Sulfur uptake in the ectomycorrhizal fungus Laccaria bicolor S238N

The importance of the ectomycorrhiza symbiosis for plant acquisition of phosphorus and nitrogen is well established whereas its contribution to sulfur nutrition is only marginally understood. In a first step to investigate the role of ectomycorrhiza in plant sulfur nutrition, we characterized sulfate and glutathione uptake in Laccaria bicolor. By studying the regulation of sulfate uptake in this ectomycorrhizal fungus, we found that in contrast to bacteria, yeast, and plants, sulfate uptake in L. bicolor was not feedback-inhibited by glutathione. On the other hand, sulfate uptake was increased by sulfur starvation as in other organisms. The activity of 3′-phosphoadenosine 5′-phosphosulfate reductase, the key enzyme of the assimilatory sulfate reduction pathway in fungi, was increased by sulfur starvation and decreased after treatment with glutathione revealing an uncoupling of sulfate uptake and reduction in the presence of reduced sulfur compounds. These results support the hypothesis that L. bicolor increases sulfate supply to the plant by extended sulfate uptake and the plant provides the ectomycorrhizal fungus with reduced sulfur.     

Agrobacterium-mediated transformation of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces commercially valuable fruit bodies, matsutake

Using agroinfection with a T-DNA vector carrying a hygromycin resistance marker, the recombinants were generated for the first time from the ectomycorrhizal basidiomycete Tricholoma matsutake, which produces commercially valuable fruit bodies, matsutake, during association with Pinus sp. plants. The transformation system may be useful in the genetic analysis of T. matsutake.     

Occurrence and distribution of endobacteria in the plant-associated mycelium of the ectomycorrhizal fungus Laccaria bicolor S238N

Fluorescence in situ hybridization, associated with confocal laser scanning microscopy or epifluorescence microscopy with deconvolution system, has allowed the detection of a community of intracellular bacteria in non-axenic samples of the ectomycorrhizal fungus Laccaria bicolor S238N. The endobacteria, mainly alpha-proteobacteria, were present in more than half of the samples, which consisted of ectomycorrhizae, fungal mats and fruit bodies, collected in the glasshouse or in the forest. Acridine orange staining suggests that the endobacteria inhabit both live and dead fungal cells. The role of these endobacteria remains to be clarified.     

High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3

Brachypodium distachyon (Brachypodium) is a small grass with biological attributes (rapid generation time, small genome, diploid accessions, small stature and simple growth requirements) that make it suitable for use as a model system. In addition, a growing list of genomic resources have been developed or are currently under development including: cDNA libraries, BAC libraries, EST sequences, BAC end sequences, a physical map, genetic markers, a linkage map and, most importantly, the complete genome sequence. To maximize the utility of Brachypodium as a model grass it is necessary to develop an efficient Agrobacterium-mediated transformation system. In this report we describe the identification of a transformable inbred diploid line, Bd21-3, and the development of a transformation method with transformation efficiencies as high as 41% of co-cultivated calluses producing transgenic plants. Conducting the co-cultivation step under desiccating conditions produced the greatest improvement in transformation efficiency.     

Transgenic cotton: factors influencing Agrobacterium-mediated transformation and regeneration

Various aspects of transformation and regeneration processes were examined in efforts to improve the efficiency of production of transgenic cotton (Gossypium hirsutum L.). Green fluorescent protein (GFP) proved to be a valuable tool in elucidating the timing and localization of transient gene expression and in visualizing conversion of transient events to stable transformation events. By day 4 after infection, there was maximal transient activity in the cells at the cut edge of Agrobacterium-infected cotyledon disks. We were able to visualize conversion of some of these events to stable transformation by day 8. The effects of Agrobacterium strains, acetosyringone, and temperature on stable transformation were also evaluated. Strain LBA4404 proved to be significantly better than EHA105. Acetosyringone increased significantly the stable transformation efficiency in cotton. Cocultivation at 21°C, compared to 25°C, consistently resulted in higher transformation frequencies. GFP expression in stably transformed callus was useful in studying the efficiency of selection during early stages of culture. We found that the survival of individual callus lines on selection medium was influenced by their original size and initial transgene expression status. Larger-size calluses and calluses expressing the transgene (GFP) had a higher rate of survival. Survival could be improved by an additional two-week culture on medium high in cytokinin and low in auxin before transfer to a medium to induce embryogenesis. However, this treatment delayed embryogenesis. Various other important aspects of the regeneration process are described and an overall scheme for producing transgenic cotton is presented.     

Ectomycorrhiza formation between Pseudotsuga menziesii seedling roots and monokaryotic and dikaryotic isolates of Laccaria bicolor

Seedling roots of Pseudotsuga menziesii were colonized with three monokaryotic isolates and one dikaryotic isolate of Laccaria bicolor to assess the effect of fungal genotype on ectomycorrhiza formation. Ectomycorrhizas resulting from colonization by the dikaryotic isolate had a multilayered mantle and a cortical Hartig net. One monokaryotic isolate (ss7) formed ectomycorrhizas comparable in anatomy to those induced by the dikaryotic isolate. Two other monokaryotic isolates (ss5, ss1) failed to form mantles or Hartig nets. Roots colonized by these isolates developed characteristics indicating an incompatible reaction.     

Agrobacterium-mediated transformation of seedling-derived maize callus

Efficient production of seedling-derived Type I callus was demonstrated for several corn genotypes including commercial inbred lines. Seeds were germinated on MS-based medium containing 10 mg l(-1) picloram and 3 mg l(-1) 6-benzylaminopurine, which induced the development of axillary buds in the area of coleoptilar node. Nodal sections of 7-10-day old seedlings were isolated, split longitudinally, and placed on callus induction medium supplemented with 2.2 mg l(-1) picloram and 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid. For lines L4 and L9 the frequency of embryogenic callus induction was 38-42% based on calli per split nodal section. Frequency of callus induction from split nodal sections of seeds germinated on media without growth regulators was 0-3%. Seedling-derived callus of five genotypes was used for Agrobacterium-mediated transformation. Two constructs containing the green fluorescence protein gene and genes for either neomycin phosphotransferase II or glyphosate selection were used in transformation experiments. Transformation frequency varied from 2 to 11% and about 60% of the T(0) plants had 1-2 copies of transgenes.     

Agrobacterium-mediated high efficiency transformation of tall fescue (Festuca arundinacea)

Tall fescue (Festuca arundinacea) is the predominant cool-season pasture grass in the USA. Embryogenic calluses were induced from seeds/caryopsis of elite tall fescue cultivars Jesup and Kentucky-31, and were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. Agrobacterium strains LBA4404 and EHA105 harboring pCAMBIA vectors or the super-binary vector pTOK233 were used to infect the embryogenic callus pieces. The number of hygromycin resistant calluses obtained per dish of infected callus pieces was in the range of 2.0-5.8, and the number of transgenic plants recovered per dish of infected callus pieces was in the range of 0.4-1.7. When transformation efficiency was calculated based on the number of transgenic plants recovered and the number of original intact calluses used, the transformation frequency was in the range of 1.9-8.7%. The use of the easily available pCAMBIA vectors could produce equivalent results as the superbinary vector pTOK233. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis using undigested and digested genomic DNA samples. Expression of the transgenes was confirmed by northern hybridization analysis, GUS staining, and detection of GFP signals. Fertile transgenic plants were obtained after vernalization in the greenhouse. Progeny analysis revealed Mendelian inheritance of the transgenes.     

Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants

A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation.     

Agrobacterium-mediated transformation of Javanica rice

Difficulties frequently encountered using direct DNA transfer methods for transformation of Javanica varieties of rice (Oryza sativa L.) have limited the application of biotechnology to these varieties. We now reportAgrobacterium-mediated transformation of Javanica cultivars Gulfmont and Jefferson that are, respectively, widely used or about to enter commercial cultivation in the southern USA. Vigorous, phenotypically normal, fertile plants expressing both the selectable marker and the gene of interest were obtained. Southern analysis showed that only one or two copies of the T-DNA insert were present. Sequence analysis of right border fragments of one line confirmed that insertion was into a coding region of rice nuclear DNA. This analysis also revealed the presence of relatively short regions of permuted T-DNA border sequences, similar to those found afterAgrobacterium-mediated transformation of dicots. Progeny analysis of lines bearing two copies showed co-segregation, indicating that they were located relatively closely on the same chromosome. The introduced genes were transmitted to the R₁ and R₂ generations in a Mendelian fashion, confirming the suitability of this approach for biotechnological improvement of elite rice cultivars.     

Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5′ gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.     

Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.     

Fungal gene expression in early symbiotic interactions between Laccaria bicolor and red pine

Ectomycorrhizas are mutualistic symbiotic organs formed by interaction between plant roots and fungi. Mycorrhizal initiation, development and functional maintenance involve morphological changes that are mediated by activation and suppression of several fungal and plant genes. During the pre-infection stage, a harmonized cross-talk takes place between the symbionts, to determine their compatibility. Upon mutual recognition, the symbionts initiate further physiological and morphological changes essential for the formation of the symbiotic organ. In order to understand the molecular mechanisms underlying these events, we developed an interaction-specific cDNA library from Laccaria bicolor that represents fungal genes regulated by its interaction with Pinus resinosa roots. Membrane array analyses of these cDNAs suggested that a wide variety of genes are involved in the pre-infection stage processes.     

Agrobacterium-mediated genetic transformation of Prunus salicina

We report Agrobacterium tumefaciens-mediated transformation of two Prunus salicina varieties, 'Angeleno' and 'Larry Anne', using a modification of the hypocotyl slice technique previously described for P. domestica. Regeneration rates on thidiazuron (TDZ) and indole-3-butyric acid (IBA) supplemented Murashige and Skoog (MS) media reached 11% for 'Angeleno' and 19% for 'Larry Anne' hypocotyl slices. Transformation using Agrobacterium tumefaciens GV3101 harboring a plasmid with the neomycin phosphotransferase II (nptII) and the green fluorescent protein (gfp) genes produced ten independent lines, six from 'Angeleno' and four from 'Larry Anne', representing transformation efficiencies of 0.8 and 0.3%, respectively, relative to the initial number of hypocotyl slices. Plants of six lines were found to produce the transgene encoded mRNAs. DNA blotting demonstrated the presence of transgene sequences in trees from five lines after 18 months of growth in the greenhouse.     

Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L⁻¹ gellan gum-solidified NDM containing 10 g L⁻¹ sucrose, 20 mg L⁻¹ hygromycin and 40 mg L⁻¹ meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 μM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.     

Population structure and mycelial phenotypic variability of the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton

Mating type allele distribution and phenotypic variability were investigated in field populations of Laccaria bicolor. Sporophores associated with Norway spruce (Picea abies), colonized by natural sources of inoculum and growing in a seed orchard, were sampled to obtain dikaryotic strains and assay their phenotypic variability for traits important to the symbiosis. Basid-iospores were also collected for mating type analysis of different mycelia. Four sporophore mating types were identified containing seven A and five B factors. Out-breeding efficiency was estimated at 73.8% and the population was slightly inbred. Crosses with previously characterized L. bicolor strains from two nearby populations identified in total six sporophore mating types and ten A and nine B factors, for an estimated outbreeding efficiency (85.7%) similar to previous studies of more spatially disparate Laccaria spp. populations. Dikaryotic strains were tested for mycelial growth rate, as a measure of their competitive ability, on agar media containing a soluble (NaH₂PO₄), or an insoluble (CaHPO₄) phosphate source. Their ability to solubilize the latter was also tested to assess their relative capacity to access insoluble, inorganic phosphate. In most cases, significant variation was detected among strains from the same site for all variables. On three sites (VC4, VC5 and VC7), each determined previously to possess a uniform mycelial genotype, phenotypic variability was likely due to epigenetic variation among different strains of the same genotype. Possible evidence for dikaryon-monokaryon crosses was observed in vivo on one sample site (VC2) where adjacent mycelia shared two mating factors. The phenotypic variability of different mycelial genotypes reflected their genetic variability observed as mating type allele diversity and underlined the importance of basidiospore dispersal in introducing new genotypes into the population. The reproductive strategies of L. bicolor are discussed and compared to those of other basidiomycete species.     

Agrobacterium-mediated transformation of pepino and regeneration of transgenic plants

Regeneration of pepino (Solanum muricatum Ait.) shoots was achieved both by organogenesis and by embryogenesis. Shoots derived via organogenesis were easily rooted and most regenerated plants appeared phenotypically normal. Transgenic plants were obtained using the binary vector pKIWI110 in the avirulent Agrobacterium tumefaciens strain LBA4404. Optimization of transformation protocols was rapidly achieved by monitoring early expression of the GUS (-D-glucuronidase) reporter gene carried on pKIWI110. Transgenic plants expressed GUS and selectable marker genes for kanamycin resistance and chlorsulfuron resistance. PCR (polymerase chain reaction) and Southern analysis provided molecular evidence for transformation.     

In situ identification of intracellular bacteria related to Paenibacillus spp. in the mycelium of the ectomycorrhizal fungus Laccaria bicolor S238N

Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 micro m in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N.     

Agrobacterium-mediated transformation protocol to overcome necrosis in elite AustralianBrassica juncea lines

In recent years,Brassica species have acquired an important position in the oilseed industry. Even thoughBrassica transformation protocols are well established,there is still a need for the development of new transformation protocols for elite AustralianB. juncea lines,because regeneration inB. juncea is highly genotype-dependent and in addition, their hypocotyl explants are susceptible to necrosis.Agrobacterium-mediated transformation protocol to overcome necrosis in elite AustralianB. juncea lines is described here. To overcome necrosis, we have adopted 2 strategies: extension of precultivation time of hypocotyl explants, and use of a 2-stage hygromycin selection process.The frequency of recovery of transformants from AustralianB. juncea andBrassica napus lines was 1.7% and 0.9%, respectively. Polymerase chain reaction tests confirmed that allBrassica plants that survived through stringent screening procedures were positive for the inserted hygromycin resistance gene,hph. Progeny from 6Brassica lines tested segregated for thehph gene, and χ² analysis suggested a 3:1 segregation ratio.This is in line with a tDNA integration into a single locus, which is an important feature of a transformation protocol for subsequent breeding purposes. Although the scientific content of this article has been reviewed,the full-text Web publication has not been edited in detail.