Extensive Intra-Kingdom Horizontal Gene Transfer Converging on a Fungal Fructose Transporter Gene

Comparative genomics revealed in the last decade a scenario of rampant horizontal gene transfer (HGT) among prokaryotes, but for fungi a clearly dominant pattern of vertical inheritance still stands, punctuated however by an increasing number of exceptions. In the present work, we studied the phylogenetic distribution and pattern of inheritance of a fungal gene encoding a fructose transporter (FSY1) with unique substrate selectivity. 109 FSY1 homologues were identified in two sub-phyla of the Ascomycota, in a survey that included 241 available fungal genomes. At least 10 independent inter-species instances of horizontal gene transfer (HGT) involving FSY1 were identified, supported by strong phylogenetic evidence and synteny analyses. The acquisition of FSY1 through HGT was sometimes suggestive of xenolog gene displacement, but several cases of pseudoparalogy were also uncovered. Moreover, evidence was found for successive HGT events, possibly including those responsible for transmission of the gene among yeast lineages. These occurrences do not seem to be driven by functional diversification of the Fsy1 proteins because Fsy1 homologues from widely distant lineages, including at least one acquired by HGT, appear to have similar biochemical properties. In summary, retracing the evolutionary path of the FSY1 gene brought to light an unparalleled number of independent HGT events involving a single fungal gene. We propose that the turbulent evolutionary history of the gene may be linked to the unique biochemical properties of the encoded transporter, whose predictable effect on fitness may be highly variable. In general, our results support the most recent views suggesting that inter-species HGT may have contributed much more substantially to shape fungal genomes than heretofore assumed.     

Citrullination Was Introduced into Animals by Horizontal Gene Transfer from Cyanobacteria

Protein posttranslational modifications add great sophistication to biological systems. Citrullination, a key regulatory mechanism in human physiology and pathophysiology, is enigmatic from an evolutionary perspective. Although the citrullinating enzymes peptidylarginine deiminases (PADIs) are ubiquitous across vertebrates, they are absent from yeast, worms, and flies. Based on this distribution PADIs were proposed to have been horizontally transferred, but this has been contested. Here, we map the evolutionary trajectory of PADIs into the animal lineage. We present strong phylogenetic support for a clade encompassing animal and cyanobacterial PADIs that excludes fungal and other bacterial homologs. The animal and cyanobacterial PADI proteins share functionally relevant primary and tertiary synapomorphic sequences that are distinct from a second PADI type present in fungi and actinobacteria. Molecular clock calculations and sequence divergence analyses using the fossil record estimate the last common ancestor of the cyanobacterial and animal PADIs to be less than 1 billion years old. Additionally, under an assumption of vertical descent, PADI sequence change during this evolutionary time frame is anachronistically low, even when compared with products of likely endosymbiont gene transfer, mitochondrial proteins, and some of the most highly conserved sequences in life. The consilience of evidence indicates that PADIs were introduced from cyanobacteria into animals by horizontal gene transfer (HGT). The ancestral cyanobacterial PADI is enzymatically active and can citrullinate eukaryotic proteins, suggesting that the PADI HGT event introduced a new catalytic capability into the regulatory repertoire of animals. This study reveals the unusual evolution of a pleiotropic protein modification.     

Biochemical Analysis of Plant Protection Afforded by a Nonpathogenic Endophytic Mutant of Colletotrichum magna

A nonpathogenic mutant of Colletotrichum magna (path-1) was previously shown to protect watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) seedlings from anthracnose disease elicited by wild-type C. magna. Disease protection was observed in stems of path-1-colonized cucurbits but not in cotyledons, indicating that path-1 conferred tissue-specific and/or localized protection. Plant biochemical indicators of a localized and systemic (peroxidase, phenylalanine ammonia-lyase, lignin, and salicylic acid) “plant-defense” response were investigated in anthracnose-resistant and -susceptible cultivars of cucurbit seedlings exposed to four treatments: (1) water (control), (2) path-1 conidia, (3) wild-type conidia, and (4) challenge conditions (inoculation into path-1 conidia for 48 h and then exposure to wild-type conidia). Collectively, these analyses indicated that disease protection in path-1-colonized plants was correlated with the ability of these plants to mount a defense response more rapidly and to equal or greater levels than plants exposed to wild-type C. magna alone. Watermelon plants colonized with path-1 were also protected against disease caused by Colletotrichum orbiculare and Fusarium oxysporum. A model based on the kinetics of plant-defense activation is presented to explain the mechanism of path-1-conferred disease protection.     

在烟草中酵母脯氨酸基因B的转化(英文)

重组质粒PYP22带有一从酵母基因文库分离到的4.6 kb DNA片段,此片段含有脯氨酸(Pro)合成途径必须的基因B(ProB)。用BamHⅠ酶解PYP22,回收ProB基因,重组入pGA471的Bg Ⅲ切口,形成含有ProB的双质粒载体PBYU4。pBYU4含有能在植物中表达的新霉素磷酸转移酶基因Ⅱ(NPT—Ⅱ),可做转基因植株的筛选标记。借助于辅助质粒pRK2013,pBYU4经过三亲结合转移到农杆菌LAB4404,在含有四环素12.5μg/ml、链霉素100μg/ml和利福平50μ/ml加的AB培养基上筛选出转接合子。提取筛选得到的农杆菌总DNA,用ECoR 1酶解,1％琼脂糖电泳,Southern转移DNA到硝酸纤维素膜上。用α—~(32)P-dCTP标记的ProB片段,与转移好的硝酸纤维素进行Southern杂交。Southern杂交证明含有ProB基因的农杆菌,在加有乙酸丁香酮(acetosyringone)125μg/ml和章鱼碱(octopine)125μg/ml的MS液体培养基中,诱导过夜。用叶圆片法转化革新1号烟草(Nicotana tabacum var.Gexin No.1),叶片与菌液共培养2～5d。从叶片分化再生出的芽转移到含有卡那霉素(K_m)80μg/ml、6—苄基腺嘌呤(6BA)1μg/ml和头孢氨噻腭钠(Cef)500μg/ml的MS培养基,筛选3周,将绿色的芽转移到含l％NaCl,6BA 1μg/ml和Cef 500μg/ml的MS培养基,进行复筛。测定经过这样筛选的再生芽的NPT—Ⅱ活性。     

The Role of Horizontal Gene Transfer by Bacteriophages in the Origin of Pathogenic Bacteria

The review considers the involvement of bacteriophages in transferring genes, which determine bacterial pathogenicity, and the increasing role of comparative genomics and genetics of bacteria and bacteriophages in detecting new cases of horizontal gene transfer. Examples of phage participation in this process proved to a different extent are described. Emphasis is placed on the original work carried out in Russia and focused on bacteriophages (temperate transposable phages and giant virulent KZ-like phages) of conditional pathogen Pseudomonas aeruginosa.Consideration is given to the possible lines of further research of the role of bacteriophages in the infection process and, in particular, the role of virulent phages, whose products are similar to those of pathogenic bacteria, in modification of clinical signs of infectious diseases and in evolution. An attempt is made to predict the possible direction of pathogen evolution associated with development of new treatment strategies and generation of new specific niches.     

Identification of Horizontal Gene Transfer from Phylogenetic Gene Trees

We suggest a new procedure to search for the genes with horizontal transfer events in their evolutionary history. The search is based on analysis of topology difference between the phylogenetic trees of gene (protein) groups and the corresponding phylogenetic species trees. Numeric values are introduced to measure the discrepancy between the trees. This approach was applied to analyze 40 prokaryotic genomes classified into 132 classes of orthologs. This resulted in a list of the candidate genes for which the hypothesis of horizontal transfer in evolution looks true.     

Homology-dependent DNA Transfer from Plants to a Soil Bacterium Under Laboratory Conditions: Implications in Evolution and Horizontal Gene Transfer

DNA transfer was demonstrated from six species of donor plants to the soil bacterium, Acinetobacter spp. BD413, using neomycin phosphotransferase (nptII) as a marker for homologous recombination. These laboratory results are compatible with, but do not prove, DNA transfer in nature. In tobacco carrying a plastid insertion of nptII, transfer was detected with 0.1 g of disrupted leaves and in oilseed rape carrying a nuclear insertion with a similar quantity of roots. Transfer from disrupted leaves occurred in sterile soil and water, without the addition of nutrients. It was detected using intact tobacco leaves and intact tobacco and Arabidopsis plants in vitro. Transfer was dose-dependent and sensitive to DNase, and mutations in the plant nptII were recovered in receptor bacteria. DNA transfer using intact roots and plants in vitro was easily demonstrated, but with greater variability. Transfer varied with plant genome size and the number of repeats of the marker DNA in the donor plant. Transfer was not detected in the absence of a homologous nptII in the receptor bacteria. We discuss these results with reference to non-coding DNA in plant genomes (e.g., introns, transposons and junk DNA) and the possibility that DNA transfer could occur in nature.     

Gene Order Phylogeny of the Genus Prochlorococcus

Background

Using gene order as a phylogenetic character has the potential to resolve previously unresolved species relationships. This character was used to resolve the evolutionary history within the genus Prochlorococcus, a group of marine cyanobacteria.

Methodology/Principal Findings

Orthologous gene sets and their genomic positions were identified from 12 species of Prochlorococcus and 1 outgroup species of Synechococcus. From this data, inversion and breakpoint distance-based phylogenetic trees were computed by GRAPPA and FastME. Statistical support of the resulting topology was obtained by application of a 50% jackknife resampling technique. The result was consistent and congruent with nucleotide sequence-based and gene-content based trees. Also, a previously unresolved clade was resolved, that of MIT9211 and SS120.

Conclusions/Significance

This is the first study to use gene order data to resolve a bacterial phylogeny at the genus level. It suggests that the technique is useful in resolving the Tree of Life.     

Epigenetic Control of Effector Gene Expression in the Plant Pathogenic Fungus Leptosphaeria maculans

Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions.     

History of the ADP/ATP-Translocase-Encoding Gene, a Parasitism Gene Transferred from a Chlamydiales Ancestor to Plants 1 Billion Years Ago

Nonmitochondrial ADP/ATP translocase is an energy parasite enzyme. Its encoding gene, tlc, is found only in Rickettsiales, Chlamydiales, and plant and alga plastids. We demonstrate the presence of tlc in Parachlamydia acanthamoebae. This gene shares more similarity with the tlc1 gene of Chlamydiaceae and the tlc of plant and alga plastids than with the tlc2 gene of Chlamydiaceae. Phylogenetic analysis, including all other tlc homologs found in GenBank, showed that tlc was duplicated in a Chlamydiales ancestor before the appearance of multicellular eukaryotes. A time scale, calibrated with seven independent time points obtained from fossil estimates and from the 16S rRNA molecular clock, was congruent with the molecular clock provided by tlc. Plant and alga plastids acquired tlc approximately when Parachlamydiaceae and Chlamydiaceae diverged, at the eucaryotic radiation time, ca. 1 billion years ago.     

Phylogenomic Analysis Demonstrates a Pattern of Rare and Ancient Horizontal Gene Transfer between Plants and Fungi

Horizontal gene transfer (HGT) describes the transmission of genetic material across species boundaries and is an important evolutionary phenomenon in the ancestry of many microbes. The role of HGT in plant evolutionary history is, however, largely unexplored. Here, we compare the genomes of six plant species with those of 159 prokaryotic and eukaryotic species and identify 1689 genes that show the highest similarity to corresponding genes from fungi. We constructed a phylogeny for all 1689 genes identified and all homolog groups available from the rice (Oryza sativa) genome (3177 gene families) and used these to define 14 candidate plant-fungi HGT events. Comprehensive phylogenetic analyses of these 14 data sets, using methods that account for site rate heterogeneity, demonstrated support for nine HGT events, demonstrating an infrequent pattern of HGT between plants and fungi. Five HGTs were fungi-to-plant transfers and four were plant-to-fungi HGTs. None of the fungal-to-plant HGTs involved angiosperm recipients. These results alter the current view of organismal barriers to HGT, suggesting that phagotrophy, the consumption of a whole cell by another, is not necessarily a prerequisite for HGT between eukaryotes. Putative functional annotation of the HGT candidate genes suggests that two fungi-to-plant transfers have added phenotypes important for life in a soil environment. Our study suggests that genetic exchange between plants and fungi is exceedingly rare, particularly among the angiosperms, but has occurred during their evolutionary history and added important metabolic traits to plant lineages.     

Evolutionary History of the Plant Pathogenic Bacterium Xanthomonas axonopodis

Deciphering mechanisms shaping bacterial diversity should help to build tools to predict the emergence of infectious diseases. Xanthomonads are plant pathogenic bacteria found worldwide. Xanthomonas axonopodis is a genetically heterogeneous species clustering, into six groups, strains that are collectively pathogenic on a large number of plants. However, each strain displays a narrow host range. We address the question of the nature of the evolutionary processes – geographical and ecological speciation – that shaped this diversity. We assembled a large collection of X. axonopodis strains that were isolated over a long period, over continents, and from various hosts. Based on the sequence analysis of seven housekeeping genes, we found that recombination occurred as frequently as point mutation in the evolutionary history of X. axonopodis. However, the impact of recombination was about three times greater than the impact of mutation on the diversity observed in the whole dataset. We then reconstructed the clonal genealogy of the strains using coalescent and genealogy approaches and we studied the diversification of the pathogen using a model of divergence with migration. The suggested scenario involves a first step of generalist diversification that spanned over the last 25 000 years. A second step of ecology-driven specialization occurred during the past two centuries. Eventually, secondary contacts between host-specialized strains probably occurred as a result of agricultural development and intensification, allowing genetic exchanges of virulence-associated genes. These transfers may have favored the emergence of novel pathotypes. Finally, we argue that the largest ecological entity within X. axonopodis is the pathovar.     

Visual Evidence of Horizontal Gene Transfer between Plants and Bacteria in the Phytosphere of Transplastomic Tobacco

Plant surfaces, colonized by numerous and diverse bacterial species, are often considered hot spots for horizontal gene transfer (HGT) between plants and bacteria. Plant DNA released during the degradation of plant tissues can persist and remain biologically active for significant periods of time, suggesting that soil or plant-associated bacteria could be in direct contact with plant DNA. In addition, nutrients released during the decaying process may provide a copiotrophic environment conducive for opportunistic microbial growth. Using Acinetobacter baylyi strain BD413 and transplastomic tobacco plants harboring the aadA gene as models, the objective of this study was to determine whether specific niches could be shown to foster bacterial growth on intact or decaying plant tissues, to develop a competence state, and to possibly acquire exogenous plant DNA by natural transformation. Visualization of HGT in situ was performed using A. baylyi strain BD413(rbcL-ΔPaadA::gfp) carrying a promoterless aadA::gfp fusion. Both antibiotic resistance and green fluorescence phenotypes were restored in recombinant bacterial cells after homologous recombination with transgenic plant DNA. Opportunistic growth occurred on decaying plant tissues, and a significant proportion of the bacteria developed a competence state. Quantification of transformants clearly supported the idea that the phytosphere constitutes a hot spot for HGT between plants and bacteria. The nondisruptive approach used to visualize transformants in situ provides new insights into environmental factors influencing HGT for plant tissues.Despite the annually increasing acreage planted with genetically modified plants worldwide, the ongoing debate on their ecological safety is controversial and gave impetus to different studies of the putative horizontal gene transfer (HGT) of recombinant DNA from plant to bacteria (12, 30). Research regarding the fate of plant transgenes in environmental microbial communities is driven by practical societal concerns related to the potential dissemination of antibiotic resistance determinants in the environment and by fundamental evolution questions about gene transfer between species and kingdoms. Different parts of a plant (globally defined as the phytosphere) support the growth of numerous and diverse bacteria that colonize the surfaces or internal tissues and display advantageous, neutral, or pathogenic functions toward the plant (1, 9, 22). However, the plant as a whole is exposed to many environmental challenges and does not always provide the same favorable conditions for bacterial growth. The latter depends on several factors, such as the presence of nutrients, moisture, shelter from desiccation and UV, and shelter from grazing and predation, all of which fluctuate rapidly and are heterogeneously distributed in and on the plant. Hence, bacterial growth seems to occur mostly in nutrient-rich, few, and localized microhabitats on plant surfaces where bacteria would form aggregates (17, 20, 21, 22, 33). The presence of large clusters of bacteria at sites of relative nutrient abundance on plant surfaces might also increase the potential for metabolic and genetic exchange (19). For example, bacterial growth and relatively high rates of transfer for a conjugative plasmid were reported to occur on plant surfaces (2, 3, 5). Similarly, availability of growth substrates, high bacterial density, and the presence of solid leaf surfaces were thought to induce gene transfer by conjugation in the phyllosphere at significantly high rates (26).Of the three mechanisms of bacterial HGT, natural transformation is considered to be the only one that could be effectively implicated in the transfer of DNA from transgenic plants to bacteria (4, 25). Although plants support bacterial growth, only putative evidence of DNA released by naturally degrading plant tissue being involved in a natural transformation process exists (23). Ceccherini et al. (6) showed, for example, that although most of the plant DNA was degraded within a short time by plant nucleases in planta during the process of plant decay, a measurable fraction escaped degradation and was still able to transform a recipient soil isolate in vitro (6).In order to assess plant-to-bacteria gene transfer, some studies have been conducted with different plant compartments. For example, the possibility for Acinetobacter baylyi strain BD413 to grow opportunistically in Ralstonia solanacearum-infected plant tissues revealed a new niche for this soil bacterium: the pathosphere. Moreover, this bacterium could be naturally transformed therein by artificially added or indigenous transgenic DNA (10, 11). Yet, other plant compartments could be as propitious to HGT; for example, the residuesphere (i.e., the naturally degrading plant material at the interface with soil) has been shown to provide conditions for growth and conjugal gene transfer between indigenous soil bacteria (7, 34). The litter and the residues of annual crops represent an important amount of final plant production, which are often left in the field after harvest and, in most cases, account for up to 60% of the world''s plant biomass (14).The assessment of natural transformation events in the soil or the phytosphere has, however, revealed several methodological challenges and biases, since quantification of transformation events has often been conducted with a cultivation-based approach requiring the plating of recipient bacteria on selective media supplemented with antibiotics. Antibiotic resistance determinants are widespread in soil environments, providing a technical intricacy in the discrimination between recipients with newly acquired traits and indigenous antibiotic-resistant flora with naturally fitted analogous genes. In addition, discrepancies between transformation frequencies determined on plates and those determined by cell densitometry revealed that the latter were usually higher by 2 orders of magnitude (37), leading to an underestimation of the phenomenon in natural settings. In addition, the uncertainty of whether each colony enumerated on a plate belongs to a single independent transformation event or is one of many clones (from one event) extracted from the sample makes transformation frequency calculations imprecise. Another drawback of the plating step is the disruptive sampling of material to rescue transformants, which averages the frequency calculations over the entire sample. Due to the spatial heterogeneity of available nutrients and biologically active DNA, localized spots, which are most conducive for HGT, might be delimited at microscale. However, to date, knowledge about the effective topology is lacking, although the heterogeneity of bacterial growth on plant surfaces has been shown (15, 20, 21, 22).The objectives of this study were to determine whether, during the natural or pathogen-induced decay of plant tissues, specific niches could be shown to foster bacterial growth, to develop a competence state, and to possibly acquire exogenous plant DNA using the Acinetobacter baylyi strain BD413 via natural transformation. Visualization of bacterial colonization plant material and detection of HGT events were performed at the leaf and bacterial scales using a cultivation-independent assay that relies upon a bioreporter tool (32). Microcosm-based experiments revealed that bacterial growth and competence development occur in different compartments of the plant. Isolation and direct visualization of transformants in situ suggest that some compartments of the phytosphere can be regarded as environmental hot spots for HGT.     

Horizontal Gene Transfer and Assortative Recombination within the Acinetobacter baumannii Clinical Population Provide Genetic Diversity at the Single carO Gene, Encoding a Major Outer Membrane Protein Channel

We described previously the presence in Acinetobacter baumannii of a novel outer membrane (OM) protein, CarO, which functions as an l-ornithine OM channel and whose loss was concomitant with increased carbapenem resistance among clonally related nosocomial isolates of this opportunistic pathogen. Here, we describe the existence of extensive genetic diversity at the carO gene within the A. baumannii clinical population. The systematic analysis of carO sequences from A. baumannii isolates obtained from public hospitals in Argentina revealed the existence of four highly polymorphic carO variants among them. Sequence polymorphism between the different A. baumannii CarO variants was concentrated in three well-defined protein regions that superimposed mostly to predicted surface-exposed loops. Polymorphism among A. baumannii CarO variants was manifested in differential electrophoretic mobilities, antigenic properties, abilities to form stable oligomeric structures, and l-ornithine influx abilities through the A. baumannii OM under in vivo conditions. Incongruence between the phylogenies of the clinical A. baumannii isolates analyzed and those of the carO variants they harbor suggests the existence of assortative (entire-gene) carO recombinational exchange within the A. baumannii population. Exchange of carO variants possessing differential characteristics mediated by horizontal gene transfer may constitute an A. baumannii population strategy to survive radically changing environmental conditions, such as the leap from inanimate sources to human hosts and vice versa, persistence in a compromised host, and/or survival in health care facilities.     

Fungal Metabolite from Members of the Genus Rhizopus

A fluorescent metabolite present in seven members of the genus Rhizopus was isolated. This compound appeared green before spray treatment and purple after spray treatment with p-anisaldehyde in visible light. Subsequent purification and structural elucidation of the isolated compound yielded 1-[2,6,10,14-tetramethyl-17-carbomethyl heptadecyl]-1-[2,6,10,14-tetramethyl-17-methanoyl heptadecyl]-benzene.     

De novo Genome Assembly of the Fungal Plant Pathogen Pyrenophora semeniperda

Pyrenophora semeniperda (anamorph Drechslera campulata) is a necrotrophic fungal seed pathogen that has a wide host range within the Poaceae. One of its hosts is cheatgrass (Bromus tectorum), a species exotic to the United States that has invaded natural ecosystems of the Intermountain West. As a natural pathogen of cheatgrass, P. semeniperda has potential as a biocontrol agent due to its effectiveness at killing seeds within the seed bank; however, few genetic resources exist for the fungus. Here, the genome of P. semeniperda isolate assembled from sequence reads of 454 pyrosequencing is presented. The total assembly is 32.5 Mb and includes 11,453 gene models encoding putative proteins larger than 24 amino acids. The models represent a variety of putative genes that are involved in pathogenic pathways typically found in necrotrophic fungi. In addition, extensive rearrangements, including inter- and intrachromosomal rearrangements, were found when the P. semeniperda genome was compared to P. tritici-repentis, a related fungal species.     

Horizontal Gene Transfer and the History of Life

Microbes acquire DNA from a variety of sources. The last decades, which have seen the development of genome sequencing, have revealed that horizontal gene transfer has been a major evolutionary force that has constantly reshaped genomes throughout evolution. However, because the history of life must ultimately be deduced from gene phylogenies, the lack of methods to account for horizontal gene transfer has thrown into confusion the very concept of the tree of life. As a result, many questions remain open, but emerging methodological developments promise to use information conveyed by horizontal gene transfer that remains unexploited today.The discovery of the existence of prokaryotic microbes dates back more than 300 years. Since then, our picture of our distant microscopic relatives has undergone several revolutions: from being the living “proofs” of the existence of spontaneous generation, they became later the “archaic” representatives of our distant ancestors, to finally be legitimately recognized as exceptionally diverse organisms, keystone to any ecosystem, including the most familiar and the most hostile environments on Earth. Similarly, although they were first seen as elementary and unbreakable bricks of life, they are now seen as genetically composite bodies, heavyweight champions of “gene robbery.” The most recent of these revolutions has indeed been the realization of their unparalleled ability to integrate genetic material coming from more or less evolutionarily distant organisms. This mechanism is called “horizontal gene transfer” as opposed to vertical transmission from mother to daughter cell.     

Cloning of Insertion Site Flanking Sequence and Construction of Transfer DNA Insert Mutant Library in Stylosanthes Colletotrichum

Stylosanthes sp. is the most important forage legume in tropical areas worldwide. Stylosanthes anthracnose, which is mainly caused by Colletotrichum gloeosporioides, is a globally severe disease in stylo production. Little progress has been made in anthracnose molecular pathogenesis research. In this study, Agrobacterium tumefaciens-mediated transformation was used to transform Stylosanthes colletotrichum strain CH008. The major factors of the genetic transformation system of S. colletotrichum were optimized as follows: A. tumefaciens’ AGL-1 concentration (OD₆₀₀), 0.8; concentration of Colletotrichum conidium, 1×10⁶ conidia/mL; acetosyringone concentration, 100 mmol/L; induction time, 6 h; co-culture temperature, 25°C; and co-culture time, 3 d. Thus, the transformation efficiency was increased to 300–400 transformants per 106 conidia. Based on the optimized system, a mutant library containing 4616 mutants was constructed, from which some mutants were randomly selected for analysis. Results show that the mutants were single copies that could be stably inherited. The growth rate, spore amount, spore germination rate, and appressorium formation rate in some mutants were significantly different from those in the wild-type strain. We then selected the most appropriate method for the preliminary screening and re-screening of each mutant’s pathogenic defects. We selected 1230 transformants, and obtained 23 strains with pathogenic defects, namely, 18 strains with reduced pathogenicity and five strains with lost pathogenicity. Thermal asymmetric interlaced PCR was used to identify the transfer DNA (T-DNA) integration site in the mutant that was coded 2430, and a sequence of 476 bp was obtained. The flanking sequence of T-DNA was compared with the Colletotrichum genome by BLAST, and a sequence of 401 bp was found in Contig464 of the Colletotrichum genome. By predicting the function of the flanking sequence, we discovered that T-DNA insertion in the promoter region of the putative gene had 79% homology with the aspartate aminotransferase gene in Magnaporthe oryzae ({"type":"entrez-protein","attrs":{"text":"XP_003719674.1","term_id":"389644084","term_text":"XP_003719674.1"}}XP_003719674.1).