RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus

Mycorrhizal symbioses are a rule in nature and may have been crucial in plant and fungal evolution. Ectomycorrhizas are mutualistic interactions between tree roots and soil fungi typical of temperate and boreal forests. The functional analysis of genes involved in developmental and metabolic processes, such as N nutrition, is important to understand the ontogeny of this mutualistic symbiosis. RNA silencing was accomplished in the model mycorrhizal fungus Laccaria bicolor by Agrobacterium‐mediated gene transfer. Promoter‐directed expression of double‐stranded RNA with a partial coding sequence of the Laccaria nitrate reductase gene resulted in fungal transgenic strains strongly affected in growth with nitrate as N source in a medium with high concentration of an utilizable C source. The phenotype correlated with a clear reduction of the target gene mRNA level and this effect was not caused by homologous recombination of the T‐DNA in the nitrate reductase locus. Transformation with the hairpin sequence resulted in specific CpG methylation of both the silenced transgene and the nitrate reductase encoding gene. The methylation in the target gene was restricted to the silencing trigger sequence and did not represent the entire genomic DNA in the dikaryon suggesting that the epigenetic changes accompanying RNA silencing affected only the transformed nucleus. Mycorrhization experiments of Populus with strongly silenced fungal strains revealed a systematic inhibition of symbiosis under mycorrhization conditions (C starvation) and nitrate as N source compared with the wild type. This inhibition of mycorrhization was reversed by an organic N source only utilizable by the fungus. These observations would indicate that the plant may be capable of monitoring and detecting the nutritional status of a potential symbiont avoiding the establishment of an unsatisfactory interaction. A probable control mechanism conducted by the plant would inhibit symbiosis when the metabolic profile of the fungal partner is not proper and mutual benefit from the symbiotic structure cannot be assured. Our results are the first report showing that the alteration of expression of a fungal gene impairs mycorrhization. Moreover, this work is the first demonstration of RNA silencing in mycorrhizal fungi and clearly shows that gene knock‐down is a powerful tool for further functional genomic studies in mycorrhizal research.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation (AMT) of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> as an efficient tool for random insertional mutagenesis

Filamentous fungus Trichoderma reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell division. The integration of the hph gene into T. reesei genome was determined by PCR and dot blot analysis. Transgenic T. reesei strains were analyzed using TAIL-PCR for their T-DNA contents. The results showed that T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination, which suggests that Agrobacterium-mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene transfer technology for T. reesei.     

Distribution and characterization of more than 1000 T‐DNA tags in the genome of Brachypodium distachyon community standard line Bd21

A collection of 4117 fertile T‐DNA lines has been generated by Agrobacterium‐mediated transformation of the diploid community standard line Bd21 of Brachypodium distachyon. The regions flanking the T‐DNA left and right borders of the first 741 transformed plants were isolated by adapter‐ligation PCR and sequenced. A total of 1005 genomic sequences (representing 44.1% of all flanking sequences retrieved) characterized 660 independent T‐DNA loci assigned to a unique location in the Brachypodium genome sequence. Seventy‐six percent of the fertile plant lines contained at least one anchored T‐DNA locus (1.17 loci per tagged line on average). Analysis of the regions flanking both borders of the T‐DNA increased the number of T‐DNA loci tagged and the number of tagged lines by approximately 50% when compared to a single border analysis. T‐DNA integration (2.4 insertions per Mb on average) was proportional to chromosome size, however, varied greatly along each chromosome with often low insertion level around centromeres. The frequency of insertion within transposable elements (5.3%) was fivefold lower than expected if random insertion would have occurred. More than half of the T‐DNAs inserted in genic regions. On average, one gene could be tagged for every second fertile plant line produced and more than one plant line out of three contained a T‐DNA insertion directly within or 500 bp around the coding sequence. Approximately, 60% of the genes tagged corresponded to expressed genes. The T‐DNA lines generated by the BrachyTAG programme are available as a community resource and have been distributed internationally since 2008 via the BrachyTAG.org web site.     

Multiple host‐cell recombination pathways act in Agrobacterium‐mediated transformation of plant cells

Using floral‐dip, tumorigenesis and root callus transformation assays of both germline and somatic cells, we present here results implicating the four major non‐homologous and homologous recombination pathways in Agrobacterium‐mediated transformation of Arabidopsis thaliana. All four single mutant lines showed similar mild reductions in transformability, but knocking out three of four pathways severely compromised Agrobacterium‐mediated transformation. Although integration of T–DNA into the plant genome is severely compromised in the absence of known DNA double‐strand break repair pathways, it does still occur, suggesting the existence of other pathways involved in T–DNA integration. Our results highlight the functional redundancy of the four major plant recombination pathways in transformation, and provide an explanation for the lack of strong effects observed in previous studies on the roles of plant recombination functions in transformation.     

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 g–1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.     

DNA cloning and screening of a partial genomic library from an arbuscular mycorrhizal fungus,Scutellospora castanea

A technique has been developed to efficiently extract purified, restrictable genomic DNA from spores of different arbuscular mycorrhizal fungi in order to begin detailed investigations of the genome of the Glomales. The protocol yielded variable amounts of DNA depending on the fungal species; for Scutellospora castanea and Gigaspora rosea it reached values of 1.5–2 ng/spore. EcoRI digests of DNA from S. castanea were cloned into pUC18 and about 1000 recombinant DNA clones were obtained. Of those screened, 50 contained inserts of 500–7000 bp. Selected inserts detected DNA sequences from S. castanea spores or roots infected by this fungus, but not from nonmycorrhizal roots. This is the first report of a partial genomic library from an arbuscular mycorrhizal fungus.     

Site‐specific T–DNA integration in Arabidopsis thaliana mediated by the combined action of CRE recombinase and ϕC31 integrase

Random T–DNA integration into the plant host genome can be problematic for a variety of reasons, including potentially variable transgene expression as a result of different integration positions and multiple T–DNA copies, the risk of mutating the host genome and the difficulty of stacking well‐defined traits. Therefore, recombination systems have been proposed to integrate the T–DNA at a pre‐selected site in the host genome. Here, we demonstrate the capacity of the ?C31 integrase (INT) for efficient targeted T–DNA integration. Moreover, we show that the iterative site‐specific integration system (ISSI), which combines the activities of the CRE recombinase and INT, enables the targeting of genes to a pre‐selected site with the concomitant removal of the resident selectable marker. To begin, plants expressing both the CRE and INT recombinase and containing the target attP site were constructed. These plants were supertransformed with a T–DNA vector harboring the loxP site, the attB sites, a selectable marker and an expression cassette encoding a reporter protein. Three out of the 35 transformants obtained (9%) showed transgenerational site‐specific integration (SSI) of this T–DNA and removal of the resident selectable marker, as demonstrated by PCR, Southern blot and segregation analysis. In conclusion, our results show the applicability of the ISSI system for precise and targeted Agrobacterium‐mediated integration, allowing the serial integration of transgenic DNA sequences in plants.     

Biolistic mediated site-specific integration in rice

Cre-lox mediated site-specific integration in tobacco or Arabidopsis used polyethylene glycol or Agrobacterium, respectively, to deliver the integrating DNA. The polyethylene glycol method is inconvenient since it requires the use of protoplasts. The Agrobacterium method is inefficient as the single-stranded T-DNA is not a substrate for Cre-lox recombination. In this study, we tested the biolistic method for the site-specific insertion of DNA into the rice genome. Two target callus lines, each harboring a single genomic lox target, were generated by Agrobacterium-mediated transformation. The target callus lines were subjected to a second round of transformation by particle bombardment with a construct designed to excise the plasmid backbone from the integrating DNA, followed by the recombination of the integrating DNA into the genomic lox target. Site-specific integration was obtained from both target callus lines. Three integrant plants were regenerated from one target line and were found to have a precise copy of the integrating DNA at the target site, although only one plant has the integrating DNA as the sole copy in the genome. Site-specific integration through the biolistic delivery of DNA can be considered for other plants that are transformable via particle bombardment.     

Termini and telomeres in T-DNA transformation

A T-DNA vector for plant transformation has been constructed in which the cloning site is located 9 bp from the right-border (RB) end and 27 bp from the left-border (LB) end. In this vector cloned DNA homologous to plant chromosomal sequences is located at the T-DNA termini, and will thus be exposed by even limited exonucleolysis in planta. The arabidopsis ADH (alcohol dehydrogenase) locus was mobilized from Agrobacterium, and integration into the recipient genome was studied. Despite the terminal location of ADH homology in this vector, the T-DNA integrated essentially at random in the Arabidopsis genome rather than at the endogenous ADH locus. T-DNA integration was blocked, however, when Arabidopsis telomeric sequences were added to the construct at each end of the ADH homology. Thus the predominant mode by which incoming T-DNA is integrated into the continuity of chromosomal DNA involves free DNA ends, but, in contrast to modes of recombination such as gap repair, does not involve extensive terminal DNA sequence homology.     

Agrobacterium T‐DNA integration into the plant genome can occur without the activity of key non‐homologous end‐joining proteins

Non‐homologous end joining (NHEJ) is the major model proposed for Agrobacterium T‐DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA‐PKcs, DNA ligase IV (LIG4), Ataxia telangiectasia mutated (ATM), and ATM‐ and Rad3‐related (ATR), play an important role in ‘classical’ (c)NHEJ. Other proteins, including histone H1 (HON1), XRCC1, and PARP1, participate in a ‘backup’ (b)NHEJ process. We examined transient and stable transformation frequencies of Arabidopsis thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, and the plant‐specific DNA LIGASE VI (LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild‐type plants, suggesting enhanced T‐DNA integration in these mutants. These results were confirmed using a promoter‐trap transformation vector that requires T‐DNA integration into the plant genome to activate a promoterless gusA (uidA) gene, by virus‐induced gene silencing (VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T‐DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, lig4, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T‐DNA integration and transgene methylation. A double mutant (ku80/parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T‐DNA integration. Thus, T‐DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration.     

Homologous recombination and allele replacement in transformants of Fusarium fujikuroi

The ascomycete Fusarium fujikuroi could be transformed stably to hygromycin resistance only when the transforming plasmid contained a fragment of DNA from the fungus. The transformation frequencies were roughly independent of the sequence of the particular fungal DNA fragment used, of its size (1.8 or 6 kb), and of whether this DNA was present only once in the fungal genome or about forty times (the genes for ribosomal RNA). The plasmid was integrated into the fungal genome by homologous recombination in the eighteen transformants tested; ectopic integration was never observed. The carB gene of F. fujikuroi was cloned and shown to complement unpigmented mutants deficient in phytoene dehydrogenase. A mutant carB allele was prepared in vitro and used to transform wild-type protoplasts; the transformants contained a genomic duplication and were heterozygous for carB; the mutant allele replaced the original wild-type allele when this was spontaneously lost in the transformants. This loss was due to gene conversion in some cases and to recombination between repeated sequences in others. Received: 5 November 1999 / Accepted: 16 March 2000     

Landscape genomics of an obligate mutualism: Concordant and discordant population structures between the leafcutter ant Atta texana and its two main fungal symbiont types

To explore landscape genomics at the range limit of an obligate mutualism, we use genotyping‐by‐sequencing (ddRADseq) to quantify population structure and the effect of host–symbiont interactions between the northernmost fungus‐farming leafcutter ant Atta texana and its two main types of cultivated fungus. Genome‐wide differentiation between ants associated with either of the two fungal types is of the same order of magnitude as differentiation associated with temperature and precipitation across the ant's entire range, suggesting that specific ant–fungus genome–genome combinations may have been favoured by selection. For the ant hosts, we found a broad cline of genetic structure across the range, and a reduction of genetic diversity along the axis of range expansion towards the range margin. This population‐genetic structure was concordant between the ants and one cultivar type (M‐fungi, concordant clines) but discordant for the other cultivar type (T‐fungi). Discordance in population‐genetic structures between ant hosts and a fungal symbiont is surprising because the ant farmers codisperse with their vertically transmitted fungal symbionts. Discordance implies that (a) the fungi disperse also through between‐nest horizontal transfer or other unknown mechanisms, and (b) genetic drift and gene flow can differ in magnitude between each partner and between different ant–fungus combinations. Together, these findings imply that variation in the strength of drift and gene flow experienced by each mutualistic partner affects adaptation to environmental stress at the range margin, and genome–genome interactions between host and symbiont influence adaptive genetic differentiation of the host during range evolution in this obligate mutualism.     

A Chitinase Encoding Gene (chit1 Gene) from the Entomopathogen Metarhizium anisopliae: Isolation and Characterization of Genomic and Full-Length cDNA

There are no reports to date of entire gene sequences coding for chitinolytic enzymes from entomopathogenic fungi, even though these enzymes act synergistically with proteolytic enzymes to solubilize insect cuticle during the key step of host penetration, having considerable importance in the biological control of some insect pests. This paper reports the complete nucleotide sequence and analysis of the chromosomal and full-length cDNA copies of the regulated gene (chit1) coding one of the chitinases produced by the biocontrol agent Metarhizium anisopliae. Degenerated primers, encompassing conserved regions of other fungal chitinases, were used to amplify a 650-bp DNA fragment, which was used to isolate genomic and cDNA clones from M. anisopliae. Albeit at least two different chitinases are characterized in this fungus, only one chit gene was isolated. The chit1 gene is interrupted by three short typical fungal introns and has a 1,521-bp ORF, which encodes a protein of 423 amino acids with a stretch of 35 amino acid residues displaying characteristics of signal peptide. The deduced sequence of the mature protein predicts a 42-kDa protein with pI of 5.8. Southern analysis of genomic DNA indicates a single copy of chit1 in the M. anisopliae genome. Received: 13 March 1998 / Accepted: 14 April 1998     

Determination of Diversity,Distribution and Host Specificity of Korean Laccaria Using Four Approaches

The genus Laccaria (Hydnangiaceae, Agaricales) plays an important role in forest ecosystems as an ectomycorrhizal fungus, contributing to nutrient cycles through symbiosis with many types of trees. Though understanding Laccaria diversity and distribution patterns, as well as its association with host plants, is fundamental to constructing a balanced plant diversity and conducting effective forest management, previous studies have not been effective in accurately investigating, as they relied heavily on specimen collection alone. To investigate the true diversity and distribution pattern of Laccaria species and determine their host types, we used four different approaches: specimen-based analysis, open database search (ODS), NGS analysis, and species-specific PCR (SSP). As a result, 14 Laccaria species have been confirmed in Korea. Results regarding the species distribution pattern were different between specimen-based analysis and SSP. However, when both were integrated, the exact distribution pattern of each Laccaria species was determined. In addition, the SSP revealed that many Laccaria species have a wide range of host types. This study shows that using these four different approaches is useful in determining the diversity, distribution, and host of ECM fungi. Furthermore, results obtained for Laccaria will serve as a baseline to help understand the role of ECM fungi in forest management in response to climate change.     

Agrobacterium‐mediated transformation of reed (Phragmites communis Trinius) using mature seed‐derived calli

Reed (Phragmites communis) is a potential bioenergy plant. We report on its first Agrobacterium‐mediated transformation using mature seed‐derived calli. The Agrobacterium strains LBA4404, EHA105, and GV3101, each harboring the binary vector pIG121Hm, were used to optimize T‐DNA delivery into the reed genome. Bacterial strain, cocultivation period and acetosyringone concentration significantly influenced the T‐DNA transfer. About 48% transient expression and 3.5% stable transformation were achieved when calli were infected with strain EHA105 for 10 min under 800 mbar negative pressure and cocultivated for 3 days in 200 μm acetosyringone containing medium. Putative transformants were selected in 25 mg l^?1 hygromycin B. PCR, and Southern blot analysis confirmed the presence of the transgenes and their stable integration. Independent transgenic lines contained one to three copies of the transgene. Transgene expression was validated by RT‐PCR and GUS staining of stems and leaves.     

Arbuscular mycorrhizal fungi change host plant DNA methylation systemically

• DNA methylation is an important epigenetic mechanism regulating gene expression in plants. DNA methylation has been shown to vary among species and also among plant tissues. However, no study has evaluated whether arbuscular mycorrhizal (AM) fungi affect DNA methylation levels in a tissue‐specific manner.
• We investigated whether symbiosis with AM fungi affects DNA methylation in the host, focusing on different plant tissues (roots versus leaves) and across time. We carried out a 6‐month pot experiment using Geranium robertianum in symbiosis with the AM fungus Funneliformis mosseae.
• Our results show that the pattern of total DNA methylation differed between leaves and roots and was related to when plants were harvested, confirming that DNA methylation is a process that occurs dynamically throughout an organism's lifetime. More importantly, the presence of AM fungus in roots of our experimental plants had a positive effect on total DNA methylation in both tissues.
• This study shows that colonisation by AM fungi can affect DNA methylation levels in their hosts and that plant DNA methylation varies in an age‐ and tissue‐specific manner.