Single T-DNA Insertion Research Articles

T-DNA characterization of genetically modified 3-R-gene late blight-resistant potato events with a novel procedure utilizing the Samplix Xdrop® enrichment technology.

Before the commercialization of genetically modified crops, the events carrying the novel DNA must be thoroughly evaluated for agronomic, nutritional, and molecular characteristics. Over the years, polymerase chain reaction-based methods, Southern blot, and short-read sequencing techniques have been utilized for collecting molecular characterization data. Multiple genomic applications are necessary to determine the insert location, flanking sequence analysis, characterization of the inserted DNA, and determination of any interruption of native genes. These techniques are time-consuming and labor-intensive, making it difficult to characterize multiple events. Current advances in sequencing technologies are enabling whole-genomic sequencing of modified crops to obtain full molecular characterization. However, in polyploids, such as the tetraploid potato, it is a challenge to obtain whole-genomic sequencing coverage that meets the regulatory approval of the genetic modification. Here we describe an alternative to labor-intensive applications with a novel procedure using Samplix Xdrop® enrichment technology and next-generation Nanopore sequencing technology to more efficiently characterize the T-DNA insertions of four genetically modified potato events developed by the Feed the Future Global Biotech Potato Partnership: DIA_MSU_UB015, DIA_MSU_UB255, GRA_MSU_UG234, and GRA_MSU_UG265 (derived from regionally important varieties Diamant and Granola). Using the Xdrop® /Nanopore technique, we obtained a very high sequence read coverage within the T-DNA and junction regions. In three of the four events, we were able to use the data to confirm single T-DNA insertions, identify insert locations, identify flanking sequences, and characterize the inserted T-DNA. We further used the characterization data to identify native gene interruption and confirm the stability of the T-DNA across clonal cycles. These results demonstrate the functionality of using the Xdrop® /Nanopore technique for T-DNA characterization. This research will contribute to meeting regulatory safety and regulatory approval requirements for commercialization with small shareholder farmers in target countries within our partnership.

Upregulation of LmHxt1 gene is associated with reduced virulence of Leptosphaeria maculans on Brassica napus

AbstractLeptosphaeria maculans is a fungal pathogen that causes heavy losses of Brassica napus crops. To develop novel means of plant protection it is necessary to understand the virulence mechanisms of pathogens. In this study we investigated a T-DNA insertion mutant of L. maculans that exhibited a hypovirulent phenotype (pHxt1ins). The mutant contains a single T-DNA insertion that affects expression of a single gene identified as a transmembrane hexose transporter (LmHxt1). This gene exhibited enhanced expression in the pHxt1ins mutant during in vitro cultivation. The excess of glucose in the cultivation medium lowered expression of LmHxt1 in the mutant line, suggesting that the gene might function as a sugar sensor. Lack of virulence of the pHxt1ins mutant in planta was observed using fluorescence microscopy in GFP labeled lines ruling out asymptomatic growth and showing inability to successfully colonize the apoplastic space of B. napus. On the other hand, in vitro growth did not differ between pHxt1ins and control lines. Interestingly, the mutant showed altered mycelium morphology and a change in conidial germination. Overall, our results suggest that the LmHxt1 gene is a novel important virulence factor for L. maculans. We also propose pHxt1ins mutant as a tool to study the role of sugar transporters in fungal metabolism and pathogenesis.

Obtaining of transgenic barrelclover plants (Medicago truncatula) producing chicken interferon gamma for veterinary use

At the Laboratory of Plant Genetic and Cellular Engineering, Department of Genetics and Biotechnology, St. Petersburg State University, five transgenic Medicago truncatula plants were obtained through Agrobacterium-mediated transformation, carrying one of the variants of the heterologous chicken interferon-gamma gene under the control of the constitutive 35S CaMV promoter. Among these, one plant harbored an unmodified gene insertion, while four had a modified gene with a deletion at the protease recognition site, providing resistance to proteolytic degradation.
 We demonstrate the application of the SWPOP-PCR “genome walking” method to determine the integration sites of T-DNA into the plant genome, identify the number of insertion copies and their orientation. Analysis of the obtained sequences revealed that only one plant exhibited a single T-DNA insertion, which represents the most optimal structure for stable expression.
 Upon self-pollination of T0 plants, 39 offspring were obtained and subjected to testing for the presence and expression of the transgene. Among them, six homozygous plants were identified using molecular methods. Quantitative assessment of transgene expression levels showed significant differences among representatives of different lines and among the offspring derived from a single transformed plant. Among the T1 and T2 progeny, the presence of the heterologous interferon protein in plant tissues was confirmed through Western blot analysis.
 The engineered barrelclover plants hold potential as bioreactors for the production of chicken interferon-gamma for veterinary applications. The use of an edible plant allows eliminating protein extraction and purification procedures, thereby resulting in a noteworthy reduction in production expenses of up to 80%.

An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

Identification of T-DNA structure and insertion site in transgenic crops using targeted capture sequencing.

The commercialization of GE crops requires a rigorous safety assessment, which includes a precise DNA level characterization of inserted T-DNA. In the past, several strategies have been developed for identifying T-DNA insertion sites including, Southern blot and different PCR-based methods. However, these methods are often challenging to scale up for screening of dozens of transgenic events and for crops with complex genomes, like potato. Here, we report using target capture sequencing (TCS) to characterize the T-DNA structure and insertion sites of 34 transgenic events in potato. This T-DNA is an 18 kb fragment between left and right borders and carries three resistance (R) genes (RB, Rpi-blb2 and Rpi-vnt1.1 genes) that result in complete resistance to late blight disease. Using TCS, we obtained a high sequence read coverage within the T-DNA and junction regions. We identified the T-DNA breakpoints on either ends for 85% of the transgenic events. About 74% of the transgenic events had their T-DNA with 3R gene sequences intact. The flanking sequences of the T-DNA were from the potato genome for half of the transgenic events, and about a third (11) of the transgenic events have a single T-DNA insertion mapped into the potato genome, of which five events do not interrupt an existing potato gene. The TCS results were confirmed using PCR and Sanger sequencing for 6 of the best transgenic events representing 20% of the transgenic events suitable for regulatory approval. These results demonstrate the wide applicability of TCS for the precise T-DNA insertion characterization in transgenic crops.

Cmcrf1, a Putative Zn2Cys6 Fungal Transcription Factor, Is Involved in Conidiation, Carotenoid Production, and Fruiting Body Development in Cordyceps militaris.

Cordyceps militaris is a high-value medicinal and edible fungus that produces many bioactive compounds, including carotenoid, and thus, improving the carotenoid productivity of C. militaris will increase its commercial value. However, little is known about the genetic regulatory mechanism of carotenoid biosynthesis in C. militaris. To further understanding the regulatory mechanism of carotenoid biosynthesis, we performed a large-scale screen of T-DNA insertional mutant library and identified a defective mutant, denoted T111, whose colonies did not change color from white to yellow upon exposure to light. Mutation analysis confirmed that a single T-DNA insertion occurred in the gene encoding a 695-amino-acid putative fungal-specific transcription factor with a predicted Zn2Cys6 binuclear cluster DNA-binding domain found uniquely in fungi. Targeted deletion of this gene, denoted C. militaris carotenogenesis regulatory factor 1 (Cmcrf1), generated the ΔCmcrf1 mutant that exhibited drastically reduced carotenoid biosynthesis and failed to generate fruiting bodies. In addition, the ΔCmcrf1 mutant showed significantly increased conidiation and increased hypersensitivity to cell-wall-perturbing agents compared with the wild-type strain. However, the Cmcrf1 gene did not have an impact on the mycelia growth of C. militaris. These results show that Cmcrf1 is involved in carotenoid biosynthesis and is required for conidiation and fruiting body formation in C. militaris.

Molecular identification and efficacy assessment of a glufosinate-tolerant and brown planthopper-resistant transgenic rice line

Insect pests and weeds are the two major biotic factors affecting crop yield in the modern agricultural system. In this study, a brown planthopper (BPH) resistance gene (BPH9) and glufosinate tolerance gene (bar) were stacked into a single T-DNA cassette and transformed into an indica rice (Oryza sativa L.) line Guangzhan 63-4S. A stable transgenic line H23 with a single T-DNA insert was generated, with the T-DNA cassette located on chromosome 3. Field resistance trial using H23 revealed high tolerance to glufosinate and excellent resistance to BPH. These results propose H23 as valuable germplasm for BPH-resistance and glufosinate-tolerance breeding in rice.

Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

Knockdown of Mitochondrial Uncoupling Proteins 1 and 2 (AtUCP1 and 2) in Arabidopsis thaliana Impacts Vegetative Development and Fertility.

Mitochondrial uncoupling proteins (UCPs) are mitochondrial inner membrane proteins that dissipate the proton electrochemical gradient generated by the respiratory chain complexes. In plants, these proteins are crucial for maintaining mitochondrial reactive oxygen species (ROS) homeostasis. In this study, single T-DNA insertion mutants for two (AtUCP1 and AtUCP2) out of the three UCP genes present in Arabidopsis thaliana were employed to elucidate their potential roles in planta. Our data revealed a significant increase in the Adenosine triphosphate (ATP)/Adenosine diphosphate (ADP) ratios of both mutants, indicating clear alterations in energy metabolism, and a reduced respiratory rate in atucp2. Phenotypic characterization revealed that atucp1 and atucp2 plants displayed reduced primary root growth under normal and stressed conditions. Moreover, a reduced fertility phenotype was observed in both mutants, which exhibited an increased number of sterile siliques and a lower seed yield compared with wild-type plants. Reciprocal crosses demonstrated that both male fertility and female fertility were compromised in atucp1, while such effect was exclusively observed in the male counterpart in atucp2. Most strikingly, a pronounced accumulation of hydrogen peroxide in the reproductive organs was observed in all mutant lines, indicating a disturbance in ROS homeostasis of mutant flowers. Accordingly, the atucp1 and atucp2 mutants exhibited higher levels of ROS in pollen grains. Further, alternative oxidase 1a was highly induced in mutant flowers, while the expression profiles of transcription factors implicated in gene regulation during female and male reproductive organ/tissue development were perturbed. Overall, these data support the important role for AtUCP1 and AtUCP2 in flower oxidative homeostasis and overall plant fertility.

Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system

BackgroundRice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria.Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants.ResultsWe introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants.ConclusionWe demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus.

Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from Cladonia macilenta. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with Agrobacterium cells, and incubation temperature, were tested to identify an optimized ATMT condition for the C. macilenta mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in C. macilenta, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.

The Salt Sensitivity Induced by Disruption of Cell Wall-Associated Kinase 1 (SlWAK1) Tomato Gene Is Linked to Altered Osmotic and Metabolic Homeostasis

Tomato cell wall-associated kinase 1 (SlWAK1) has only been studied in biotic stress response and hence its function in abiotic stress remains unknown. In a screening under salinity of an insertional mutant collection of tomato (Solanum lycopersicum L.), a mutant exhibiting lower degree of leaf chlorosis than wild type (WT) together with reduced leaf Na+ accumulation was selected. Genetic analysis of the mutation revealed that a single T-DNA insertion in the SlWAK1 gene was responsible of the mutant phenotype. Slwak1 null mutant reduced its shoot growth compared with WT, despite its improved Na+ homeostasis. SlWAK1 disruption affected osmotic homeostasis, as leaf water content was lower in mutant than in WT under salt stress. In addition, Slwak1 altered the source-sink balance under salinity, by increasing sucrose content in roots. Finally, a significant fruit yield reduction was found in Slwak1 vs. WT under long-term salt stress, mainly due to lower fruit weight. Our results show that disruption of SlWAK1 induces a higher sucrose transport from source leaf to sink root, negatively affecting fruit, the main sink at adult stage.

Generation of Epichloë Strains Expressing Fluorescent Proteins Suitable for Studying Host-Endophyte Interactions and Characterisation of a T-DNA Integration Event.

Methods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as reporter gene products, allowing non-invasive detection in living cells. This study reports the introduction of genes for two fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein, DsRed, into the genomes of two distinct perennial ryegrass (Lolium perenne L.)-associated Epichloë endophyte strains using A. tumefaciens-mediated transformation. Comprehensive characterisation of reporter gene-containing endophyte strains was performed using molecular genetic, phenotypic, and bioinformatic tools. A combination of long read and short read sequencing of a selected transformant identified a single complex T-DNA insert of 35,530 bp containing multiple T-DNAs linked together. This approach allowed for comprehensive characterisation of T-DNA integration to single-base resolution, while revealing the unanticipated nature of T-DNA integration in the transformant analysed. These reporter gene endophyte strains were able to establish and maintain stable symbiotum with the host. In addition, the same endophyte strain labelled with two different fluorescent proteins were able to cohabit the same plant. This knowledge can be used to provide the basis to develop strategies to gain new insights into the host-endophyte interaction through independent and simultaneous monitoring in planta throughout its life cycle in greater detail.

A Similar Secretome Disturbance as a Hallmark of Non-pathogenic Botrytis cinerea ATMT-Mutants?

The gray mold fungus Botrytis cinerea is a necrotrophic pathogen able to infect hundreds of host plants, including high-value crops such as grapevine, strawberry and tomato. In order to decipher its infectious strategy, a library of 2,144 mutants was generated by random insertional mutagenesis using Agrobacterium tumefaciens-mediated transformation (ATMT). Twelve mutants exhibiting total loss of virulence toward different host plants were chosen for detailed analyses. Their molecular characterization revealed a single T-DNA insertion in different loci. Using a proteomics approach, the secretome of four of these strains was compared to that of the parental strain and a common profile of reduced lytic enzymes was recorded. Significant variations in this profile, notably deficiencies in the secretion of proteases and hemicellulases, were observed and validated by biochemical tests. They were also a hallmark of the remaining eight non-pathogenic strains, suggesting the importance of these secreted proteins in the infection process. In the twelve non-pathogenic mutants, the differentiation of infection cushions was also impaired, suggesting a link between the penetration structures and the secretion of proteins involved in the virulence of the pathogen.

Advancing Functional Genetics Through Agrobacterium-Mediated Insertional Mutagenesis and CRISPR/Cas9 in the Commensal and Pathogenic Yeast Malassezia.

Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study, we applied a recently-developed Agrobacterium tumefaciens-mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in Malassezia furfur A total of 767 transformants were screened for sensitivity to 10 different stresses, and 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: CDC55 and PDR10 This system is based on cotransformation of M. furfur mediated by A. tumefaciens, to deliver both a CAS9-gRNA construct that induces double-strand DNA breaks and a gene replacement allele that serves as a homology-directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens-mediated transformation to aid in the identification of gene functions in M. furfur, through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.

OsTSD2-mediated cell wall modification affects ion homeostasis and salt tolerance.

Salt stress is a major environmental threat to meeting the food demands of an increasing global population. The identification and exploitation of salt adaption mechanisms in plants are therefore vital for crop breeding. We here define the rice mutant (sstm1) whose salt sensitivity was unambiguously assigned to a single T-DNA insertion through segregational analysis following backcrossing to the wild type line. Insertion was within OsTSD2, which encoded a pectin methyltransferase. The sstm1 and allelic mutants, collectively known as tsd2, displayed higher content of Na+ and lower level of K+ in the shoot, which is likely to lead to reduced salt tolerance. Molecular analysis revealed reduced expression of the genes maintaining K+ /Na+ homeostasis in tsd2, including OsHKT1;5, OsSOS1, and OsKAT1. Furthermore, OsTSD2 influenced ion distribution between the hull and the rice seed, which could improve food safety with heavy metal pollution. Amino acid levels tended to be increased in tsd2 mutants, implicating a role of pectin in the regulation of metabolism. Taken together, we have demonstrated an important facet of salt tolerance, which implicated OsTSD2-mediated cell wall pectin modification as a key component that could be widely applied in crop science.

TADEA-PCR is a highly efficient method of amplifying unknown flanking fragments of T-DNA transformants.

Forward genetic analysis, widely used to find new gene functions, benefits from the availability of mutants. At present, based on Agrobacterium-mediated plant transformation technology, many transfer (T)-DNA transformants have been created. However, cloning their T-DNA insertion sites, which enables identification of the mutated genes, is still challenging. In this study, we improved adapter ligation-mediated polymerase chain reaction (A-PCR), which mainly utilizes the Thermal Asymmetric interlaced reaction and Degenerate sequence-recognizing restriction Endonucleases (TADE). Using the new method TADE-mediated A-PCR (TADEA-PCR), we successfully cloned 22 of all the 24 junction sites in 10 Arabidopsis thaliana L. transformants that contained 12 T-DNA insertions in total, giving a success rate of 91.7%. In most cases, the two junction sites resulting from a single T-DNA insertion were simultaneously cloned. In addition, TADEA-PCR was able to clone more than two junction sites present in one transformant containing several T-DNA insertions. Overall, TADEA-PCR is a powerful technique for cloning T-DNA insertion sites.

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus.

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

Feedback Microtubule Control and Microtubule-Actin Cross-talk in Arabidopsis Revealed by Integrative Proteomic and Cell Biology Analysis of KATANIN 1 Mutants

Microtubule organization and dynamics are critical for key developmental processes such as cell division, elongation, and morphogenesis. Microtubule severing is an essential regulator of microtubules and is exclusively executed by KATANIN 1 in Arabidopsis In this study, we comparatively studied the proteome-wide effects in two KATANIN 1 mutants. Thus, shotgun proteomic analysis of roots and aerial parts of single nucleotide mutant fra2 and T-DNA insertion mutant ktn1-2 was carried out. We have detected 42 proteins differentially abundant in both fra2 and ktn1-2 KATANIN 1 dysfunction altered the abundance of proteins involved in development, metabolism, and stress responses. The differential regulation of tubulins and microtubule-destabilizing protein MDP25 implied a feedback microtubule control in KATANIN 1 mutants. Furthermore, deregulation of profilin 1, actin-depolymerizing factor 3, and actin 7 was observed. These findings were confirmed by immunoblotting analysis of actin and by microscopic observation of actin filaments using fluorescently labeled phalloidin. Results obtained by quantitative RT-PCR analysis revealed that changed protein abundances were not a consequence of altered expression levels of corresponding genes in the mutants. In conclusion, we show that abundances of several cytoskeletal proteins as well as organization of microtubules and the actin cytoskeleton are amended in accordance with defective microtubule severing.

Generation and Screening of T-DNA Insertion Mutants Mediated by Agrobacterium tumefaciens in the Garden Asparagus Stem Blight Pathogen Phomopsis asparagi

The garden asparagus stem blight caused by filamentous fungus Phomopsis asparagi exposes a serious threat on asparagus production globally. However, to present, we understand poorly about the molecular mechanisms of fungal pathogenicity. To facilitate functional genomics research of P. asparagi, here we developed a highly efficient and stable Agrobacterium tumefaciens-mediated transformation approach which yielded 150-200 transformants per 1×106 conidia. Our results indicated that 25°C, acetosyringone concentration of 150μmol/L, and 72h were recommended as optimal co-cultivation conditions for the transformation. Using this transformation approach, we constructed a T-DNA insertion mutant library containing 1253 strains. Twenty randomly selected T-DNA insertion mutants were able to grow on 0.2×PDA selective media after five successive subcultures without selective pressure, indicating that the exogenous T-DNA was stably integrated into the P. asparagi genome. We confirmed several randomly selected mutants using PCR with primers specific to the hph gene. Southern blots suggested that three out of the five selected mutants have a single T-DNA insertion. Interestingly, multiple mutant candidates with growth defects were obtained from the growth assay. Moreover, several mutants were selected for further analysis on the T-DNA flanking sequences through TAIL-PCR analysis. A sequence comparison of total junction fragments implied that the insertion of T-DNA within P. asparagi genome appeared to be a random event. The transformation technology and genetic resources developed here will facilitate studies of pathogenic mechanisms in this devastating filamentous fungal pathogen of garden asparagus.