Plant defensin antibacterial mode of action against Pseudomonas species

Background: Though many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5- lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. Also, a plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments. Results: Plant defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. A defensin from Medicago truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. The ability for MtDef4 to damage bacterial outer membranes was also verified visually through fluorescent microscopy. Another defensin from M. truncatula, MtDef5, failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. The transposon insertion site on MtDef4 resistant P. syringae pv. syringae mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to plant defensin treatments. Conclusions: MtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. MtDef5, appears to have a different antibacterial MOA. Additionally, the MtDef4 antibacterial mode of action may also involve inhibition of translation

Modifying crops to increase cell wall digestibility

Improving digestibility of roughage cell walls will improve ruminant animal performance and reduce loss of nutrients to the environment. The main digestibility impediment for dicotyledonous plants is highly lignified secondary cell walls, notably in stem secondary xylem, which become almost non-digestible. Digestibility of grasses is slowed severely by lignification of most tissues, but these cell walls remain largely digestible. Cell wall lignification creates an access barrier to potentially digestible wall material by rumen bacteria if cells have not been physically ruptured. Traditional breeding has focused on increasing total dry matter digestibility rather than cell wall digestibility, which has resulted in minimal reductions in cell wall lignification. Brown midrib mutants in some annual grasses exhibit small reductions in lignin concentration and improved cell wall digestibility. Similarly, transgenic approaches down-regulating genes in monolignol synthesis have produced plants with reduced lignin content and improved cell wall digestibility. While major reductions in lignin concentration have been associated with poor plant fitness, smaller reductions in lignin provided measurable improvements in digestibility without significantly impacting agronomic fitness. Additional targets for genetic modification to enhance digestibility and improve roughages for use as biofuel feedstocks are discussed; including manipulating cell wall polysaccharide composition, novel lignin structures, reduced lignin/polysaccharide cross-linking, smaller lignin polymers, enhanced development of non-lignified tissues, and targeting specific cell types. Greater tissue specificity of transgene expression will be needed to maximize benefits while avoiding negative impacts on plant fitness.cauliflower mosiac virus (CaMV) 35S promote

Transgenic expression of Medicago truncatula PR10 and PR5 promoters in alfalfa shows pathogen induced up-regulation of transgene expression

Poster presentation at the 2016 North American Alfalfa Improvement Conference.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

Plant defensins inhibit growth of pathogens in the alfalfa crown rot disease complex

Poster presentation at the 2016 American Phytopathological Society Annual Meeting.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

Physiological and molecular characterization of aluminum resistance in Medicago truncatula

<p>Abstract</p> <p>Background</p> <p>Aluminum (Al) toxicity is an important factor limiting crop production on acid soils. However, little is known about the mechanisms by which legumes respond to and resist Al stress. To explore the mechanisms of Al toxicity and resistance in legumes, we compared the impact of Al stress in Al-resistant and Al-sensitive lines of the model legume, <it>Medicago truncatula </it>Gaertn.</p> <p>Results</p> <p>A screen for Al resistance in 54 <it>M. truncatula </it>accessions identified eight Al-resistant and eight Al-sensitive lines. Comparisons of hydroponic root growth and root tip hematoxylin staining in an Al-resistant line, T32, and an Al-sensitive line, S70, provided evidence that an inducible Al exclusion mechanism occurs in T32. Transcriptional events associated with the Al resistance response were analyzed in T32 and S70 after 12 and 48 h Al treatment using oligonucleotide microarrays. Fewer genes were differentially regulated in response to Al in T32 compared to S70. Expression patterns of oxidative stress-related genes, stress-response genes and microscopic examination of Al-treated root tips suggested a lower degree of Al-induced oxidative damage to T32 root tips compared to S70. Furthermore, genes associated with cell death, senescence, and cell wall degradation were induced in both lines after 12 h of Al treatment but preferentially in S70 after 48 h of Al treatment. A multidrug and toxin efflux (MATE) transporter, previously shown to exude citrate in <it>Arabidopsis</it>, showed differential expression patterns in T32 and S70.</p> <p>Conclusion</p> <p>Our results identified novel genes induced by Al in Al-resistant and sensitive <it>M. truncatula </it>lines. In T32, transcription levels of genes related to oxidative stress were consistent with reactive oxygen species production, which would be sufficient to initiate cell death of Al-accumulating cells thereby contributing to Al exclusion and root growth recovery. In contrast, transcriptional levels of oxidative stress-related genes were consistent with excessive reactive oxygen species accumulation in S70 potentially resulting in necrosis and irreversible root growth inhibition. In addition, a citrate-exuding MATE transporter could function in Al exclusion and/or internal detoxification in T32 based on Al-induced transcript localization studies. Together, our findings indicate that multiple responses likely contribute to Al resistance in <it>M. truncatula</it>.</p

Characterization of the bacterial stem blight pathogen of alfalfa, Pseudomonas syringae pv. syringae ALF3

Poster presentation at the 2014 meeting of the American Phytopathological Society,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

Antibacterial Activity of Plant Defensins Against Alfalfa Crown Rot Pathogens

Poster presentation at the 2017 meeting of the American Society for Microbiology.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

Antimicrobial Activity of Brassica rapa Nectar Lipid Transfer Protein

Poster presentation at the 2017 Mycological Society of America Annual Meeting.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 resistanc

Spatio-temporal expression patterns of Arabidopsis thaliana and Medicago truncatula defensin-like genes

Plant genomes contain several hundred defensin-like (DEFL) genes that encode short cysteine-rich proteins resembling defensins, which are well known antimicrobial polypeptides. Little is known about the expression patterns or functions of many DEFLs because most were discovered recently and hence are not well represented on standard microarrays. We designed a custom Affymetrix chip consisting of probe sets for 317 and 684 DEFLs from Arabidopsis thaliana and Medicago truncatula, respectively for cataloging DEFL expression in a variety of plant organs at different developmental stages and during symbiotic and pathogenic associations. The microarray analysis provided evidence for the transcription of 71% and 90% of the DEFLs identified in Arabidopsis and Medicago, respectively, including many of the recently annotated DEFL genes that previously lacked expression information. Both model plants contain a subset of DEFLs specifically expressed in seeds or fruits. A few DEFLs, including some plant defensins, were significantly up-regulated in Arabidopsis leaves inoculated with Alternaria brassicicola or Pseudomonas syringae pathogens. Among these, some were dependent on jasmonic acid signaling or were associated with specific types of immune responses. There were notable differences in DEFL gene expression patterns between Arabidopsis and Medicago, as the majority of Arabidopsis DEFLs were expressed in inflorescences, while only a few exhibited root-enhanced expression. By contrast, Medicago DEFLs were most prominently expressed in nitrogen-fixing root nodules. Thus, our data document salient differences in DEFL temporal and spatial expression between Arabidopsis and Medicago, suggesting distinct signaling routes and distinct roles for these proteins in the two plant species