Isolation and characterization of antifungal peptides from plants

De Beer, Abre (Stellenbosch : University of Stellenbosch, 2008-03)

Thesis

ENGLISH ABSTRACT: Over the last decade research has shown the importance of small antimicrobial peptides in the innate immunity of plants. These peptides do not only play a critical role in the multilayered defense systems of plants, but have proven valuable in the engineering of disease resistant food crops towards the ultimate aim of reducing the dependency on chemical fungicides. As the lists of isolated and characterized peptides grew, it became clear that other biological activities, in addition to the antimicrobial capacity, could be linked to some of these peptides; these alternative activities could have important applications in the field of medicine. This has made the defensin encoding genes prime targets for the agricultural and medical biotechnology sectors. To this end we set out to evaluate South African flora for the presence of plant defensin sequences and to isolate plant defensin genes that might be useful in biotechnology applications. Moreover, by isolating and characterizing these novel peptides, also in an in planta environment and in interaction with fungal pathogens, important knowledge will be gained of the biological role and importance of the peptides in the plant body. The plant host targets were South Africa Brassicaceae species including indigenous species, as well as Vitis vinifera, as the most important fruit crop in the world and since no defensins have been isolated from this economically important crop plant. The Brassicaceae family has been shown to be abundant in defensin peptides and several of the best characterized peptides with potent activity have been isolated from this family. Based on initial activity screens conducted on selected South African Brassicaceae spp. we concluded that these spp. contain promising antifungal peptide activities, warranting further efforts to isolate the genes and encoding peptides and to characterize them further. The preliminary activity screens used a peptide-enrichment isolation strategy that favored the isolation of basic, heat-stable peptides; these properties are characteristic features of plant antimicrobial peptides. These peptide fractions showed strong antifungal activities against the test organisms. A PCR-amplification strategy was subsequently designed and implemented, leading to the isolation of 14 novel defensin peptide encoding genes from four South African Brassicaceae spp., including the indigenous South African species Heliophila coronopifolia. Amino acid sequence analysis of these peptides revealed that they are diverse in amino acid composition and share only 42% homology at amino acid level. This divergence in amino acid composition is important for the identification of new biological activities within closely related plant defensins. Single amino acid changes have been contributed with the divergent biological activities observed in closely related plant defensin peptides. Phylogenetic analysis conducted on the deduced amino acid sequences revealed that all the new defensins share a close relationship to other Brassicaceae members of the plant defensin superfamily and was furthest removed from the defensins isolated from the families Solanaceae and Poaceae. Classification analysis of these peptides showed that they belong to subgroup A3 of the defensin superfamily. A putative defensin sequence was also isolated from V. vinifera cultivar, Pinotage, and termed Vv-AMP1. Genetic characterization showed that only a single gene copy of this peptide is present within the V. vinifera genome, situated on chromosome 1. Genetic characterization of this peptide encoding gene within the Vitis genus showed that this gene has stayed conserved throughout the divergent evolution of the Vitis genus. Expression studies of Vv-AMP1 revealed that this gene is expressed in a tissue specific and developmentally regulated manner, being only expressed in grape berries and only at the onset of vèraison. Induction of Vv-AMP1 in grapevine leaf material could never be achieved through the external application of hormones, osmotic stress, wounding, or pathogen infection by Botrytis cinerea. Deduced amino acid analysis showed that Vv-AMP1 encoded for a 77 amino acid peptide consisting of a 30 amino acid signal peptide and a 47 amino acid mature peptide, with putative antifungal activity. The Vv-AMP1 peptide grouped with the subclass B type defensins, which have been documented to have both antifungal and antibacterial activities. The Vv-AMP1 signal peptide directed the green fluorescent protein (GFP) reporter gene to the apoplastic regions in cells with high levels of accumulation in the vascular tissue and the guard cells of the stomata. Recombinant Vv-AMP1 peptide was successfully purified from a bacterial host and shown to have a size of 5.495 kDa. Recombinant Vv-AMP1 showed strong antifungal activity at low concentrations against a broad spectrum of fungal pathogens, which included Verticillium dahliae (IC50 of 1.8 μg mL-1) and the necrotrophic pathogen Botrytis cinerea (IC50 of 12-13 μg mL-1). Antifungal activity of Vv-AMP1 did not induce morphological changes in fungal hyphae, but its activity was associated with induced membrane permeabilization in treated hyphae. Vv-AMP1 was successfully introduced into Nicotiana tabacum as confirmed by Southern blot analysis and 20 individual lines were generated. Genetic characterization confirmed the integration and expression of the gene in the heterologous tobacco environment. The peptide was under control of its native signal sequence which has been shown to direct its product to the apoplastic regions of cells. The transgenic lines were analyzed to determine the presence and activity of the grapevine defensin peptide. Western blot analyses of partially purified plant extracts detected a signal of the expected size in both the untransformed control and the transgenic lines. Comprehensive analysis of EST databases identified three highly homologous sequences from tobacco that probably caused the background signal in the control. These crude protein extracts were able to inhibit the growth of V. dahliae in vitro when tested in a microtiter plate assay, but the inhibition could not be conclusively linked to the presence of the transgenic peptide, since non-expressing transgenic lines, included as controls, also showed inhibition. Similar results were obtained with infection studies, clearly showing that despite successful integration and expression of the transgene, the peptides was either not functional in the heterologous environment, or perhaps unstable under the particular regulatory conditions. This peptide belongs to a subclass of peptides known for associated activities that might activate tight control by plant hosts if threshold levels are reached. These aspects need further investigation, specifically since it is in stark contrast to previous results obtained with defensins from a different subclass. This study has also yielded significant other related resources that would be instrumental for further possible biotechnology exploitation of some of the novel peptides, but also to provide genetic constructs and plant material that would be invaluable to address fundamentally important questions such as the regulation and mode of action of defensin peptides, specifically in interaction with pathogen hosts. The novel peptides have been transformed to various hosts, including grapevine and these transgenic populations are available to facilitate the next rounds of research into this extremely promising group of antifungal peptides.

AFRIKAANSE OPSOMMING: In die laaste dekade het navorsing die belangrike rol van klein antimikrobiese peptiede in plantweerstandsmeganismes beklemtoon. Hierdie peptiede speel nie alleenlik 'n belangrike rol in die komplekse lae van plantweerstandstelsels nie, maar het ook hulle ekonomiese potensiaal getoon in die manipulering van siekteweerstandbiedendheid in voedselgewasse met die oorkoepelende doel om landbougewasse minder afhanklik van chemiese spuitstowwe te maak. Soos wat die hoeveelheid geïsoleerde en gekarakteriseerde peptiede toeneem, het dit duidelik geword dat ander biologiese aktiwiteite, bykomend tot die antimikrobiese kapasiteit, met sommige van dié peptiede verbind kan word; hierdie alternatiewe aktiwiteite het belangrike toepassing in veral die mediese veld. Dit het die defensin-koderende gene kernteikens vir die landbou- en mediese biotegnologiesektore gemaak. In die studie is daar begin om die Suid-Afrikaanse blommeryk te evalueer vir die teenwoordigheid van plantdefensingene en om dié gene te isoleer wat van ekonomiese belang vir die biotegnologiebedryf kan wees. Deur die in vitro- én in planta karakterisering van die unieke plantdefensinpeptiede word daar gemik daarna om belangrike inligting in te win oor die biologiese rol van die peptiede binne die plantligggaam. Die plantgashere wat geteiken is sluit in die Suid-Afrikaanse Brassicaceae-spesies, insluitende inheemse spesies, asook Vitis vinifera, wat as die belangrikste vrugtegewas ter wêreld beskou word. Die Brassicaceae-familie is welbekend daarvoor dat dit 'n ryk bron van plantdefensinpeptiede is en verskeie van die bes gekarakteriseerde antifungiese defensinpeptiede is van dié familie afkomstig. Aanvanklike aktiwiteitstoetse het getoon dat die Suid-Afrikaanse Brassicaceae-spesies belowende antifungiese aktiwiteit toon, wat die verdere isolering en karakterisering van dié gene en hul peptiedprodukte regverdig. Die aanvanklike aktiwiteitstoetse het 'n selektiewe peptiedverrykingstrategie gevolg wat die isolering van basiese, hittestabiele peptiede bevoordeel het; hierdie eienskappe is baie kenmerkend van plant-antimikrobiese peptiede. Die peptiedfraksies wat met hierdie metode geïsoleer is, het sterk antifungiese aktiwiteit teen die toetsorganismes getoon. Die resultate het gelei tot die ontwikkeling en toepassing van 'n polimerasekettingreaksie-strategie, wat daartoe gelei het dat 14 nuwe defensingene van vier Suid-Afrikaanse Brassicaceae-genera, insluitend die inheemse spesie Heliophila coronopifolia, geïsoleer kon word. Afgeleide aminosuurvolgorde-analises van die nuwe defensinpeptiede het gewys dat hulle slegs 42% homologie het. Hierdie diversiteit in aminosuurvolgorde is belangrik vir die identifisering van nuwe biologiese aktiwitiete binne die groep van verwante peptiede. Navorsing het verder getoon dat enkel-aminosuurverskille bydra tot die diverse spektrum van biologiese aktiwiteite binne 'n groep van verwante defensinpeptiede. Filogenetiese analise van die aminosuurvolgordes het getoon dat al die nuwe defensinpeptiede 'n sterk verwantskap met plantdefensinpeptiede, wat van ander Brassicaceae-spesies geïsoleer is, toon. Daarteenoor het dit die kleinste verwantskap getoon met plantdefensinpeptiede wat van die Solanaceae- en Poaceae-families geïsoleer is. Klassifikasiestudies het bewys dat die nuwe peptiede saam met subgroep A3 van die plantdefensin-superfamilie groepeer. 'n Moontlike plantdefensingeen, genaamd Vv-AMP1, is ook van die V vinifera-kultivar, Pinotage, geïsoleer. Genetiese karakterisering het aangedui dat slegs 'n enkele kopie van die geen in die V. vinifera-genoom teenwoordig en op chromosoom 1 geleë is. Genetiese karakterisering van Vv-AMP1 binne die Vitus-genus het gewys dat die geen binne die genus evolusionêr gekonserveerd is. Uitdrukkingstudies van Vv-AMP1 het verder bewys dat die geen uitgedruk word op 'n weefselspesifieke, ontwikkelingsgekoppelde wyse, naamlik slegs in druiwekorrels en slegs tydens rypwording. Vv-AMP1-uitdrukking kon nooit geïnduseer word in wingerdblare deur die uitwendige toediening van hormone, osmotiese stres, wonding of patogeeninfeksie deur Botrtys cinerea nie. Ontleding van die afgeleide aminosuurvolgorde het gewys dat Vv-AMP1 kodeer vir 'n 77-aminosuurpeptied, wat uit 'n 30-aminosuurseinpeptied en 'n 47-aminosuur-aktiewe peptied met voorspelde antifungiese aktiwiteit bestaan. Die Vv-AMP1-peptied is gegroepeer met subgroep B van die plantdefensin-superfamilie, 'n subgroep wat vir beide antifungiese en antibakteriese aktiwiteit gedokumenteer is. Die Vv-AMP1-seinpeptied het die groen fluoressensie-indikatorproteïen (GFP) na die apoplastiese areas van die plantselle gelei, met hoë vlakke van lokalisering in die vaatbundelweefsel en sluitselle van die huidmondjies. Die rekombinante Vv-AMP1-peptied is suksesvol geproduseer en uit 'n bakteriese produksieras gesuiwer, en het 'n molekulêre massa van 5.495 kDa gehad. Die gesuiwerde peptide het by lae konsentrasies 'n sterk aktiwiteit getoon teen 'n breë spektrum van fungiese patogene, wat Verticllium dahliae (IC50 van 1.8 μg mL-1) en die nekrotrofiese patogeen, B. cinerea (IC50 van 12-13 μg mL-1), ingesluit het. Vv-AMP1-aktiwiteit het geen ooglopende morfologiese veranderinge in die fungi-hifes veroorsaak nie, maar hulle aktiwiteit is verbind met 'n verhoogde membraandeurdringbaarheid in behandelde fungi-hifes. Suksesvolle intergrasie van Vv-AMP1 in die Nicotiana tabacum-genoom is deur Southern-kladontledings bevestig en 20 individuele transgeniese lyne is ontwikkel. Genetiese karakterisering van die transgeniese lyne het gewys dat Vv-AMP1 suksesvol geïntegreer is en ook in die transgeniese tabakomgewing uitgedruk word. Die peptied is uitgedruk onder beheer van sy eie seinpeptied, wat die aktiewe produk na die apoplastiese areas van die plantselle teiken. Die transgeniese tabaklyne is ook ontleed om te bepaal of die wingerdpeptied suksesvol geproduseer word en sy aktiwiteit in die transgeniese omgewing behou. Western-kladanalise van semi-gesuiwerde plantproteïenekstrakte het 'n positiewe sein gelewer in beide die kontroleplante en die transgeniese plantlyne. Bestudering van tabakgeenuitdrukkings-databasisse het drie nukleotiedvolgordes opgelewer wat homologie met Vv-AMP1 toon en moontlik verantwoordelik kan wees vir die positiewe sein in die ongetransformeerde kontroleplante. Kru proteïenekstrakte van die transgeniese tabaklyne het in vitro-aktiwiteit teen V. dahliae getoon. Geen oortuigende ooreenkoms kon egter gevind word tussen V. dahliae-inhibisie en die teenwoordigheid van die transgeniese Vv-AMP1-peptied nie, aangesien kontroleplante wat Southern-klad-positief is, maar nie geenuitdrukking toon nie, ook inhibisie van V. dahliae veroorsaak het. Soortgelyke resultate is met infeksiestudies verkry. Alle resultate dui daarop dat, al is daar suksesvolle integrasie en uitdrukking van die geen in tabak verkry, dat die Vv-AMP1 peptied óf onaktief óf onstabiel in die transgeniese tabakomgewing is. Die peptied behoort aan 'n subgroep peptiede met aktiwiteite wat, sodra sekere vlakke van peptied oorskry word, die moontlik streng kontrole op proteïenvlak in die gasheerplant kan uitlok. Sekere aspekte van die studie sal verder bestudeer moet word, aangesien die data teenstrydig is met data wat verkry is met soortgelyke plantdefensinpeptiede wat aan 'n ander subgroep behoort. Die studie het baie hulpbronne gegenereer wat vir die biotegnologiesektor belangrik kan wees, veral op ekonomiese gebied. Verder is die geenkonstrukte en plantlyne wat ontwikkel is waardevol om fundamentele vrae rondom die regulering en meganisme van aksie van defensinpeptiede, spesifiek plantpatogeeninteraksie, te beantwoord. Die nuwe plantdefensingene is na verskeie gasheerplante, insluitende wingerd, getransformeer waar die transgeniese lyne die volgende rondte van navorsing oor die bestudering oor die belangrike groep van antifungiese peptiede, sal aanvul.

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