Green Fluorescent Protein Transformants Research Articles

Isolates of the Nematophagous Fungus Pochonia chlamydosporia Are Endophytic in Banana Roots and Promote Plant Growth

The biocontrol fungus Pochonia chlamydosporia colonizes banana roots endophytically. Root hairs and root surface were colonized by a stable GFP (green fluorescent protein) transformant of the fungus. Hyphal penetration in root cells was also observed. Spores of P. chlamydosporia 123, significantly increase root and leaf length and weight in banana plantlets (Musa acuminata cv. ‘Dwarf Cavendish’) in growth chamber experiments 30 days post-inoculation. In greenhouse 8-L pot experiments, P. chlamydosporia 123 spore inoculation significantly increases root, corm and leaf length, and leaf weight in banana plants (75 days post-inoculation). Spore inoculation of P. chlamydosporia strains from diverse origin (Pc21, Pc123, Pc399, and Pccat), significantly increase root, corm and leaf length and weight in banana plantlets. Pc21 from Italy was the best colonizer of banana roots. Consequently, this strain significantly increases banana root and leaf length most. Root colonization by P. chlamydosporia was also detected using cultural techniques and qPCR.

Detecting the colonization of ericoid mycorrhizal fungi in Vaccinium uliginosum using in situ polymerase chain reaction and green fluorescent protein

BackgroundEricoid mycorrhizal fungi (EMF) play important roles in mineral cycling and plant nutrient acquisition, and they increase plant survival in nutrient-poor environments. In this study, we detected the colonization of EMF using a green fluorescent protein (GFP) expression method and in situ PCR.ResultsGenetic transformants of Cryptosporiopsis ericae and Sordariomycetes sp. expressing GFP were obtained via Agrobacterium tumefaciens-mediated transformation. GFP transformants were able to infect Vaccinium uliginosum, and their fluorescence was visible in the hair roots. Both in situ PCR and the GFP-expressing method indicated that EMF could colonize the hair roots of V. uliginosum 2 weeks after inoculation.ConclusionsThis research represents the first attempt to detect ericoid mycorrhizal colonization using in situ PCR. A GFP-expressing method is an excellent system for detecting the colonization of EMF, but it is dependent on the successful transformation and expression of the gfp gene. In situ PCR and the GFP expression may be developed as new tools to study the interactions of EMF both with ericaceous plants and with the environment.

Green Fluorescent Protein Transformation Sheds More Light on a Widespread Mycoparasitic Interaction.

Powdery mildews, ubiquitous obligate biotrophic plant pathogens, are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. Some Ampelomyces strains are commercialized biocontrol agents of crop pathogenic powdery mildews. Using Agrobacterium tumefaciens-mediated transformation (ATMT), we produced stable Ampelomyces transformants that constitutively expressed green fluorescent protein (GFP) to (i) improve the visualization of the mildew-Ampelomyces interaction and (ii) decipher the environmental fate of Ampelomyces fungi before and after acting as a mycoparasite. Detection of Ampelomyces structures, and especially hyphae, was greatly enhanced when diverse powdery mildew, leaf, and soil samples containing GFP transformants were examined with fluorescence microscopy compared with brightfield and differential interference contrast optics. We showed for the first time, to our knowledge, that Ampelomyces strains can persist up to 21 days on mildew-free host plant surfaces, where they can attack powdery mildew structures as soon as these appear after this period. As saprobes in decomposing, powdery mildew-infected leaves on the ground and also in autoclaved soil, Ampelomyces strains developed new hyphae but did not sporulate. These results indicate that Ampelomyces strains occupy a niche in the phyllosphere where they act primarily as mycoparasites of powdery mildews. Our work has established a framework for a molecular genetic toolbox for the genus Ampelomyces using ATMT.

Green Fluorescent Protein (GFP) as a Reporter Gene for the Plant Pathogenic Oomycete Phytophthora ramorum

Transgenic Phytophthora ramorum strains that produce green fluorescent protein (GFP) constitutively were obtained after stable DNA integration using a polyethylene glycol and CaCl₂-based transformation protocol. Green fluorescent protein production was studied in developing colonies and in different propagules of the pathogen to evaluate its use in molecular and physiological studies. About 12% of the GFP transformants produced GFP to a level detectable by a confocal laser scanning microscope. Green fluorescent protein could be visualized in structures with vital protoplasm, such as hyphal tips and germinating cysts. In infection studies with Rhododendron, one of the GFP expressing strains showed aggressiveness equal to that of the corresponding non-labelled isolate. Thus, GFP could be used as a reporter gene in P. ramorum. Limitations of the technology are discussed.

Real‐time PCR quantification and live‐cell imaging of endophytic colonization of barley (Hordeum vulgare) roots byFusarium equisetiandPochonia chlamydosporia

*New tools were developed for the study of the endophytic development of the fungal species Fusarium equiseti and Pochonia chlamydosporia in barley (Hordeum vulgare) roots. These were applied to monitor the host colonization patterns of these potential candidates for biocontrol of root pathogens. * Molecular beacons specific for either F. equiseti or P. chlamydosporia were designed and used in real-time polymerase chain reaction (PCR) quantification of fungal populations in roots. Genetic transformation of isolates with the green fluorescent protein (GFP) gene was carried out using an Agrobacterium tumefaciens-mediated transformation protocol, and spatial patterns of root colonization were investigated by laser confocal microscopy. * Quantification of endophytes by real-time PCR in roots of barley gave similar results for all fungi, and was more accurate than culturing methods. Conversely, monitoring of root colonization by GFP-expressing transformants showed differences in the endophytic behaviours of the two species, and provided evidence of a plant response against endophyte colonization. * Both F. equiseti and P. chlamydosporia colonized barley roots endophytically, escaping attempts by the host to prevent fungal growth within root tissues. This strongly supports a balanced antagonism between the virulence of the colonizing endophyte and the plant defence response. Development of real-time PCR techniques and GFP transformants of these fungal species will facilitate future work to determine their biocontrol capacity.

Reporter gene transformation of the trunk disease pathogenPhaeomoniella chlamydosporaand biological control agentTrichoderma harzianum

The economically important trunk disease pathogen Phaeomoniella chlamydospora causes Petri disease in Vitis vinifera and is also associated with the Esca trunk disease complex. Not much is known about the pathogen’s epidemiology and interactions with the grapevine host, other trunk disease pathogens and biological control agents such as Trichoderma harzianum. Reporter gene labelling of plant pathogens and biocontrol agents can facilitate host-pathogen-biocontrol interaction studies. Therefore, the first aim of this study was to stably transform T. harzianum strain T77 and a South African isolate of P. chlamydospora with the Green fluorescent protein (GFP) and the Red fluorescent protein (DsRed-Express) reporter genes. Several stably transformed reporter gene labelled transformants were obtained for both fungi. The second aim was to evaluate these transformants and their respective wild type isolates for several in vitro biological characteristics, virulence (P. chlamydospora) and host colonisation (T. harzianum) on hydroponically grown grapevine cuttings. Additionally, a previously described New Zealand P. chlamydospora GFP transformant (pCT74-P7) and its wild type isolate were also biologically characterised. In vitro biological and virulence characteristics of P. chlamydospora transformants did not vary much from their respective wild type isolates. In vitro biological characterisation of T. harzianum transformants showed that one of the transformants exhibited a faster growth rate at lower temperatures. Nonetheless, characterisation of its host colonisation ability on grapevine cuttings showed no difference when compared with another transformant and the wild type isolate. All Trichoderma isolates showed differential colonisation of grapevine cultivars Chenin Blanc and Merlot.

Characterization of Serine Proteinase Expression in Agaricus bisporus and Coprinopsis cinerea by Using Green Fluorescent Protein and the A. bisporus SPR1 Promoter

The Agaricus bisporus serine proteinase 1 (SPR1) appears to be significant in both mycelial nutrition and senescence of the fruiting body. We report on the construction of an SPR promoter::green fluorescent protein (GFP) fusion cassette, pGreen_hph1_SPR_GFP, for the investigation of temporal and developmental expression of SPR1 in homobasidiomycetes and to determine how expression is linked to physiological and environmental stimuli. Monitoring of A. bisporus pGreen_hph1_SPR_GFP transformants on media rich in ammonia or containing different nitrogen sources demonstrated that SPR1 is produced in response to available nitrogen. In A. bisporus fruiting bodies, GFP activity was localized to the stipe of postharvest senescing sporophores. pGreen_hph1_SPR_GFP was also transformed into the model basidiomycete Coprinopsis cinerea. Endogenous C. cinerea proteinase activity was profiled during liquid culture and fruiting body development. Maximum activity was observed in the mature cap, while activity dropped during autolysis. Analysis of the C. cinerea genome revealed seven genes showing significant homology to the A. bisporus SPR1 and SPR2 genes. These genes contain the aspartic acid, histidine, and serine residues common to serine proteinases. Analysis of the promoter regions revealed at least one CreA and several AreA regulatory motifs in all sequences. Fruiting was induced in C. cinerea dikaryons, and fluorescence was determined in different developmental stages. GFP expression was observed throughout the life cycle, demonstrating that serine proteinase can be active in all stages of C. cinerea fruiting body development. Serine proteinase expression (GFP fluorescence) was most concentrated during development of young tissue, which may be indicative of high protein turnover during cell differentiation.

Import-associated translational inhibition: novel in vivo evidence for cotranslational protein import into Dictyostelium discoideum mitochondria.

To investigate protein import into the mitochondria of Dictyostelium discoideum, green fluorescent protein (GFP) was fused as a reporter protein either to variable lengths of the N-terminal region of chaperonin 60 (the first 23, 40, 80, 97, and 150 amino acids) or to the mitochondrial targeting sequence of DNA topoisomerase II. The fusion proteins were expressed in AX2 cells under the actin-15 promoter. Fluorescence images of GFP transformants confirmed that Dictyostelium chaperonin 60 is a mitochondrial protein. The level of the mitochondrially targeted GFP fusion proteins was unexpectedly much lower than the nontargeted (cytoplasmic) forms. The distinction between targeted and nontargeted protein activities was investigated at both the transcriptional and translational levels in vivo. We found that targeting GFP to the mitochondria results in reduced levels of the fusion protein even though transcription of the fusion gene and the stability of the protein are unaffected. [(35)S]methionine labeling and GFP immunoprecipitation confirmed that mitochondrially targeted GFP is translated at much slower rates than nontargeted GFP. The results indicate a novel phenomenon, import-associated translational inhibition, whereby protein import into the mitochondria limits the rate of translation. The simplest explanation for this is that import of the GFP fusion proteins occurs cotranslationally, i.e., protein synthesis and import into mitochondria are coupled events. Consistent with cotranslational import, Northern analysis showed that the GFP mRNA is associated with isolated mitochondria. This association occurred regardless of whether the GFP was fused to a mitochondrial leader peptide. However, the presence of an import-competent leader peptide stabilized the mRNA-mitochondria association, rendering it more resistant to extensive EDTA washing. In contrast with GFP, the mRNA of another test protein, aequorin, did not associate with the mitochondria, and its translation was unaffected by import of the encoded polypeptide into the mitochondria.

Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes

To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (“super-promoter”), mas-CaMV35s, and CAB]. GFP was tested with six A.␣tumefaciens strains, one plasmid (pLC101) and the doubleCaMV35s (dCaMV35s) promoter. The CaMV35s promoter produced more GUS expression than the CAB promoter. A. tumefaciens strains EHA105 and LBA4404 harboring the same plasmid (pBIN19) differed in their effects on GUS expression suggesting an interaction between A. tumefaciens strain and plasmid. A combination of A. tumefaciens EHA105, plasmid pBIN19 and the CaMV35s promoter produced the highest rates of transformation in peach epicotyl internodes (56.8%), cotyledons (52.7%), leaves (20%), and embryonic axes (46.7%) as evaluated by the percentage of explants expressing GUS 14 days after co-cultivation. GFP expression under the control of the dCaMV35s promoter was highest for internode explants but only reached levels of 18–19%. When GFP-containing plasmid pCL101 was combined with each of five A. tumefaciens strains the highest levels of transformation were 20–21% (internode and cotyledons, respectively). When nine peach genotypes were co-cultivated with A. tumefaciens strain EHA105 and GFP-containing plasmid pCL101 the highest levels of transformation were 26–28% (cotyledons and internodes, respectively). While GFP represents a potentially useful transformation marker that allows the non-destructive evaluation of transformation, rates of GFP transformation under the conditions of this study were low. It will be necessary to optimize expression of this marker gene in peach.

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex ( S. rolfsii) and inter-cellular growth in the medulla ( S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.

GUS and GFP transformation of the biocontrol strain Clonostachys rosea IK726 and the use of these marker genes in ecological studies

Marker genes were introduced in the biocontrol strain Clonostachys rosea IK726 (IBT 9371) as a tool for monitoring the strain in ecological studies. The β-glucuronidase (GUS) reporter gene and a gene encoding the green fluorescent protein (GFP) were, in separate experiments, integrated into the genome of IK726 using the Hygromycin B (HygB) resistance gene as selective marker. In order to select GUS and GFP transformants that resembled the wildtype strain, growth rate, production of enzymes and of metabolites of four GUS positive and six GFP positive transformants were tested in vitro. In addition, the biocontrol efficacy against disease caused by seed-borne Fusarium culmorum was evaluated on barley grown in sand. Compared to the wildtype, two selected GUS and GFP transformants, IK726c5 and IK726d11, did not vary in physiological properties. Both maintained the ability to colonize barley roots, and to reduce efficiently the severity of F. culmorum without affecting plant emergence. Quantification of GUS activity of IK726c5 in peat and vermiculite and on seeds was carried out. The GFP transformant, IK726d11, was visualized by epifluorescence and confocal scanning laser microscopy directly in soil, vermiculite, on carrot seed and roots, and on barley leaves. It was shown that C. rosea can thrive in very different niches. Conidia germination, colonization and conidiogenesis were demonstrated in vivo in all four environments. This is the first report on transformation of Clonostachys rosea with marker genes.

Influence of Soil Microbial Biomass on Growth and Biocontrol Efficac of Trichoderma harzianum

The hyphal growth and biocontrol efficacy of Trichodemo harzianum in soil may depend on its interactions with biotic components of the soil environment. The effect of soil microbial biomass on growth and biocontrol efficacy of T. hanianum isolate ThzIDl-M3 (green fluorescent protein transformant) was investigated using artificially prepared different levels of soil microbial biomass (153,328, or 517ug biomass carbon per g of dry soil; BC). The hyphal growth of T. harzanum was significantly inhibited in the soil with 328 or 517 <TEX>$\mu$</TEX>g BC compared with 153 ug BC. When ThzIDl-M3 was added to the soils as an alginate pellet formulation, the recoverable population of ThzIDl-M3 varied, but the highest population occurred in 517ug BC. Addition of alginate pellets of ThzIDl-M3 to the soils (10 per 50 g) resulted in increased indigenous microbial populations (total fungi, bacterial fluorescent Pseudomonas app., and actinomycetes). Furthermore, colonizing ability of ThzIDl-M3 on sclerotia of Sclerotinia sclerotiorum was significantly reduced in the soil with high revel of BC. These results suggest that increased soil microbial biomass contributes to increased interactions between introduced T. harzianum and soil microorganisms, consequently reducing the biocontrol efficacy of 1T. harzianum.

Microscopic analysis of the effect of azoxystrobin treatments on Mycosphaerella graminicola infection using green fluorescent protein (GFP)-expressing transformants.

Green fluorescent protein (GFP)-expressing transformants were used to investigate the effects of strobilurin fungicide azoxystrobin on Mycosphaerella graminicola infection. Azoxystrobin treatments (125 or 250 g AI ha-1) were applied at various stages of the infection process under controlled conditions. GFP transformants showed conserved in vitro sensitivity to azoxystrobin and pathogenicity. Azoxystrobin controlled over 90% of M graminicola infections when applied before or during penetration of the pathogen (15% of the incubation phase). Azoxystrobin also impaired the growth of intercellular hyphae in M graminicola post-penetration infection stages when applied at up to 50% of the incubation phase. Incubating infections observed in treated leaves were viable, but their growth was impaired and they did not induce necrosis under controlled conditions. Reduction by half of azoxystrobin dosage had little or no effect on azoxystrobin efficiency in controlling M graminicola. The contribution of post-penetration fungistatic effect to azoxystrobin curative properties toward M graminicola in a field situation is discussed.

Exploring infection of wheat and carbohydrate metabolism in Mycosphaerella graminicola transformants with differentially regulated green fluorescent protein expression.

A Mycosphaerella graminicola strain transformed with the green fluorescent protein (GFP) downstream of either a carbon source-repressed promoter or a constitutive promoter was used to investigate in situ carbohydrate uptake during penetration of the fungus in wheat leaves. The promoter region of the acu-3 gene from Neurospora crassa encoding isocitrate lyase was used as a carbon source-repressed promoter. The promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase was used as a constitutive promoter. Fluorometric measurement of GFP gene expression in liquid cultures of acu-3-regulated transformants indicated that the N. crassa acu-3 promoter functions in M. graminicola as it does in N. crassa, i.e., acetate induced and carbon source repressed. Glucose, fructose, and saccharose triggered the repression, whereas mannitol, xylose, and cell wall polysaccharides did not. Monitoring the GFP level during fungal infection of wheat leaves revealed that acu-3 promoter repression occurred after penetration until sporulation, when newly differentiated pycnidiospores fluoresced. The use of GFP transformants also allowed clear visualization of M. graminicola pathogenesis. No appressoria were formed, but penetration at cell junctions was observed. These results give new insight into the biotrophic status of M. graminicola.

Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora.

Transgenic Phytophthora palmivora strains that produce green fluorescent protein (GFP) or beta-glucuronidase (GUS) constitutively were obtained after stable DNA integration using a polyethylene-glycol and CaCl2-based transformation protocol. GFP and GUS production were monitored during several stages of the life cycle of P. palmivora to evaluate their use in molecular and physiological studies. 40% of the GFP transformants produced the GFP to a level detectable by a confocal laser scanning microscope, whereas 75% of the GUS transformants produced GUS. GFP could be visualised readily in swimming zoospores and other developmental stages of P. palmivora cells. For high magnification microscopic studies, GFP is better visualised and was superior to GUS. In contrast, for macroscopic examination, GUS was superior. Our findings indicate that both GFP and GUS can be used successfully as reporter genes in P. palmivora.

Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora

Transgenic Phytophthora palmivora strains that produce green fluorescent protein (GFP) or β-glucuronidase (GUS) constitutively were obtained after stable DNA integration using a polyethylene-glycol and CaCl 2-based transformation protocol. GFP and GUS production were monitored during several stages of the life cycle of P. palmivora to evaluate their use in molecular and physiological studies. 40% of the GFP transformants produced the GFP to a level detectable by a confocal laser scanning microscope, whereas 75% of the GUS transformants produced GUS. GFP could be visualised readily in swimming zoospores and other developmental stages of P. palmivora cells. For high magnification microscopic studies, GFP is better visualised and was superior to GUS. In contrast, for macroscopic examination, GUS was superior. Our findings indicate that both GFP and GUS can be used successfully as reporter genes in P. palmivora.