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The article titled "Correction" in the Plant Journal provides updates and corrections to figures in a previous article titled "Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs." The corrections include the removal of accidental duplications, correction of gel splicing errors, and the inclusion of additional data. The corrected figures show the interaction of HAB1 with PYL5, PYL8, and PYL6, the subcellular localization of PYL5 and its interaction with HAB1, and the ABA-hypersensitive inhibition of germination and root growth in PYL5-overexpressing lines. The authors apologize for the errors. [Extracted from the article]

SUMMARY: Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant–pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)‐GFP vector and Agrobacterium‐mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV‐GFP system, we initially tested it with well‐described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV‐GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV‐GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV‐GFP system in identifying apoplastic effectors. The easy operation, time‐saving nature, broad effectiveness, and low technical requirements of the TMV‐GFP system make it a promising approach for high‐throughput screening of effectors with immune interference activity from various pathogens. Significance Statement: The development of a robust high‐throughput functional screening assay for pathogen effectors will facilitate the study in plant‐pathogen interactions. We established a novel screening system using the TMV‐GFP vector, enabling the rapid identification of effectors that interfere with plant immunity by observing GFP fluorescence under a UV lamp within a few days. The TMV‐GFP system is a promising approach for high‐throughput screening of effectors from diverse pathogens. [ABSTRACT FROM AUTHOR]

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N‐terminal photosensory chromophore module (PCM); the C‐terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time‐resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto‐488 was attached to each mutant, and time‐resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full‐length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr‐to‐Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr. [ABSTRACT FROM AUTHOR]

This article discusses a strategy called the T-circle vector strategy that increases the frequency of site-specific integration (SSI) in soybean plants. The authors used a non-homologous end joining (NHEJ) repair pathway to insert the entire T-DNA into a specific target site in the soybean genome. They tested different vector designs and found that a vector with two target sites and a split selectable marker gene resulted in a higher frequency of SSI events. The T-circle vector strategy has the potential to reduce costs and improve the efficiency of plant production. [Extracted from the article]

SUMMARY: Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium‐mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant‐microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long‐read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium‐mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large‐scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants. Significance Statement: Agrobacterium‐mediated transformation (AMT) of plants is used extensively as a tool to create new genetic variants for research purposes or agricultural needs. In this work, we summarize the molecular mechanisms involved in AMT and the effects on plant genomes resulting from the integration of exogenous DNA through this method. [ABSTRACT FROM AUTHOR]

The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops. Here, we established efficient Agrobacterium‐mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated nuclease 9 (Cas9) genome editing in a perennial, stress‐tolerant forage grass, sheepgrass (Leymus chinensis). By screening for active single‐guide RNAs (sgRNAs), accessions that regenerate well, suitable Agrobacterium strains, and optimal culture media, and co‐expressing the morphogenic factor TaWOX5, we achieved 11% transformation and 5.83% editing efficiency in sheepgrass. Knocking out Teosinte Branched1 (TB1) significantly increased tiller number and biomass. This study opens avenues for studying gene function and breeding in sheepgrass. [ABSTRACT FROM AUTHOR]

Dr. Steven H. Strauss, a University Distinguished Professor of Forest Biotechnology at Oregon State University, discusses his interest in plant science and his motivation to pursue a career in research. He credits his mentors during his PhD at the University of California at Berkeley for being his role models. Dr. Strauss highlights some of his favorite papers from the New Phytologist journal, including one on the genetic variation in amenability to genetic transformation and regeneration, another on the determination of crown phenology in quaking aspen, and a third on lignin modification in poplar. He also expresses his fondness for poplars, especially aspens and cottonwoods, due to their beauty and amenability to biotech methods. [Extracted from the article]

Summary: Agrobacterium tumefaciens microbe‐associated molecular pattern elongation factor Tu (EF‐Tu) is perceived by orthologs of the Arabidopsis immune receptor EFR activating pattern‐triggered immunity (PTI) that causes reduced T‐DNA‐mediated transient expression. We altered EF‐Tu in A. tumefaciens to reduce PTI and improved transformation efficiency.A robust computational pipeline was established to detect EF‐Tu protein variation in a large set of plant bacterial species and identified EF‐Tu variants from bacterial pathogen Pseudomonas syringae pv. tomato DC3000 that allow the pathogen to escape EFR perception. Agrobacterium tumefaciens strains were engineered to substitute EF‐Tu with DC3000 variants and examined their transformation efficiency in plants.Elongation factor Tu variants with rarely occurred amino acid residues were identified within DC3000 EF‐Tu that mitigates recognition by EFR. Agrobacterium tumefaciens strains were engineered by expressing DC3000 EF‐Tu instead of native agrobacterial EF‐Tu and resulted in decreased plant immunity detection.These engineered A. tumefaciens strains displayed an increased efficiency in transient expression in both Arabidopsis thaliana and Camelina sativa. The results support the potential application of these strains as improved vehicles to introduce transgenic alleles into members of the Brassicaceae family. [ABSTRACT FROM AUTHOR]

Tissue engineering strategies using cell‐laden constructs have shown promising results in the treatment of various types of damaged tissues. However, inadequate oxygen delivery to the macroscale 3D cell‐constructs for regenerating skeletal muscle tissue has remained a multiplex issue owing to the pivotal factors including cell metabolism and several regulatory intercellular pathways that eventually influence various cellular activities and determines cell phenotype. To overcome this issue, a photosynthetic cyanobacterium (Synechococcus elongatus) is employed in a methacrylated gelatin bioink. Furthermore, to effectively induce cell alignment in the bioink, in situ electric field stimulation is used in a bioprinting system to fabricate cell‐laden scaffolds for regenerating skeletal muscle tissue. Owing to the synergistic effects of the bioactive microenvironment that rescues cells from hypoxic conditions and activations of voltage‐gated ion channels, highly aligned, multi‐nucleated myofibers are obtained as well as significant upregulation (7–10‐fold) of myogenic‐related genes compared with conventionally prepared cell‐constructs. In addition, in vivo studies using a mouse volumetric muscle loss model demonstrate considerable restoration of muscle functionality and regeneration. [ABSTRACT FROM AUTHOR]

Summary: The Potyviridae are the largest family of plant‐pathogenic viruses. Members of this family are the soil‐borne bymoviruses barley yellow mosaic virus (BaYMV) and barley mild mosaic virus (BaMMV), which, upon infection of young winter barley seedlings in autumn, can cause yield losses as high as 50%. Resistance breeding plays a major role in coping with these pathogens. However, some viral strains have overcome the most widely used resistance. Thus, there is a need for novel sources of resistance. In ancient landraces and wild relatives of cultivated barley, alleles of the susceptibility factor PROTEIN DISULFIDE ISOMERASE LIKE 5–1 (PDIL5‐1) were identified to confer resistance to all known strains of BaYMV and BaMMV. Although the gene is highly conserved throughout all eukaryotes, barley is thus far the only species for which PDIL5‐1‐based virus resistance has been reported. Whereas introgression by crossing to the European winter barley breeding pool is tedious, time‐consuming and additionally associated with unwanted linkage drag, the present study exemplifies an approach to targeted mutagenesis of two barley cultivars employing CRISPR‐associated endonuclease technology to induce site‐directed mutations similar to those described for PDIL5‐1 alleles that render certain landraces resistant. Homozygous primary mutants were produced in winter barley, and transgene‐free homozygous M2 mutants were produced in spring barley. A variety of mutants carrying novel PDIL5‐1 alleles were mechanically inoculated with BaMMV, by which all frameshift mutations and certain in‐frame mutations were demonstrated to confer resistance to this virus. Under greenhouse conditions, virus‐resistant mutants showed no adverse effects in terms of growth and yield. [ABSTRACT FROM AUTHOR]

SUMMARY: Transient transgenic expression accelerates pharming and facilitates protein studies in plants. One embodiment of the approach involves leaf infiltration of Agrobacterium strains whose T‐DNA is engineered with the gene(s) of interest. However, gene expression during 'agro‐infiltration' is intrinsically and universally impeded by the onset of post‐transcriptional gene silencing (PTGS). Nearly 20 years ago, a simple method was developed, whereby co‐expression of the tombusvirus‐encoded P19 protein suppresses PTGS and thus enhances transient gene expression. Yet, how PTGS is activated and suppressed by P19 during the process has remained unclear to date. Here, we address these intertwined questions in a manner also rationalizing how vastly increased protein yields are achieved using a minimal viral replicon as a transient gene expression vector. We also explore, in side‐by‐side analyses, why some proteins do not accumulate to the expected high levels in the assay, despite vastly increased mRNA levels. We validate that enhanced co‐expression of multiple constructs is achieved within the same transformed cells, and illustrate how the P19 system allows rapid protein purification for optimized downstream in vitro applications. Finally, we assess the suitability of the P19 system for subcellular localization studies – an originally unanticipated, yet increasingly popular application – and uncover shortcomings of this specific implement. In revisiting the P19 system using contemporary knowledge, this study sheds light onto its hitherto poorly understood mechanisms while further illustrating its versatility but also some of its limits. Significance Statement: Since its disclosure in 2003, agro‐infiltration used in combination with the P19 RNA silencing suppressor has been a popular method to achieve high levels of transient gene expression in Nicotiana benthamiana, though the underlying molecular mechanisms have remained unclear. By shedding light on these mechanisms, the present contemporary reassessment of the method allows improvements to protein yields and further illustrates its versatility, but also exposes and rationalizes some of its limits. [ABSTRACT FROM AUTHOR]

BACKGROUND: Carboxyspermidine (C‐Spd) is a potentially valuable polyamine carboxylate compound and an excellent building block for spermidine synthesis, which is a critical polyamine with significant implications for human health and longevity. C‐Spd can also be used to prepare multivalent cationic lipids and modify nucleoside probes. Because of these positive effects on human health, C‐Spd is of considerable interest as a food additive and pharmaceutical target. RESULTS: A putative gene afcasdh from Agrobacterium fabrum str. C58, encoding carboxyspermidine dehydrogenase with C‐Spd biosynthesis activity, was synthesized and transformed into Escherichia coli BL21 (DE3) for overexpression. The recombinant AfCASDH was purified and fully characterized. The optimum temperature and pH for the recombinant enzyme were 30 °C and 7.5, respectively. The coupled catalytic strategy of AfCASDH and various NADPH regeneration systems were developed to enhance the efficient production of C‐Spd compound. Finally, the maximum titer of C‐Spd production successfully achieved 1.82 mmol L−1 with a yield of 91% by optimizing the catalytic conditions. CONCLUSION: A novel AfCASDH from A. fabrum str. C58 was characterized that could catalyze the formation of C‐Spd from putrescine and l‐aspartate‐β‐semialdehyde (L‐Asa). A whole‐cell catalytic strategy coupled with NADPH regeneration was established successfully for C‐Spd biosynthesis for the first time. The coupled system indicated that AfCASDH might provide a feasible method for the industrial production of C‐Spd. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Concanavalin A (ConA), a mannose (Man)‐specific leguminous lectin isolated from the jack bean (Canavalia ensiformis) seed extracts, was discovered over a century ago. Although ConA has been extensively applied in various life science research, recombinant mature ConA expression has not been fully established. Here, we aimed to produce recombinant ConA (rConA) in lettuce (Lactuca sativa) using an Agrobacterium tumefaciens‐mediated transient expression system. rConA could be produced as a fully active form from soluble fractions of lettuce leaves and purified by affinity chromatography. From 12 g wet weight of lettuce leaves, 0.9 mg rConA could be purified. The glycan‐binding properties of rConA were then compared with that of the native ConA isolated from jack bean using glycoconjugate microarray and frontal affinity chromatography. rConA demonstrated a glycan‐binding specificity similar to nConA. Both molecules bound to N‐glycans containing a terminal Man residue. Consistent with previous reports, terminal Manα1‐6Man was found to be an essential unit for the high‐affinity binding of rConA and nConA, while bisecting GlcNAc diminished the binding of rConA and nConA to Manα1‐6Man‐terminated N‐glycans. These results demonstrate that the fully active rConA could be produced using the A. tumefaciens‐mediated transient expression system and used as a recombinant substitute for nConA. [ABSTRACT FROM AUTHOR]

Keywords: sweet potato; genetic engineering; naturally transgenic plant; Agrobacterium; transformation EN sweet potato genetic engineering naturally transgenic plant Agrobacterium transformation 671 673 3 03/28/23 20230401 NES 230401 Sweet potato ( I Ipomoea batatas i [L.] I Agrobacterium rhizogenes i contains two transferable T-DNA regions: TR-DNA corresponds to IbT-DNA1 (harbouring the auxin biosynthesis genes I iaaH i and I iaaM i ) and TL-DNA (harbouring the various I Rol i genes) to IbT-DNA2 (Figure 1c). [Extracted from the article]

Summary: We have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium‐derived vir and T‐DNA‐mediated transformation. Oh H1 is a unique, non‐phytopathogenic species, categorized as a BSL‐1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1‐8 strain containing a binary vector system. Oh H1‐8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy‐. Oh H1‐8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1‐8 generated high‐quality, transgenic events with single‐copy, plasmid backbone‐free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock‐inducible excision system resulting in marker‐free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null‐segregant lines to wild‐type controls. We have demonstrated that Oh H1‐8, combined with spectinomycin selection, is an efficient, rapid, marker‐free and yield‐neutral transformation system for elite soybean. [ABSTRACT FROM AUTHOR]

Summary: Sorghum bicolor (L.) Moench, the fifth most important cereal worldwide, is a multi‐use crop for feed, food, forage and fuel. To enhance the sorghum and other important crop plants, establishing gene function is essential for their improvement. For sorghum, identifying genes associated with its notable abiotic stress tolerances requires a detailed molecular understanding of the genes associated with those traits. The limits of this knowledge became evident from our earlier in‐depth sorghum transcriptome study showing that over 40% of its transcriptome had not been annotated. Here, we describe a full spectrum of tools to engineer, edit, annotate and characterize sorghum's genes. Efforts to develop those tools began with a morphogene‐assisted transformation (MAT) method that led to accelerated transformation times, nearly half the time required with classical callus‐based, non‐MAT approaches. These efforts also led to expanded numbers of amenable genotypes, including several not previously transformed or historically recalcitrant. Another transformation advance, termed altruistic, involved introducing a gene of interest in a separate Agrobacterium strain from the one with morphogenes, leading to plants with the gene of interest but without morphogenes. The MAT approach was also successfully used to edit a target exemplary gene, phytoene desaturase. To identify single‐copy transformed plants, we adapted a high‐throughput technique and also developed a novel method to determine transgene independent integration. These efforts led to an efficient method to determine gene function, expediting research in numerous genotypes of this widely grown, multi‐use crop. [ABSTRACT FROM AUTHOR]

Genetic manipulation for generating knock‐out experiments is essential in deciphering the precise function of a gene. However, dikaryotic fungi pose the inherent challenge of having two allelic versions of each gene, one in each nucleus. In addition, they often are slow‐growing and do not withstand protoplasting, which is why Agrobacterium tumefaciens‐mediated transformation has been adapted. To obtain knock‐out strains, however, is not feasible with a mere deletion construct transformation and screening for deletions in both nuclear copies. Hence, a convenient method using chemically synthesized dicer substrate interfering RNA (DsiRNA) for posttranscriptional interference of targeted mRNA was developed, based on the fungal dicer/argonaute system inherent in fungi for sequence recognition and degradation. A proof‐of‐principle using this newly established method for knock‐down of the volatile geosmin is presented in the dikaryotic fungus Tricholoma vaccinum that is forming ectomycorrhizal symbiosis with spruce trees. The gene ges1, a terpene synthase, was transcribed with a 50‐fold reduction in transcript levels in the knockdown strain. The volatile geosmin was slightly reduced, but not absent in the fungus carrying the knockdown construct pointing at low specificity in other terpene synthases known for that class of enzymes. [ABSTRACT FROM AUTHOR]

The intracellular bacterial pathogen Legionella pneumophila utilizes the Dot/Icm type IV secretion system to translocate approximately 300 effector proteins to establish a replicative niche known as the Legionella‐containing vacuole. The Dot/Icm system is classified as a type IVB secretion system, which is evolutionarily closely related to the I‐type conjugation systems and is distinct from type IVA secretion systems, such as the Agrobacterium VirB/D4 system. Although both type IVA and IVB systems directly transport nucleic acids or proteins into the cytosol of recipient cells, the components and architecture of type IVB systems are much more complex than those of type IVA systems. Taking full advantage of rapidly developing cryo‐electron microscopy techniques, the structural details of the transport apparatus and coupling complexes in the Dot/Icm system have been clarified in the past few years. In this review, we summarize recent progress in the structural studies of the L. pneumophila type IVB secretion system and the insights gained into the mechanisms of substrate recognition and transport. [ABSTRACT FROM AUTHOR]

Summary: Agrobacterium tumefaciens colonizes the galls (plant tumors) it causes, and the roots of host and nonhost plants.Transposon‐sequencing (Tn‐Seq) was used to discover A.tumefaciens genes involved in reproductive success (fitness genes) on Solanum lycopersicum and Populus trichocarpa tumors and S.lycopersicum and Zea mays roots.The identified fitness genes represent 3–8% of A. tumefaciens genes and contribute to carbon and nitrogen metabolism, synthesis and repair of DNA, RNA and proteins and envelope‐associated functions. Competition assays between 12 knockout mutants and wild‐type confirmed the involvement of 10 genes (trpB, hisH, metH, cobN, ntrB, trxA, nrdJ, kamA, exoQ, wbbL) in A.tumefaciens fitness under both tumor and root conditions. The remaining two genes (fecA, noxA) were important in tumors only. None of these mutants was nonpathogenic, but four (hisH, trpB, exoQ, ntrB) exhibited impaired virulence. Finally, we used this knowledge to search for chemical and biocontrol treatments that target some of the identified fitness pathways and report reduced tumorigenesis and impaired establishment of A.tumefaciens on tomato roots using tannic acid or Pseudomonas protegens, which affect iron assimilation.This work revealed A.tumefaciens pathways that contribute to its competitive survival in plants and highlights a strategy to identify plant protection approaches against this pathogen. [ABSTRACT FROM AUTHOR]

Assessing immune responses and cell death in Nicotiana benthamiana leaf agro‐infiltration assays is a powerful and widely used experimental approach in molecular plant pathology. Here, we describe a reliable high‐throughput protocol to quantify strong, macroscopically visible cell death responses in N. benthamiana agro‐infiltration assays. The method relies on measuring the reduction of leaf autofluorescence in the red spectrum upon cell death induction and provides quantitative data suitable for straightforward statistical analysis. Two different well‐established model nucleotide‐binding and leucine‐rich repeat domain proteins (NLRs) were used to ensure the genericity of the approach. Its accuracy and versatility were compared to visual scoring of the cell death response and standard methods commonly used to characterize NLR activities in N. benthamiana. A discussion of the advantages and limitations of our method compared to other protocols demonstrates its robustness and versatility and provides an effective means to select the best‐suited protocol for a defined experiment. [ABSTRACT FROM AUTHOR]

SUMMARY: Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration. By integrating a single copy of the lux operon into the genome, we generated a stable 'AgroLux' strain, which is bioluminescent without affecting Agrobacterium growth in vitro and in planta. To illustrate its versatility, we used AgroLux to demonstrate that high light intensity post infiltration suppresses both Agrobacterium luminescence and protein expression. We also discovered that AgroLux can detect Avr/Cf‐induced immune responses before tissue collapse, establishing a robust and rapid quantitative assay for the hypersensitive response (HR). Thus, AgroLux provides a non‐destructive, versatile and easy‐to‐use imaging tool to monitor both Agrobacterium and plant responses. Significance Statement: We generated a genome‐integrated bioluminescent Agrobacterium strain that grows normally in vitro and in planta. We illustrate its broad versatility by correlating reduced bioluminescence with protein expression upon high light stress, and by establishing the early and sensitive detection of immunity induced by the coexpression of matching Avr/Cf gene combinations. [ABSTRACT FROM AUTHOR]

Summary: The plant‐growth‐promoting bacterium Azospirillum brasilense is able to associate with the microalgae Chlorella sorokiniana. Attachment of A. brasilense increases the metabolic performances of the microalgae. Recent genome analyses have revealed that the A. brasilense Az39 genome contains two complete sets of genes encoding type VI secretion systems (T6SS), including the T6SS1 that is induced by the indole‐3‐acetic acid (IAA) phytohormone. The T6SS is a multiprotein machine, widespread in Gram‐negative bacteria, that delivers protein effectors in both prokaryotic and eukaryotic cells. Here we show that the A. brasilense T6SS is required for Chlorella‐Azospirillum synthetic mutualism. Our data demonstrate that the T6SS is an important determinant to promote production of lipids, carbohydrates and photosynthetic pigments by the microalgae. We further show that this is likely due to the role of the T6SS during the attachment stage and for the production of IAA phytohormones. Finally, we demonstrate that the A. brasilense T6SS provides antagonistic activities against a number of plant pathogens such as Agrobacterium, Pectobacterium, Dickeya and Ralstonia species in vitro, suggesting that, in addition to promoting growth, A. brasilense might confer T6SS‐dependent bio‐control protection to microalgae and plants against bacterial pathogens. [ABSTRACT FROM AUTHOR]

Summary: CRISPR‐Cas is a powerful DNA double‐strand break technology with wide‐ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knockouts in multiple crops, precise nucleotide replacement and especially gene insertion into a pre‐defined genomic location remain highly challenging. Here, we report an efficient, selectable marker‐free site‐specific gene insertion in maize using Agrobacterium infection. Advancements in maize transformation and new vector design enabled increase of targeted insertion frequencies by two orders of magnitude in comparison to conventional Agrobacterium‐mediated delivery. Importantly, these advancements allowed not only a significant improvement of the frequency, but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium‐mediated transformation. [ABSTRACT FROM AUTHOR]

Establishing a transformation system is an essential first step for functional studies. Numerous transformation systems have been developed and optimized for filamentous ascomycetes, including Agrobacterium‐mediated transformation. Recently, such a system was developed for the African tree pathogen Ceratocystisalbifundus. This fungus is part of the family Ceratocystidaceae, which includes many tree and plant pathogens of economic importance. Despite the many advantages associated with Agrobacterium‐mediated transformation, establishing and optimizing this system within a species are extremely arduous due to the many species‐specific parameters that must be optimized. This study aimed to apply the existing C. albifundusAgrobacterium‐mediated transformation protocol to eight diverse Ceratocystidaceae species from eight different genera. Minor adjustments were made to the already established protocol to increase transformant yield before it was used to successfully produce transformants for six of the eight targeted species without the need for any further species‐specific optimizations. Although the number of transformants obtained was less than for C. albifundus, this study proves that protocols for Agrobacterium‐mediated transformation might be more transferable than previously thought. It also provides a useful benchmark for future transformation studies in the Ceratocystidaceae and will make species‐specific optimization easier. [ABSTRACT FROM AUTHOR]

SUMMARY: Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium‐mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium‐mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas‐mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies. Significance Statement: The tissue culture and transformation technology developed herein represents a significant advance in Agrobacterium‐mediated transformation and CRISPR/Cas‐mediated genome editing of an important orphan crop, cowpea (Vigna unguiculata (L.) Walp.). The principles established in this study have the potential to improve the transformation and editing efficiencies not only for cowpea, but also for other legume species, such as soybean (Glycine max) and common bean (Phaseolus vulgaris). [ABSTRACT FROM AUTHOR]

Summary: Integration of Agrobacterium tumefaciens transferred DNA (T‐DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T‐DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end‐joining (NHEJ) pathways. Recent evidence suggests that in Arabidopsis, DNA polymerase θ (PolQ) may be a crucial enzyme involved in T‐DNA integration.We conducted quantitative transformation assays of wild‐type and polQ mutant Arabidopsis and rice, analyzed T‐DNA/plant DNA junction sequences, and (for Arabidopsis) measured the amount of integrated T‐DNA in mutant and wild‐type tissue.Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency of polQ mutants was c. 20% that of wild‐type plants. T‐DNA/plant DNA junctions from these transformed rice and Arabidopsis polQ mutants closely resembled those from wild‐type plants, indicating that loss of PolQ activity does not alter the characteristics of T‐DNA integration events. polQ mutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation.We suggest that either multiple redundant pathways function in T‐DNA integration, and/or that integration requires some yet unknown pathway. See also the Commentary on this article by Faure, 229: 2386–2388. [ABSTRACT FROM AUTHOR]

Lawson cypress (Chamaecyparis lawsoniana), an important landscape tree, is widely planted in gardens and parks throughout Iran. Crown gall disease on Lawson cypress trees was observed in Sari and Juybar Counties, Mazandaran province, northern Iran, in 2017. Isolation from galls on potato dextrose agar (PDA) containing CaCO3 yielded bacterial colonies, the predominant types of which were purified and selected for characterization. The isolates were Gram‐negative, oxidase positive, able to grow in 2% NaCl and produced 3‐ketolactose. They hydrolysed esculin, casein and arbutin but not starch, gelatin or Tween 80. Two representative isolates were selected for PCR amplification and sequencing of DNA gyrase subunit B (gyrB) gene. In the phylogenetic tree based on the partial sequence of the gyrB gene, isolates KH1 and KH2 clustered with Agrobacterium pusense. The pathogenicity of all isolates was confirmed by inoculation on Jimsonweed (Datura stramonium) and carrot discs (Daucus carota). Confirmation of the presence of genes involved in pathogenicity was made by performing PCR with the virD2A/virD2C and VCF/VCR primer pairs which resulted in amplification of the expected 224 and 730 bp fragments in all studied isolates, respectively. A. pusense was therefore identified as the causal agent of crown and stem gall of Lawson cypress. This appears to be the first report on the natural occurrence of crown gall disease on Lawson cypress and the first record of a plant disease caused by A. pusense. [ABSTRACT FROM AUTHOR]

Summary: The lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth, and that transient depletion of a host immune gene and transient expression of a type‐III effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure−function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers a reliable approach for further comprehensive analysis of plant immunity. [ABSTRACT FROM AUTHOR]

Transformation of sugar beet with genes conferring tolerance against biotic stresses might accelerate improvement of new hybrid varieties with respect to root yield. In the present study, the optimization of transformation procedure and the integration of four agronomically important genes were assessed in two sugar beet inbred lines. First, the transformation system was optimized with the Agrobacterium tumefacienes LBA4404 and GV3101 strains harboring a recombinant plasmid vector. The vector was constructed based on pBI121 plasmid containing the cryIA105, cryIIIAa, CP4‐epsps and gox genes. The effects of four factors including the strains and growth density of Agrobacterium, inoculation time, and plant lines on the frequency of regeneration were assessed. The adventitious bud excised leaflets were used as explants for tissue culture. A factorial experiment based on completely randomized design was adopted as statistical model. Analysis of variance showed that simple effect of Agrobacterium strain, growth density and inoculation time on regeneration of transgenic plantlets, as well as growth rate × inoculation time interaction had significant effects on regeneration of transgenic plantlets. Means comparison analyses indicated that OD600 = 0.6 and inoculation time of 15 min were the best conditions for transformation. Totally, more than 330 putative transgenic plantlets were regenerated during transformation. The presence of transgenes in the putative transgenic plants was confirmed by polymerase chain reaction (PCR) analysis. Subsequently, transcription of transgenes was confirmed by Reverse Transcription‐PCR analysis. Overall, our results revealed that the modified protocol for transformation could be used successfully to introduce new gene cassettes into sugar beet lines. [ABSTRACT FROM AUTHOR]

An efficient biocatalytic approach for enantio‐ and regioselective ring‐opening of styrene oxides with cyanate was developed by using the halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1, generating the corresponding chiral 5‐aryl‐2‐oxazolidinones in up to 47% yield and 90% ee. Additionally, the origin of enantioselectivity and regioselectivity of the HheC‐catalyzed cyanate‐mediated ring‐opening process was uncovered by single enantiomer bioconversions and molecular docking study. [ABSTRACT FROM AUTHOR]

Site‐specific recombinase enzymes function in heterologous cellular environments to initiate strand‐switching reactions between unique DNA sequences termed recombinase binding sites. Depending on binding site position and orientation, reactions result in integrations, excisions, or inversions of targeted DNA sequences in a precise and predictable manner. Here, we established five different stable recombinase expression lines in maize through Agrobacterium‐mediated transformation of T‐DNA molecules that contain coding sequences for Cre, R, FLPe, phiC31 Integrase, and phiC31 excisionase. Through the bombardment of recombinase activated DsRed transient expression constructs, we have determined that all five recombinases are functional in maize plants. These recombinase expression lines could be utilized for a variety of genetic engineering applications, including selectable marker removal, targeted transgene integration into predetermined locations, and gene stacking. [ABSTRACT FROM AUTHOR]

Beta‐glucan is currently under consideration as an alternative to in‐feed antibiotics. The aim of the study was to investigate Agrobacterium sp. ZX09 beta‐glucan on intestinal morphology, cytokine concentration, mucin expression and microbial populations of weaning piglets. Pigs were randomly assigned to one of five dietary treatments supplemented with 0, 25, 50, 100 and 200 mg/kg beta‐glucan. Data showed an increase in ADG at the 100 mg/kg group (p = .03). A significant increase in villus height and reduction in crypt depth were fund in ileal tissue at the 100 mg/kg inclusion level (p < .05). Dietary supplementation of 100 mg/kg beta‐glucan enhanced IL‐10 concentration (p = .04) and gene expression of MUC1 and MUC2 (p < .05) in the jejunum. Dietary supplementation of 100 mg/kg beta‐glucan provoked the up‐regulation of Lactobacillus counts and down‐regulation of Escherichia coli counts in the caecum (p = .05). Data suggested that improved growth performance in response to beta‐glucan supplementation at 100 mg/kg in weaned piglets may be explained by the improved intestinal function. [ABSTRACT FROM AUTHOR]

Summary: An efficient Agrobacterium‐mediated site‐specific integration (SSI) technology using the flipase/flipase recognition target (FLP/FRT) system in elite maize inbred lines is described. The system allows precise integration of a single copy of a donor DNA flanked by heterologous FRT sites into a predefined recombinant target line (RTL) containing the corresponding heterologous FRT sites. A promoter‐trap system consisting of a pre‐integrated promoter followed by an FRT site enables efficient selection of events. The efficiency of this system is dependent on several factors including Agrobacterium tumefaciens strain, expression of morphogenic genes Babyboom (Bbm) and Wuschel2 (Wus2) and choice of heterologous FRT pairs. Of the Agrobacterium strains tested, strain AGL1 resulted in higher transformation frequency than strain LBA4404 THY‐ (0.27% vs. 0.05%; per cent of infected embryos producing events). The addition of morphogenic genes increased transformation frequency (2.65% in AGL1; 0.65% in LBA4404 THY‐). Following further optimization, including the choice of FRT pairs, a method was developed that achieved 19%–22.5% transformation frequency. Importantly, >50% of T0 transformants contain the desired full‐length site‐specific insertion. The frequencies reported here establish a new benchmark for generating targeted quality events compatible with commercial product development. [ABSTRACT FROM AUTHOR]

Summary: Plasmids are mobile DNAs that can adjust host cell functions for their own amplification and dissemination. We identified Quorum sensing flagella small RNA regulator (QfsR), a small RNA, transcribed from the virulence tumour‐inducing (Ti) plasmid in the phytopathogen Agrobacterium fabrum. QfsR is widely conserved throughout RepABC plasmids carried by Rhizobiaceae. Target prediction, expression analysis and site‐direct mutagenesis experiments showed that QfsR directly pairs within polycistronic mRNAs transcribed from chromosome (genes involved in flagella apparatus) and Ti plasmid (genes involved in conjugative transfer). QfsR leads to a coordinated expression of whole polycistronic mRNA molecules. Whereas a lack of QfsR represses motility, its overproduction increases the quorum sensing signal accumulation and the Ti plasmid conjugative transfer. Based on these observations, we propose QfsR as a hub connecting regulatory networks of motility and plasmid conjugative transfer. To our knowledge, QfsR is the first example of a plasmid‐encoded sRNA that controls chromosomal polycistronic gene expression. [ABSTRACT FROM AUTHOR]

Summary: Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T‐DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus‐based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next‐generation virus‐based tools will be indispensable landmarks in the synthetic biology roadmap to better crops. [ABSTRACT FROM AUTHOR]

Prokaryotic (6‐4) photolyases branch at the base of the evolution of cryptochromes and photolyases. Prototypical members contain an iron–sulphur cluster which was lost in the evolution of the other groups. In the Agrobacterium (6‐4) photolyase PhrB, the repair of DNA lesions containing UV‐induced (6‐4) pyrimidine dimers is stimulated by Mg2+. We propose that Mg2+ is required for efficient lesion binding and for charge stabilization after electron transfer from the FADH− chromophore to the DNA lesion. Furthermore, two highly conserved Asp residues close to the DNA‐binding site are essential for the effect of Mg2+. Simulations show that two Mg2+ bind to the region around these residues. On the other hand, DNA repair by eukaryotic (6‐4) photolyases is not increased by Mg2+. In these photolyases, structurally overlapping regions contain no Asp but positively charged Lys or Arg. During the evolution of photolyases, the role of Mg2+ in charge stabilization and enhancement of DNA binding was therefore taken over by a postiviely charged amino acid. Besides PhrB, another prokaryotic (6‐4) photolyase from the marine cyanobacterium Prochlorococcus marinus, PromaPL, which contains no iron–sulphur cluster, was also investigated. This photolyase is stimulated by Mg2+ as well. The evolutionary loss of the iron–sulphur cluster due to limiting iron concentrations can occur in a marine environment as a result of iron deprivation. However, the evolutionary replacement of Mg2+ by a positively charged amino acid is unlikely to occur in a marine environment because the concentration of divalent cations in seawater is always sufficient. We therefore assume that this transition could have occurred in a freshwater environment. [ABSTRACT FROM AUTHOR]

Summary: Aided by translocated virulence proteins, Agrobacterium tumefaciens transforms plant cells with oncogenic T‐DNA. In the host cells the virulence protein VirD5 moves to the nucleus, where it becomes localized at the kinetochores, and disturbs faithful chromosome segregation, but the molecular mechanism underlying this remains unknown.To gain more insight, we screened amongst the kinetochore proteins for VirD5 interactors using bimolecular fluorescence complementation assays, and tested chromosome segregation in yeast cells.We found that VirD5 interacts with the conserved mitotic Aurora kinase Ipl1 in yeast and likewise with plant Aurora kinases. In vitro VirD5 was found to stimulate the activity of Ipl1. Phosphorylation of substrates by Ipl1 in vivo is known to result in the detachment between kinetochore and spindle microtubule. This is necessary for error correction, but increased Ipl1/Aurora kinase activity is known to cause spindle instability, explaining enhanced chromosome mis‐segregation seen in the presence of VirD5. That activation of the Ipl1/Aurora kinase at least partially underlies the toxicity of VirD5 became apparent by artificial boosting the activity of the specific counteracting phosphatase Glc7 in vivo, which relieved the toxicity.These findings reveal a novel mechanism by which a pathogenic bacterium manipulates host cells. [ABSTRACT FROM AUTHOR]

During bacterial conjugation, plasmid DNA is transferred from cell to cell. In Agrobacterium fabrum, conjugation is regulated by the phytochrome photoreceptors Agp1 and Agp2. Both contribute equally to this regulation. Agp1 and Agp2 are histidine kinases, but, for Agp2, we found no autophosphorylation activity. A clear autophosphorylation signal, however, was obtained with mutants in which the phosphoaccepting Asp of the C‐terminal response regulator domain is replaced. Thus, the Agp2 histidine kinase differs from the classical transphosphorylation pattern. We performed size exclusion, photoconversion, dark reversion, autophosphorylation, chromophore assembly kinetics and fluorescence resonance energy transfer measurements on mixed Agp1/Agp2 samples. These assays pointed to an interaction between both proteins. This could partially explain the coaction of both phytochromes in the cell. [ABSTRACT FROM AUTHOR]

In the present study, I Agrobacterium i -mediated transformation of I Indica i rice ASD16 with two genes involved in tocopherol biosynthesis, I viz i ., I TC i and I HPT, i was carried out and the transgenic plants were analysed for the vitamin E ( -tocopherol) content. Keywords: Agrobacterium; rice; vitamin E; HPLC EN Agrobacterium rice vitamin E HPLC 1083 1085 3 06/15/21 20210601 NES 210601 Micronutrient deficiency results in malnutrition, which is prevalent all over the world and may lead to premature death in women and children (White and Broadley, 2009). [Extracted from the article]

This article is a Commentary on Nishizawa‐Yokoi et al. (2021), 229: 2859–2872. [ABSTRACT FROM AUTHOR]

This article is a Commentary on Xu et al., 226: 1213–1221. [ABSTRACT FROM AUTHOR]

Summary: The adaptation of the Agrobacterium‐mediated floral‐dipping technique is limited, to date, to a small number of plants. In this paper, we present the efficient transformation of one of the leading plants in the cut flower industry, lisianthus (Eustoma grandiflorum). This method is approximately 18 months shorter than the known tissue culture‐based transformation. The Excalibur Pink cultivar and two additional breeding lines, X‐1042 and X‐2541, were transformed using three different marker genes (benzyl alcohol acetyltransferase (BEAT) originating from Clarkia breweri, the feedback‐insensitive bacterial gene AroG*, and the empty pART27 vector expressing a kanamycin‐resistance cassette (nptII)). Genomic transformation was successful in all tested cases with transformation efficiency ranked from 0.2 to 2.9%, which is well in the range of results from Arabidopsis studies. Unlike Arabidopsis, in which floral‐dipping transformation was efficient only at a pre‐anthesis stage before ovary sealing, lisianthus flowers were transformed when dipping occurred 4 days pre‐anthesis or 3−5 days post‐anthesis with 1.5 and 3.7% efficiencies, respectively. Post‐anthesis transformation occurred when the flower ovaries were sealed. Flower dipping of Excalibur Pink flowers with fluorescent Agrobacterium containing a GFP marker gene demonstrated Agrobacterium entrance into the sealed flower ovary through the open stigma and style tube. In this study, we demonstrated floral‐dipping transformation of a commercial plant, lisianthus Excalibur Pink, occurring after sealing of the ovaries, probably via the stigma and wide open style tunnel. Significance Statement: Here we present successful Agrobacterium‐mediated genetic transformation of lisianthus via flower‐dipping. A unique finding of this study, is that successful transformation was achieved post‐anthesis, when lisianthus flower ovules are completely sealed, in contrast to the findings in Arabidopsis, suggesting possible entrance of the Agrobacterium through the flower style tube. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Agrobacterium tumefaciens can genetically transform plants by translocating a piece of oncogenic DNA, called T‐DNA, into host cells. Transfer is mediated by a type IV secretion system (T4SS). Besides the T‐DNA, which is transferred in a single‐stranded form and at its 5′ end covalently bound to VirD2, several other effector proteins (VirE2, VirE3, VirD5, and VirF) are translocated into the host cells. The fate and function of the translocated proteins inside the host cell are only partly known. Therefore, several studies were conducted to visualize the translocation of the VirE2 protein. As GFP‐tagged effector proteins are unable to pass the T4SS, other approaches like the split GFP system were used, but these require specific transgenic recipient cells expressing the complementary part of GFP. Here, we investigated whether use can be made of the photostable variant of LOV, phiLOV2.1, to visualize effector protein translocation from Agrobacterium to non‐transgenic yeast and plant cells. We were able to visualize the translocation of all five effector proteins, both to yeast cells, and to cells in Nicotiana tabacum leaves and Arabidopsis thaliana roots. Clear signals were obtained that are easily distinguishable from the background, even in cases in which by comparison the split GFP system did not generate a signal. Significance Statement: Although A. tumefaciens is widely used for genetic transformation of numerous different plant species, many aspects of the transformation process are still unclear. Here, we made use of the phiLOV2.1 fluorescent protein to directly visualize effector protein translocation into host cells. In contrast to previous GFP‐based methodologies, this method does not rely on special transgenic host cells. We successfully visualized protein translocation into A. thaliana root, tobacco leaf, and yeast cells. [ABSTRACT FROM AUTHOR]

Tragopogon (Asteraceae) is an excellent natural system for studies of recent polyploidy. Development of an efficient CRISPR/Cas9‐based genome editing platform in Tragopogon will facilitate novel studies of the genetic consequences of polyploidy. Here, we report our initial results of developing CRISPR/Cas9 in Tragopogon. We have established a feasible tissue culture and transformation protocol for Tragopogon. Through protoplast transient assays, use of the TragCRISPR system (i.e. the CRISPR/Cas9 system adapted for Tragopogon) was capable of introducing site‐specific mutations in Tragopogon protoplasts. Agrobacterium‐mediated transformation with Cas9‐sgRNA constructs targeting the phytoene desaturase gene (TraPDS) was implemented in this model polyploid system. Sequencing of PCR amplicons from the target regions indicated simultaneous mutations of two alleles and four alleles of TraPDS in albino shoots from Tragopogon porrifolius (2x) and Tragopogon mirus (4x), respectively. The average proportions of successfully transformed calli with the albino phenotype were 87% and 78% in the diploid and polyploid, respectively. This appears to be the first demonstration of CRISPR/Cas9‐based genome editing in any naturally formed neopolyploid system. Although a more efficient tissue culture system should be developed in Tragopogon, application of a robust CRISPR/Cas9 system will permit unique studies of biased fractionation, the gene‐balance hypothesis and cytonuclear interactions in polyploids. In addition, the CRISPR/Cas9 platform enables investigations of those genes involved in phenotypic changes in polyploids and will also facilitate novel functional biology studies in Asteraceae. Our workflow provides a guide for applying CRISPR/Cas9 to other nongenetic model plant systems. [ABSTRACT FROM AUTHOR]

Summary: Biotechnology provides a means for the rapid genetic improvement of plants. Although single genes have been important in engineering herbicide and pest tolerance traits in crops, future improvements of complex traits like yield and nutritional quality will likely require the introduction of multiple genes. This research reports a system (GAANTRY; Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY) for the flexible, in vivo stacking of multiple genes within an Agrobacterium virulence plasmid Transfer‐DNA (T‐DNA). The GAANTRY system utilizes in vivo transient expression of unidirectional site‐specific recombinases and an alternating selection scheme to sequentially assemble multiple genes into a single transformation construct. To demonstrate GAANTRY's capabilities, 10 cargo sequences were sequentially stacked together to produce a 28.5‐kbp T‐DNA, which was used to generate hundreds of transgenic events. Approximately 90% of the events identified using a dual antibiotic selection screen exhibited all of the introduced traits. A total of 68% of the tested lines carried a single copy of the selection marker transgene located near the T‐DNA left border, and only 8% contained sequence from outside the T‐DNA. The GAANTRY system can be modified to easily accommodate any method of DNA assembly and generate high‐quality transgenic plants, making it a powerful, yet simple to use tool for plant genetic engineering. [ABSTRACT FROM AUTHOR]

Summary: Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T‐DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O‐antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef‐Tu and flagellin pathogen‐associated molecular pattern‐response and glycerol‐3‐phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host‐mediated quorum‐quenching. Finally, construction of knock‐out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host–pathogen interactions. [ABSTRACT FROM AUTHOR]

Summary: Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi‐arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium‐mediated transformation of sorghum. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free ‘quality events’ ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84‐7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9‐mediated gene knockouts in Tx430. [ABSTRACT FROM AUTHOR]

Summary: T‐DNA transfer from Agrobacterium to its host plant genome relies on multiple interactions between plant proteins and bacterial effectors. One such plant protein is the Arabidopsis VirE2 interacting protein (AtVIP1), a transcription factor that binds Agrobacterium tumefaciens C58 VirE2, potentially acting as an adaptor between VirE2 and several other host factors. It remains unknown, however, whether the same VirE2 protein has evolved to interact with multiple VIP1 homologues in the same host, and whether VirE2 homologues encoded by different bacterial strains/species recognize AtVIP1 or its homologues. Here, we addressed these questions by systematic analysis (using the yeast two‐hybrid and co‐immunoprecipitation approaches) of interactions between VirE2 proteins encoded by four major representatives of known bacterial species/strains with functional T‐DNA transfer machineries and eight VIP1 homologues from Arabidopsis and tobacco. We also analysed the determinants of the VirE2 sequence involved in these interactions. These experiments showed that the VirE2 interaction is degenerate: the same VirE2 protein has evolved to interact with multiple VIP1 homologues in the same host, and different and mutually independent VirE2 domains are involved in interactions with different VIP1 homologues. Furthermore, the VIP1 functionality related to the interaction with VirE2 is independent of its function as a transcriptional regulator. These observations suggest that the ability of VirE2 to interact with VIP1 homologues is deeply ingrained into the process of Agrobacterium infection. Indeed, mutations that abolished VirE2 interaction with AtVIP1 produced no statistically significant effects on interactions with VIP1 homologues or on the efficiency of genetic transformation. [ABSTRACT FROM AUTHOR]