activity. A spread plate method was used to quantify antibacterial activity of defensins. Bacteria were grown out to an OD600 value of 0.1, and a 200 μL culture was incubated with shaking for 3 hours with concentrations of defensin peptide up to 30 μg/mL. The bacteria were serially diluted, and 100 μL was plated on to NBY plates. After 48 hours of incubation, the bacterial colonies were counted. The amount of defensin needed to inhibit growth of pathogen strains by 50% (IC50) was calculated. The core motif of MtDef4 was shown to be the most effective truncated peptide with IC50 values of 3.4 μM against Pseudomonas syringae pv. syringae and 4.52 μM against Xanthomonas alfalfae. Also, the corresponding full length MtDef4 peptide was found to be active against P. syringae pv. syringae and X. alfalfae with IC50 values of 0.43 μM and 0.68 μM, respectively. These experiments show the previously overlooked high biological activity of plant defensins against bacterial pathogens. Additionally, these results indicate that the γ-core-motif can be used to predict biological activity of the full-length defensin, and that transgenic expression of plant defensins in alfalfa has the potential to lead to improved crown rot resistance.
Characterization of the bacterial stem blight pathogen of alfalfa, Pseudomonas syringae pv. syringae ALF3
(2014) Samac, Deborah A; Studholme, David J; Ao, Samadangla
Bacterial stem blight of alfalfa occurs sporadically in the central and western U.S. Yield losses of up to 50% of the first harvest can occur with some cultivars. Developing resistant cultivars is hampered by lack of information on the pathogen and a standard test for evaluating plant germplasm. Bacteria producing a fluorescent pigment were isolated on King’s B agar from alfalfa with symptoms of bacterial stem blight from near Cheyenne, WY. The strain ALF3 was tentatively identified as Pseudomonas syringae pv. syringae based on 16S rDNA sequence and PCR amplification of syrB for lipodepsinonapeptide toxin production. Multilocus sequence analysis indicated that ALF3 falls within a clade containing strains of P. syringae pv. syringae with closest affinity to FF5 from pear. Comparison of a draft whole-genome sequence of ALF3 further confirmed that ALF3 most closely resembles FF5 (~96% sequence identity) and P. syringae pv. aptata DSM50252 from beet. Approximately 60 genes were unique to ALF3, including several predicted genes in the T3SS cluster such as a type III helper protein HrpZ (Pto) and phage-associated genes. ALF3 was highly pathogenic to snapbean pods but caused only mild symptoms on leaves of snapbean, pear, and sugarbeet. A standardized method for evaluating disease resistance in alfalfa was developed. Cultivars with fall dormancy ratings of 1 and 2 had higher percentages of resistant plants than cultivars with fall dormancy ratings of 8-11.
Variances in nutritient content and yield of alfalfa protein concentrate processed with five mthods from high yielding biomass type alfalfa
(2017) Coburn, Jessica; Wells, Scott; Samac, Deborah A
The demand for protein is growing with increased populations and world affluence. A sustainable and affordable protein source is needed to support the growing aquaculture industry worldwide. Alfalfa produces high levels of protein and provides numerous environmental services, potentially making it an ideal feedstock in aquaculture. This project evaluates five methods of alfalfa protein concentrate (APC) coagulation for yield and nutritional content. A biomass-type alfalfa was harvested at full flower with a flail mower, fresh material homogenized in a laboratory blender, juice expressed by hand, and protein coagulated using five methods. The methods were: (1) heating in an 80°C water bath for 30 minutes, (2) freezing the juice at -15°C, (3) lowering the pH of the juice to 4.0 with hydrochloric acid, (4) lowering the pH of juice chilled in refrigeration with chilled hydrochloric acid, (5) first, raising the pH of the juice to 10.0 with sodium hydroxide, letting it set for 15 minutes, then lowering the pH down to 4.0 with hydrochloric acid. The temperature and pH based methods resulted in significant differences in protein yield and concentration. While the acid based coagulation methods resulted in the highest yield and lowest fiber content, temperature methods resulted in higher concentrations of protein. Extraction methods also impacted crude fat, sugar, and specific amino acids. Acid coagulation resulted in decreased fiber and fat content, which may be beneficial for aquaculture uses. Heat treatment resulted in increased concentration of limiting amino acids and lower sugar content compared to the other methods. These findings show that APC producers can adjust production methods for a specialized APC product meant for specific diets or uses.
Identification of markers associated with race-specific resistance to Aphanomyces root rot in alfalfa, Poster at the 2017 APS meeting
(2017) Samac, Deborah; Bucciarelli, Bruna; Dornbusch, Melinda; Miller, Susan; Yu, Long-Xi
Aphanomyces root rot, caused by Aphanomyces euteiches, is one of the most important diseases of alfalfa in the United States. Two races of the pathogen are recognized and although most cultivars are resistant to race 1, fewer have resistance to race 2, the predominant race in North America. Molecular markers are needed to facilitate breeding for resistance and to clarify race/resistance gene structure. Resistant and susceptible seedlings were identified from three resistant cultivars, WAPH1, WAPH5 and 53V52, and used as parents to produce F1 populations. Severity of symptoms corresponded with amount of pathogen DNA and oospore density in roots. Race-specific resistance involves a hypersensitive response of individual epidermal or cortical cells upon pathogen attack followed by suberization of cells surrounding the stele and strong autofluorescence in cortical cells, indicating the presence of phenolic compounds. Segregation ratios of F1 populations suggested that resistance to race 1 in WAPH1 is conditioned by a single gene but resistance to race 1 is multigenic in WAPH5 and 53V52, and resistance to race 2 is multigenic in all three cultivars. Segregation for resistance to seven strains of A. euteiches in 70 F1 full-sib plants derived from 53V52 suggested the presence of clustered resistance genes and multiple race types. Identification of resistance gene loci is in progress using genotyping by sequencing and genetic mapping of F1 populations.