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Extracellular vesicles (EVs) represent a system for the coordinated secretion of a variety of molecular cargo including proteins, lipids, nucleic acids, and metabolites. They have an essential role in intercellular communication in multicellular organisms and have more recently been implicated in host-pathogen interactions. Study of the role for EVs in fungal biology has focused on pathogenic yeasts that are major pathogens in humans. In this study we have expanded the investigation of fungal EVs to plant pathogens, specifically the major cotton pathogen Fusarium oxysporum f. sp. vasinfectum. EVs isolated from F. oxysporum f. sp. vasinfectum culture medium have a morphology and size distribution similar to EVs from yeasts such as Candida albicans and Cryptococcus neoformans. A unique feature of the EVs from F. oxysporum f. sp. vasinfectum is their purple color, which is predicted to arise from a napthoquinone pigment being packaged into the EVs. Proteomic analysis of F. oxysporum f. sp. vasinfectum EVs revealed that they are enriched in proteins that function in synthesis of polyketides as well as proteases and proteins that function in basic cellular processes. Infiltration of F. oxysporum f. sp. vasinfectum EVs into the leaves of cotton or N. benthamiana plants led to a phytotoxic response. These observations lead to the hypothesis that F. oxysporum f. sp. vasinfectum EVs are likely to play a crucial role in the infection process.

SummaryExpression of the plant defensin NaD1 in transgenic cotton plants increases plant survival, disease tolerance, and yield when grown in soil naturally infested with Fusarium oxysporum and Verticillium dahliae. Importantly, transgenic plants did not exhibit any detrimental agronomic features.

Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans found in all higher plants. Although the precise function(s) of any single AGP is unknown, they are implicated in diverse developmental roles such as differentiation, cell-cell recognition, embryogenesis and programmed cell death. DNA sequencing projects have made possible the identification of the genes encoding a large number of putative AGP protein backbones. In contrast, our understanding of how AGPs undergo extensive post-translational modification is poor and it is important to understand these processes since they are likely to be critical for AGP function. Genes believed to be responsible for post-translational modification of an AGP protein backbone, include prolyl hydroxylases, glycosyl transferases, proteases and glycosylphosphatidylinositol-anchor synthesising enzymes. Here we examine models for proteoglycan function in animals and yeast to highlight possible strategies for determining the function(s) of individual AGPs in plants.

Pathogenic microbes are developing resistance to established antibiotics, making the development of novel antimicrobial molecules paramount. One major resource for discovery of antimicrobials is the arsenal of innate immunity molecules that are part of the first line of pathogen defense in many organisms. Gene encoded cationic antimicrobial peptides are a major constituent of innate immune arsenals. Many of these peptides exhibit potent antimicrobial activity in vitro . However, a major hurdle that has impeded their development for use in the clinic is the loss of activity at physiological salt concentrations, attributed to weakening of the electrostatic interactions between the cationic peptide and anionic surfaces of the microbial cells in the presence of salt. Using plant defensins we have investigated the relationship between the charge of an antimicrobial peptide and its activity in media with elevated salt concentrations. Plant defensins are a large class of antifungal peptides that have remarkable stability at extremes of pH and temperature as well as resistance to protease digestion. A search of a database of over 1200 plant defensins identified ZmD32, a defensin from Zea mays , with a predicted charge of +10.1 at pH 7, the highest of any defensin in the database. Recombinant ZmD32 retained activity against a range of fungal species in media containing elevated concentrations of salt. In addition, ZmD32 was active against Candida albicans biofilms as well as both Gram negative and Gram-positive bacteria. This broad spectrum antimicrobial activity, combined with a low toxicity on human cells make ZmD32 an attractive lead for development of future antimicrobial molecules.

BackgroundPlant defensins are small (45–54 amino acids), basic, cysteine-rich proteins that have a major role in innate immunity in plants. Many defensins are potent antifungal molecules and are being evaluated for their potential to create crop plants with sustainable disease resistance. Defensins are produced as precursor molecules which are directed into the secretory pathway and are divided into two classes based on the absence (class I) or presence (class II) of an acidic C-terminal propeptide (CTPP) of about 33 amino acids. The function of this CTPP had not been defined.ResultsBy transgenically expressing the class II plant defensin NaD1 with and without its cognate CTPP we have demonstrated that NaD1 is phytotoxic to cotton plants when expressed without its CTPP. Transgenic cotton plants expressing constructs encoding the NaD1 precursor with the CTPP had the same morphology as non-transgenic plants but expression of NaD1 without the CTPP led to plants that were stunted, had crinkled leaves and were less viable. Immunofluorescence microscopy and transient expression of a green fluorescent protein (GFP)-CTPP chimera were used to confirm that the CTPP is sufficient for vacuolar targeting. Finally circular dichroism and NMR spectroscopy were used to show that the CTPP adopts a helical confirmation.ConclusionsIn this report we have described the role of the CTPP on NaD1, a class II defensin from Nicotiana alata flowers. The CTPP of NaD1 is sufficient for vacuolar targeting and plays an important role in detoxification of the defensin as it moves through the plant secretory pathway. This work may have important implications for the use of defensins for disease protection in transgenic crops.

logical interactions between crop components and emphasize species diversity rather than only crop yield (Mat-Integrating trees into pasture may increase pasture production and son et al., 1997; Garrett and McGraw, 2000). improve nutritive value by altering both species composition and productivity. Our objective was to determine forage yield and botani-Silvopastoralism is one such agroforestry practice that cal composition in response to tree species, tree density, and slope po-intentionally integrates trees, forage crops, and livestock sition in an emulated silvopasture (the site had no animals). In 1995, into a structural practice of planned interactions (Clason black walnut (Juglans nigra L.) and honey locust (Gleditisia triaand Sharrow, 2000). Greater forage production, nutritive canthos L.) trees were planted within plots (r ϭ 3) of predominantly value, and digestibility are reported for pastures grown tall fescue (Festuca arundinacea Schreb.) pasture. Trees were planted under trees (Smith, 1942; Garrett and Kurtz, 1983; Burner down slopes in rows to create low, medium, and high tree densities and Brauer, 2003) relative to open sites, and this may at shoulder-, mid-, and toe-slope positions. Sampling sites (n ϭ 54) reflect changes in botanical composition. For example, under field treatment combinations were harvested May to October Brooks (1951) noted reduced species of poor forage merit at 35-d intervals in 2002 and 2003. Before spring, summer, and fall such as broomsedge (Andropogon virginicus L.) and A.L. Buergler, Oregon State Univ., Corvallis, OR 97331;

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