Agrobacterium Tumefaciens Research Articles

Agrobacterium-mediated transfer of the Fusarium graminearum Tri6 gene into barley using mature seed-derived shoot tips as explants

Key messageWe transferred the Tri6 gene into the elite barley GemCraft via new transformation method through shoot organogenesis and identified the rearrangements of transgenes and phenotypic variations in the transgenic plants.Despite its agronomic and economic importance, barley transformation is still very challenging for many elite varieties. In this study, we used direct shoot organogenesis to transform the elite barley cultivar GemCraft with the RNAi constructs containing Tri6 gene of Fusarium graminearum, which causes fusarium head blight (FHB). We isolated 4432 shoot tips and co-cultured these explants with Agrobacterium tumefaciens. A total of 25 independent T0 transgenic plants were generated including 15 events for which transgene-specific PCR amplicons were observed. To further determine the presence of transgenes, the T1 progenies of all 15 T0 plants were analyzed, and the expected PCR products were obtained in 10 T1 lines. Droplet digital (dd) PCR analysis revealed various copy numbers of transgenes in the transgenic plants. We determined the insertion site of transgenes using long-read sequencing data and observed the rearrangements of transgenes. We found phenotypic variations in both T1 and T2 generation plants. FHB disease was evaluated under growth chamber conditions, but no significant differences in disease severity or deoxynivalenol accumulation were observed between two Tri6 transgenic lines and the wildtype. Our results demonstrate the feasibility of the shoot tip transformation and may open the door for applying this system for genetic improvement and gene function research in other barley genotypes.

Microbial activity of dark-chestnut soil in winter wheat crops depending on fertiliser application

Preventing soil degradation is an important task of modern agrarian science. Preservation and increase in beneficial soil microflora are a precondition for satisfactory ecological functioning of soil and its fertility. The purpose of this study was to investigate the dynamics of beneficial soil microflora in winter wheat crops under the impact of mineral fertilisers and Agrobacterium radiobacter on the lands of the south of Ukraine. The study was conducted in threefold replication in the conditions of the Kherson region in 2016- 2020 in the experimental fields of the Institute of Climate-Smart Agriculture of the National Academy of Agrarian Sciences of Ukraine. The study used a systematic design, and the following factors were investigated: no fertiliser and Agrobacterium radiobacter; N120P90; N90P60 + Agrobacterium radiobacter; N120P90 + Agrobacterium radiobacter. The study on the microflora composition in the soil layer at 0-30 cm was carried out in the main stages of winter wheat growth and development through the inoculation of nutritious environment. Statistical analysis was performed using the methods of analysis of variance, correlation, and regression analysis. Dynamic changes in the soil biota composition under the influence of the studied factor were established. The slightest response to mineral fertilisers and bacterial preparation application was in ammonifying bacteria, while the strongest response was in cellulosedegrading microorganisms. The winter wheat harvesting revealed a decrease in the number of ammonifying bacteria in the soil, while the number of nitrifying and cellulose-degrading microflora increased significantly. Regression models for the prediction of the number of nitrifying bacteria and cellulose-degrading microorganisms, developed based on experimental data, have great accuracy (the error is 3.78% and 7.79%), and allow determining the adverse effect of phosphorus fertiliser on the microflora of dark-chestnut soil. The study has no analogues in Ukraine and expands theoretical knowledge about the influence of mineral fertilisers and bacterial preparation, containing Agrobacterium radiobacter, on the composition of beneficial microflora of the dark-chestnut soil

A simple and efficient in planta transformation method based on the active regeneration capacity of plants

Plant genetic transformation strategies serve as essential tools for the genetic engineering and advanced molecular breeding of plants. However, the complicated operational protocols and low efficiency of current transformation strategies restrict the genetic modification of most plant species. This paper describes the development of the regenerative activity–dependent in planta injection delivery (RAPID) method based on the active regeneration capacity of plants. In this method, Agrobacterium tumefaciens is delivered to plant meristems via injection to induce transfected nascent tissues. Stable transgenic plants can be obtained by subsequent vegetative propagation of the positive nascent tissues. The method was successfully used for transformation of plants with strong regeneration capacity, including different genotypes of sweet potato (Ipomoea batatas), potato (Solanum tuberosum), and bayhops (Ipomoea pes-caprae). Compared with traditional transformation methods, RAPID has a much higher transformation efficiency and shorter duration, and it does not require tissue culture procedures. The RAPID method therefore overcomes the limitations of traditional methods to enable rapid in planta transformation and can be potentially applied to a wide range of plant species that are capable of active regeneration.

Volatile organic compounds of the soil bacterium Bacillus halotolerans suppress pathogens and elicit defense-responsive genes in plants

Volatile organic compounds (VOCs) produced by bacteria play an important, yet relatively unexplored role in interactions between plants and phytopathogens. In this study, the soil bacterium Bacillus halotolerans NYG5 was identified as a potent biocontrol agent against several phytopathogenic fungi (Macrophomina phaseolina, Rhizoctonia solani, Pythium aphanidermatum, and Sclerotinia sclerotiorum) through the production of VOCs. NYG5-emitted VOCs also inhibited the growth of bacterial pathogens (Agrobacterium tumefaciens, Xanthomonas campestris, Clavibacter michiganensis, and Pseudomonas syringae). When cultured in various growth media, NYG5 produced a variety of VOCs. Five distinct VOCs (2-methylbutanoic acid, 5-methyl-2-hexanone, 2,3-hexanedione, 2-ethyl-1-hexanol, and 6-methyl-2-heptanone) were identified using headspace GC-MS. 2,3-Hexanedione exhibited potent lethal effects on the tested phytopathogens and nematicidal activity against Meloidogyne javanica at a concentration of 50 ppm. In addition, 0.05 ppm 2,3-hexanedione stimulated the expression of pathogenesis-related genes 1 and 2 in Arabidopsis thaliana. Interestingly, 2,3-hexanedione is used as a food additive at higher concentrations than those tested in this study. Hence, 2,3-hexanedione is a promising biologically active compound that might serve as a sustainable alternative to common chemical pesticides and an elicitor of plant defense.

Transcriptomic Evidence of a Link between Cell Wall Biogenesis, Pathogenesis, and Vigor in Walnut Root and Trunk Diseases.

Crown gall disease (Agrobacterium tumefaciens), crown/root rot disease (Phytophthora spp.), root lesion disease (Pratylenchus vulnus) and tree vigor are key traits affecting the productivity and quality of walnuts in California. Unchallenged hybrid rootstocks were analyzed by RNA-seq to examine pre-formed factors affecting these traits. Enrichment analysis of the differentially expressed genes revealed that the increased expression of cell wall biogenesis-related genes plays a key role in susceptibility to A. tumefaciens, susceptibility to Phytophthora spp. and increased vigor. Analysis of the predicted subcellular loci of the encoded proteins revealed that many gene products associated with vigor and susceptibility were targeted to the plasma membrane and extracellular space, connecting these traits to sustaining barrier function. We observed that RNA processing and splicing, along with predicted nuclear targeting, were associated with resistance to A. tumefaciens, resistance to Phytophthora spp. and low vigor. Four genes within the J. microcarpa QTL region for resistance to A. tumefaciens and Phytophthora spp. were represented among our transcripts, with two of the genes being differentially expressed in association with resistance to A. tumefaciens and decreased vigor. No differential expression related to Phytophthora spp. or P. vulnus resistance was observed in this region. Additionally, the J. microcarpa haplotype expressed more transcripts associated with resistance to A. tumefaciens, Phytophthora spp. and low vigor, but not P. vulnus, than the J. regia haplotype. We also report unique and shared hormone and defense responses associated with each trait. This research suggests a link between cell wall biogenesis, vigor and critical root diseases of walnut.

The potential of PGPR and Trichoderma-based bioproducts and resistant cultivars as tools to manage clubroot disease in cruciferous crops.

The objective of this research was to determine the potential use of eco-friendly technologies to reduce the clubroot disease caused by Plasmodiophora brassicae, the main constraint of cruciferous crops worldwide. Two commercial bioproducts were evaluated in susceptible broccoli, one based on the PGPR consortium (Bacillus amyloliquefaciens, Bacillus pumilus, and Agrobacterium radiobacter K84) and the other one based on Trichoderma koningiopsis Th003 (Tricotec® WG). Additionally, the resistant broccoli cv. Monclano® was tested under two concentrations of resting spores (RS) of P. brassicae, 1 × 103 and 1 × 105 RS g-1 of soil. The first phase of evaluations with broccoli was carried out under a greenhouse, while susceptible broccoli, cauliflower, and red cabbage were included in a subsequent field phase. Tebuconazole + Trifloxystrobin mixture and Fluazinam were included as positive controls. The effectiveness of the bioproducts depended on the nature of the biocontrol agent, the concentration of P. brassicae, and the dose of treatment. Tricotec® showed consistent plant growth promotion but no biocontrol effect against clubroot, and the rhizobacteria-based bioproduct significantly reduced the disease in both greenhouse and field experiments. Higher disease severity was observed with the higher dose of Tricotec®. Under field conditions, the rhizobacteria reduced the incidence progress by 26%, 39%, and 57% under high, medium, and low pressure of the pathogen, respectively. However, no reduction of clubroot severity under high pressure of the pathogen was observed. Complete inhibition of club formation in roots was achieved via the fungicide, but a phytotoxic effect was observed under greenhouse conditions. Fungicides reduced the incidence progress of clubroot, but not the severity under high inoculum pressure in the field. The fungicides, the bacterial treatment, and the combination of bioproducts tended to delay the progress of the disease compared with the negative control and Tricotec alone. The resistant broccoli showed a low level of disease under high concentrations of P. brassicae (less than 10% incidence and up to 2% severity). These results suggested the overall potential of commercial tools based on the PGPR consortium and plant resistance to control P. brassicae. The integration of control measures, the role of Trichoderma spp. in P. brassicae-cruciferous pathosystems, and the need to recover highly infested soils will be discussed.

CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

Chemical components of different solvent extracts of Asclepias curassavica L. and antibacterial effect of the extracts on tomato pathogens

The use of environmentally friendly and sustainable agricultural methods in the control of diseases and pests is of great importance. In both conventional and organic agricultural production systems, the utilization of various plant extracts as part of integrated pest management has gained significance in recent years. The chemical constituents of various solutions derived from the flowers, leaves, and roots of the Asclepias curassavica L. plant were investigated, along with the possibilities of utilizing these solutions in the control of tomato plant pathogenic bacteria. As a result of the analysis, acetic acid in 60% and 80% ethanol solutions, as well as acetic acid methy ester in 60% and 80% methanol solutions, were detected in the flowers, leaves, and roots. The effects of A. curassavica flower, leaf, and root extracts, prepared using three different solvents (water, methanol, and ethanol), were investigated on eight different pathogenic bacteria (Agrobacterium tumefaciens, Clavibacter michiganensis, Dickeya zeae, Pectobacterium caratovorum, Pseudomonas phaseolicola, Pseudomonas tomato, Pseudomonas viridiflava, and Xanthomonas euvesicatoria) that cause diseases in plants. Several main compounds such as acetic acid, acetic acid. methyl ester, Furfural, 2-Furanmethanol, 4H-Pyran-4-one. 2.3-dihydro-3.5-dihydroxy-6-methyl-, Glycerin, Benzo furan. 2.3-dihydro- and 5-Hydroxy methyl furfural were identified as analyzed by GC-MS with different concentrations of ethanol and methanol solutions used for the flower, leaves and root of A. curassavica plants. The flower extract prepared with 80% ethanol exhibited a higher inhibition zone (ranging from 1.5 mm to 5.3 mm) in all pathogens, compared to other applications. The successful suppression effect of A. curassavica flower extracts on this disease is promising, especially in organic farming areas. Additionally, since it is environmentally friendly and sustainable, it can be included in integrated control methods to prevent the loss of productivity caused by diseases.

Anti-Agrobacterium tumefactions sesquiterpene derivatives from the marine-derived fungus Trichoderma effusum.

Agrobacterium tumefaciens can harm various fruit trees, leading to significant economic losses in agricultural production. It is urgent to develop new pesticides to effectively treat this bacterial disease. In this study, four new sesquiterpene derivatives, trichoderenes A-D (1-4), along with six known compounds (5-10), were obtained from the marine-derived fungus Trichoderma effusum. The structures of 1-4 were elucidated by extensive spectroscopic analyses, and the calculated ECD, ORD, and NMR methods. Structurally, the hydrogen bond formed between the 1-OH group and the methoxy group enabled 1 to adopt a structure resembling that of resorcylic acid lactones, thereby producing the ECD cotton effect. Compound 3 represents the first example of C12 nor-sesquiterpene skeleton. Compounds 1-10 were tested for their antimicrobial activity against A. tumefactions. Among them, compounds 1-3 and 8-10 exhibited inhibitory activity against A. tumefactions with MIC values of 3.1, 12.5, 12.5, 6.2, 25.0, and 12.5 μg/mL, respectively.

Promoter Prediction in Agrobacterium tumefaciens Strain C58 by Using Artificial Intelligence Strategies.

Promoters are the genomic regions upstream of genes that RNA polymerase binds in order to initiate gene transcription. Understanding the regulation of gene expression depends on being able to identify promoters, because they are the most important component of gene expression. Agrobacterium tumefaciens (A. tumefaciens) strain C58 was the subject of this study with the goal of creating a machine learning-based model to predict promoters. In this study, nucleotide density (ND), k-mer, and one-hot were used to encode the promoter sequence. Support vector machine (SVM) on fivefold cross-validation with incremental feature selection (IFS) was used to optimize the generated features. These improved characteristics were then used to distinguish promoter sequences by feeding them into the random forest (RF) classifier. Tenfold cross-validation (CV) analysis revealed that the projected model has the ability to produce an accuracy of 84.22%.

The effect of bacterial products and mineral fertilizers on growth and productivity of Brassica juncea Czern

The paper reports the results of field experiments that analyzed changes in growth indicators and productivity of Sarepta mustard (Brassica juncea Czern.) of the Start variety (k-4259) in response to inoculation and increasing doses of mineral nitrogen. Morphometric parameters and productivity of dry mass were measured during the stage of harvesting maturity, i.e., the full flowering stage. The seeds were inoculated with the following bacterial products: Agrofil (Agrobacterium radiobacter, strain 10), Mizorin (Arthrobacter mysorens, strain 7), Mobilin (Pseudomonas fluorescens, strain PG-5), and Flavobakterin (Flavobacterium sp., strain 30). The amount of nitrogen ranged from N30 to N120. It was enhanced with the application of PK at the rate of 60 kg/ha. The experiments showed that the application of bacterial products and increasing doses of mineral nitrogen has a stimulating effect on plants. The fertilizer formulation N120P60K60 and rhizobacteria strains Flavobacterium sp., strain 30, and Pseudomonas fluorescens, strain PG-5, proved to be the most effective. The obtained data were roughly consistent with the application of N90 and N120. The use of mineral fertilizers resulted in the highest productivity of dry shoot mass.

Isolation and characterization of bacteriophages for controlling Rhizobium radiobacter - causing stem and crown gall of highbush blueberry.

Stem and crown gall disease caused by the plant pathogen Rhizobium radiobacter has a significant impact on highbush blueberry (Vaccinium corymbosum) production. Current methods for controlling the bacterium are limited. Lytic phages, which can specifically target host bacteria, have been widely gained interest in agriculture. In this study, 76 bacteriophages were recovered from sewage influent and screened for their inhibitory effect against Rhizobium spp. The phages were genetically characterized through whole-genome sequencing, and their lytic cycle was confirmed. Five potential candidate phages (isolates IC12, IG49, AN01, LG08, and LG11) with the ability to lyse a broad range of hosts were chosen and assessed for their morphology, environmental stability, latent period, and burst size. The morphology of these selected phages revealed a long contractile tail under transmission electron microscopy. Single-step growth curves displayed that these phages had a latent period of 80-110 min and a burst size ranging from 8 to 33 phages per infected cell. None of these phages contained any antimicrobial resistance or virulence genes in their genomes. Subsequently, a combination of two-, three- and four-phage cocktails were formulated and tested for their efficacy in a broth system. A three-phage cocktail composed of the isolates IC12, IG49 and LG08 showed promising results in controlling a large number of R. radiobacter strains in vitro. In a soil/peat-based model, the three-phage cocktail was tested against R. radiobacter PL17, resulting in a significant reduction (p < 0.05) of 2.9 and 1.3 log10 CFU/g after 24 and 48 h of incubation, respectively. These findings suggest that the three-phage cocktail (IC12, IG49 and LG08) has the potential to serve as a proactive antimicrobial solution for controlling R. radiobacter on blueberry.

Construction and validation of co-expression vector for rice alpha tubulin and microtubule associated protein respectively fused with fluorescent proteins.

Microtubule (MT) consists of α-tubulin and β-tubulin. The dynamic instability regulated by various microtubule associated proteins (MAPs) is essential for MT functions. To analyze the interaction between tubulin/MT and MAP in vivo, we usually need tubulin and MAP co-expressed. Here, we constructed a dual-transgene vector expressing rice (Oryza sativa) α-tubulin and MAP simultaneously. To construct this vector, plant expression vector pCambia1301 was used as the plasmid backbone and Gibson assembly cloning technology was used. We first fused and cloned the GFP fragment, α-tubulin open reading frame (ORF), and NOS terminator into the vector pCambia1301 to construct the p35S::GFP-α-tubulin vector that expressed GFP-α-tubulin fusion protein. Subsequently, we fused and cloned the CaMV 35S promoter, mCherry fragment, and NOS terminator into the p35S::GFP-α-tubulin vector to generate the universal dual-transgene expression vector (p35S::GFP-α-tubulin-p35S::mCherry vector). With the p35S::GFP-α-tubulin-p35S::mCherry vector, MAP ORF can be cloned into the site of 5' or 3' terminus of mCherry to co-express GFP-α-tubulin and MAP-mCherry/mCherry-MAP. To validate the availability and universality of the dual-transgene expression vector, a series of putative rice MAP genes including GL7, OsKCBP, OsCLASP, and OsMOR1 were cloned into the vector respectively, transformed into Agrobacterium tumefaciens strain, and expressed in Nicotiana benthamiana leaves. The results indicated that all of the MAPs were co-expressed with α-tubulin and localized to MTs, validating the availability and universality of the vector and that GL7, OsKCBP, OsCLASP, and OsMOR1 might be MAPs. The application of the co-expression vector constructed by us would facilitate studies on the interaction between tubulin/MT and MAP in tobacco transient expression systems or transgenic rice.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

Capture-and-release of a sulfoquinovose-binding protein on sulfoquinovose-modified agarose.

The solute-binding protein (SBP) components of periplasmic binding protein-dependent ATP-binding cassette (ABC)-type transporters often possess exquisite selectivity for their cognate ligands. Maltose binding protein (MBP), the best studied of these SBPs, has been extensively used as a fusion partner to enable the affinity purification of recombinant proteins. However, other SBPs and SBP-ligand based affinity systems remain underexplored. The sulfoquinovose-binding protein SmoF, is a substrate-binding protein component of the ABC transporter cassette in Agrobacterium tumefaciens involved in importing sulfoquinovose (SQ) and its derivatives for SQ catabolism. Here, we show that SmoF binds with high affinity to the octyl glycoside of SQ (octyl-SQ), demonstrating remarkable tolerance to extension of the anomeric substituent. The 3D X-ray structure of the SmoF·octyl-SQ complex reveals accommodation of the octyl chain, which projects to the protein surface, providing impetus for the synthesis of a linker-equipped SQ-amine using a thiol-ene reaction as a key step, and its conjugation to cyanogen bromide modified agarose. We demonstrate the successful capture and release of SmoF from SQ-agarose resin using SQ as competitive eluant, and selectivity for release versus other organosulfonates. We show that SmoF can be captured and purified from a cell lysate, demonstrating the utility of SQ-agarose in capturing SQ binding proteins from complex mixtures. The present work provides a pathway for development of 'capture-and-release' affinity resins for the discovery and study of SBPs.

Transmission and Management of Pathogenic Agrobacterium tumefaciens and Rhodococcus fascians in Select Ornamentals.

Pathogenic Agrobacterium tumefaciens and Rhodococcus fascians are phytobacteria that induce crown gall and leafy gall disease, respectively, resulting in undesirable growth abnormalities. When present in nurseries, plants infected by either bacterium are destroyed, resulting in substantial losses for growers, especially those producing plants valued for their ornamental attributes. There are many unanswered questions regarding pathogen transmission on tools used to take cuttings for propagation and whether products used for bacterial disease control are effective. We investigated the ability to transmit pathogenic A. tumefaciens and R. fascians on secateurs and the efficacy of registered control products against both bacteria in vitro and in vivo. Experimental plants used were Rosa × hybrida, Leucanthemum × superbum, and Chrysanthemum × grandiflorum for A. tumefaciens and Petunia × hybrida and Oenothera 'Siskiyou' with R. fascians. In separate experiments, we found secateurs could convey both bacteria in numbers sufficient to initiate disease in a host-dependent manner and that bacteria could be recovered from secateurs after a single cut through an infected stem. In in vivo assays, none of six products tested against A. tumefaciens prevented crown gall disease, although several products appeared promising in in vitro trials. Likewise, four compounds trialed against R. fascians failed to prevent disease. Sanitation and clean planting material remain the primary means of disease management.

Drought stress enhances the efficiency of floral dip method of Agrobacterium-mediated transformation in Arabidopsis thaliana.

The Agrobacterium-mediated floral dip protocol is the most extensively used transformation method for a model plant Arabidopsis thaliana. Several useful methods for Agrobacterium tumefaciens-mediated transformations of Arabidopsis are existing, but they are time consuming and with low transformation efficiency. Here, we developed a transgenic Arabidopsis lines TET12p::TET12-RFP in a short period of time and enhanced transformation efficiency by using a modified transformation method by applying drought stress after floral dip. In this protocol, Agrobacterium cells carrying TET12p::TET12-RFP recombinant vectors were resuspended in a solution of 5% sucrose, 0.05% (v/v) silwet L-77 to transform female gametes of developing Arabidopsis inflorescences. Treated Arabidopsis were then applied with different levels of drought stresses to stimulate plants for the utilization of maximum plant energy in seed maturation process. The applied stresses achieved the fast maturation of already treated inflorescences while stopped the growing of newly arising untreated inflorescence, thus decreased the chances of wrong collection of untransformed seeds. Consequently, the collected seeds were mostly transgenic with a transformation frequency of at least 10%, thus the screening for positive transformants selection was more advantageous on a selective medium as compared to a classical floral dip method. Within 2-3 months, two hundred of individual transgenic plants were produced from just 10 infiltrated plants. This study concludes that application of drought stresses in a specific stage of plant is a beneficial strategy for achieving the transgenic Arabidopsis in a short period of time with high transformation efficiency.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

Design and characterization of hyperactive mutants of the Agrobacterium tumefaciens telomere resolvase, TelA.

Telomere resolvases are a family of DNA cleavage and rejoining enzymes that produce linear DNAs terminated by hairpin telomeres from replicated intermediates in bacteria that possess linear replicons. The telomere resolvase of Agrobacterium tumefaciens, TelA, has been examined at the structural and biochemical level. The N-terminal domain of TelA, while not required for telomere resolution, has been demonstrated to play an autoinhibitory role in telomere resolution, conferring divalent metal responsiveness on the reaction. The N-terminal domain also inhibits the competing reactions of hp telomere fusion and recombination between replicated telomere junctions. Due to the absence of the N-terminal domain from TelA/DNA co-crystal structures we produced an AlphaFold model of a TelA monomer. The AlphaFold model suggested the presence of two inhibitory interfaces; one between the N-terminal domain and the catalytic domain and a second interface between the C-terminal helix and the N-core domain of the protein. We produced mutant TelA's designed to weaken these putative interfaces to test the validity of the modeled interfaces. While our analysis did not bear out the details of the predicted interfaces the model was, nonetheless, extremely useful in guiding design of mutations that, when combined, demonstrated an additive activation of TelA exceeding 250-fold. For some of these hyperactive mutants stimulation of telomere resolution has also been accompanied by activation of competing reactions. However, we have also characterized hyperactive TelA mutants that retain enough autoinhibition to suppress the competing reactions.

Standardization of reference genes for real-time PCR analysis of Vigna radiata L. under Agrobacterium tumefaciens infection

Gene expression analysis using real-time polymerase chain reaction (PCR) requires the validation of a stable reference gene for normalization. Previously reported candidates may not be suitable for novel crops. Vigna radiata is a protein-rich legume crop used in many countries across the world. As this crop is recalcitrant to transformation such as most legumes, we were interested in V. radiata-Agrobacterium interaction studies. Toward this end, we wanted to identify a suitable reference for normalization under Agrobacterium-infections conditions. We selected seven candidate genes (Actin, Tubulin, Glyceraldehyde 3-phosphate dehydrogenase, Ubiquitin 10, Cyclophilin, 18S Ribosomal RNA, and F-box protein) and performed temporal-expression analysis using real-time PCR at 3 h, 24 h, and 4-day post-infection. When their Ct values were compared using the routinely used four algorithms, Normfinder, geNorm, Bestkeeper, and RefFinder, each suggested a different candidate gene. We narrowed-down to Cyclophilin by taking the statistical approach of using analysis of variance and Tukey honestly significant difference. Thus, our report signifies the use of additional statistical analyses in case of situations of discrepancies due to variable suggestions by software.