Optimized Transformation and Gene Editing of the B104 Public Maize Inbred by Improved Tissue Culture and Use of Morphogenic Regulators.

Plant transformation is a bottleneck for the application of gene editing in plants. In Zea mays (maize), a breakthrough was made using co-transformation of the morphogenic transcription factors BABY BOOM (BBM) and WUSCHEL (WUS) to induce somatic embryogenesis. Together with adapted tissue culture media, this was shown to increase transformation efficiency significantly. However, use of the method has not been reported widely, despite a clear need for increased transformation capacity in academic settings. Here, we explore use of the method for the public maize inbred B104 that is widely used for transformation by the research community. We find that only modifying tissue culture media already boosts transformation efficiency significantly and can reduce the time in tissue culture by 1 month. On average, production of independent transgenic plants per starting embryo increased from 1 to 4% using BIALAPHOS RESISTANCE (BAR) as a selection marker. In addition, we reconstructed the BBM-WUS morphogenic gene cassette and evaluated its functionality in B104. Expression of the morphogenic genes under tissue- and development stage-specific promoters led to direct somatic embryo formation on the scutellum of zygotic embryos. However, eight out of ten resulting transgenic plants showed pleiotropic developmental defects and were not fertile. This undesirable phenotype was positively correlated with the copy number of the morphogenic gene cassette. Use of constructs in which morphogenic genes are flanked by a developmentally controlled Cre/LoxP recombination system led to reduced T-DNA copy number and fertile T0 plants, while increasing transformation efficiency from 1 to 5% using HIGHLY-RESISTANT ACETOLACTATE SYNTHASE as a selection marker. Addition of a CRISPR/Cas9 module confirmed functionality for gene editing applications, as exemplified by editing the gene VIRESCENT YELLOW-LIKE (VYL) that can act as a visual marker for gene editing in maize. The constructs, methods, and insights produced in this work will be valuable to translate the use of BBM-WUS and other emerging morphogenic regulators (MRs) to other genotypes and crops.

Efficient CRISPR-mediated base editing in Agrobacterium spp.

Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of "engineering the engineer," leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.

Ectopic Expression of a Self-Incompatibility Module Triggers Growth Arrest and Cell Death in Vegetative Cells.

Self-incompatibility (SI) is used by many angiosperms to reject self-pollen and avoid inbreeding. In field poppy (Papaver rhoeas), SI recognition and rejection of self-pollen is facilitated by a female S-determinant, PrsS, and a male S-determinant, PrpS PrsS belongs to the cysteine-rich peptide family, whose members activate diverse signaling networks involved in plant growth, defense, and reproduction. PrsS and PrpS are tightly regulated and expressed solely in pistil and pollen cells, respectively. Interaction of cognate PrsS and PrpS triggers pollen tube growth inhibition and programmed cell death (PCD) of self-pollen. We previously demonstrated functional intergeneric transfer of PrpS and PrsS to Arabidopsis (Arabidopsis thaliana) pollen and pistil. Here, we show that PrpS and PrsS, when expressed ectopically, act as a bipartite module to trigger a self-recognition:self-destruct response in Arabidopsis independently of its reproductive context in vegetative cells. The addition of recombinant PrsS to seedling roots expressing the cognate PrpS resulted in hallmark features of the P rhoeas SI response, including S-specific growth inhibition and PCD of root cells. Moreover, inducible expression of PrsS in PrpS-expressing seedlings resulted in rapid death of the entire seedling. This demonstrates that, besides specifying SI, the bipartite PrpS-PrsS module can trigger growth arrest and cell death in vegetative cells. Heterologous, ectopic expression of a plant bipartite signaling module in plants has not been shown previously and, by extrapolation, our findings suggest that cysteine-rich peptides diversified for a variety of specialized functions, including the regulation of growth and PCD.

CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis.

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function.

Genome Editing-Based Engineering of CESA3 Dual Cellulose-Inhibitor-Resistant Plants.

The rapid appearance of herbicide-resistant weeds combined with a lack of novel herbicides being brought to market reduces crop production, thereby threatening food security worldwide. Here, we report on the use of the previously identified cellulose biosynthesis-inhibiting chemical compound C17 as a potential herbicide. Toxicity tests showed that C17 efficiently inhibits the growth of various weeds and widely cultivated dicotyledonous crops, whereas only slight or no growth inhibition was observed for monocotyledonous crops. Surprisingly, when exposed to a mixture of C17 and one of two well-known cellulose biosynthesis inhibitors (CBIs), isoxaben and indaziflam, an additive growth inhibition was observed, demonstrating that C17 has a different mode of action that can be used to sensitize plants toward known CBIs. Moreover, we demonstrate that a C17-resistant CESA3 allele can be used as a positive transformation selection marker and that C17 resistance can be obtained through genome engineering of the wild-type CESA3 allele using clustered regularly interspaced short palindromic repeats-mediated base editing. This editing system allowed us to engineer C17 tolerance in an isoxaben-resistant line, resulting in double herbicide-resistant plants.

POLAR-guided signalling complex assembly and localization drive asymmetric cell division.

Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD), which is necessary for plant survival1-4. In Arabidopsis thaliana, the GLYCOGEN SYNTHASE KINASE3 (GSK3)/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) phosphorylates both the mitogen-activated protein kinase (MAPK) signalling module5,6 and its downstream target, the transcription factor SPEECHLESS (SPCH)7, to promote and restrict ACDs, respectively, in the same stomatal lineage cell. However, the mechanisms that balance these mutually exclusive activities remain unclear. Here we identify the plant-specific protein POLAR as a stomatal lineage scaffold for a subset of GSK3-like kinases that confines them to the cytosol and subsequently transiently polarizes them within the cell, together with BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), before ACD. As a result, MAPK signalling is attenuated, enabling SPCH to drive ACD in the nucleus. Moreover, POLAR turnover requires phosphorylation on specific residues, mediated by GSK3. Our study reveals a mechanism by which the scaffolding protein POLAR ensures GSK3 substrate specificity, and could serve as a paradigm for understanding regulation of GSK3 in plants.

Inulin chain length modification using a transgenic approach opening new perspectives for chicory.

Chicory capable of synthesizing long-chain inulin is of great interest. During the growing season, the sucrose-sucrose 1-fructosyltransferase (1-SST) activity is vital for production of long-chain inulin in chicory. With the purpose to increase inulin chain length, we employed Agrobacterium-mediated transformation method. Transgenic chicory plants (Cichorium intybus L. var. sativum) cv. 'Melci' has been developed to overexpress sucrose-sucrose 1-fructosyltransferase (1-SST) under the control of the CaMV 35S promoter. The integration of the T-DNA into the plant genome was confirmed by PCR on genomic DNA using gene-specific primers. Quantification of the 1-SST transcript expression level revealed that transgenic plants showed higher 1-SST expression than those in non-transgenic plants. Further analyses proved that the fructan content of the roots significantly increased in the transgenic plants. These results revealed that overexpression of the 1-SST, the key gene in inulin biosynthesis in chicory, might serve as a novel approach to develop plants with the long-chain inulin content.

NAC Transcription Factors ANAC087 and ANAC046 Control Distinct Aspects of Programmed Cell Death in the Arabidopsis Columella and Lateral Root Cap.

Programmed cell death in plants occurs both during stress responses and as an integral part of regular plant development. Despite the undisputed importance of developmentally controlled cell death processes for plant growth and reproduction, we are only beginning to understand the underlying molecular genetic regulation. Exploiting the Arabidopsis thaliana root cap as a cell death model system, we identified two NAC transcription factors, the little-characterized ANAC087 and the leaf-senescence regulator ANAC046, as being sufficient to activate the expression of cell death-associated genes and to induce ectopic programmed cell death. In the root cap, these transcription factors are involved in the regulation of distinct aspects of programmed cell death. ANAC087 orchestrates postmortem chromatin degradation in the lateral root cap via the nuclease BFN1. In addition, both ANAC087 and ANAC046 redundantly control the onset of cell death execution in the columella root cap during and after its shedding from the root tip. Besides identifying two regulators of developmental programmed cell death, our analyses reveal the existence of an actively controlled cell death program in Arabidopsis columella root cap cells.

Disruption of the Pathogenicity and Sex Ratio of the Beet Cyst Nematode Heterodera schachtii by Host-Delivered RNA Interference.

The beet cyst nematode (BCN) Heterodera schachtii causes serious damage and yield losses in numerous important crops worldwide. This study examines the efficacy of three types of transgenic Arabidopsis RNA interference (RNAi) lines to decrease the biological activity of this devastating nematode. The first RNAi construct (E1E2-RNAi) targets two nematode endoglucanase genes, which are involved in BCN pathogenicity, the second construct (MSP-RNAi) contains a fragment corresponding to the major sperm protein transcript necessary for BCN development and reproduction, and the third construct (E1E2MSP-RNAi) comprises all three target fragments. Transcript expression profiles of the target genes in all biological stages of the nematode were determined for the initial inoculated population and the resulting progeny. Bioassay data under indoor aseptic cultivation indicated that feeding on these RNAi lines did not affect pathogenic activity and reproductive capacity of the initial population, whereas inoculating the progeny into new transgenic plants corresponding with the lines from which they were recovered reduced the nematode penetration and the number of eggs per cyst. In addition, the male/female ratio increased more than the double, and the effects of RNAi continued in the second generation of the nematodes, because the progeny derived from E1E2-RNAi and E1E2MSP-RNAi lines showed an impaired ability to infect wild-type plants.

KIRA1 and ORESARA1 terminate flower receptivity by promoting cell death in the stigma of Arabidopsis.

Flowers have a species-specific functional life span that determines the time window in which pollination, fertilization and seed set can occur. The stigma tissue plays a key role in flower receptivity by intercepting pollen and initiating pollen tube growth toward the ovary. In this article, we show that a developmentally controlled cell death programme terminates the functional life span of stigma cells in Arabidopsis. We identified the leaf senescence regulator ORESARA1 (also known as ANAC092) and the previously uncharacterized KIRA1 (also known as ANAC074) as partially redundant transcription factors that modulate stigma longevity by controlling the expression of programmed cell death-associated genes. KIRA1 expression is sufficient to induce cell death and terminate floral receptivity, whereas lack of both KIRA1 and ORESARA1 substantially increases stigma life span. Surprisingly, the extension of stigma longevity is accompanied by only a moderate extension of flower receptivity, suggesting that additional processes participate in the control of the flower's receptive life span.

Directional auxin transport mechanisms in early diverging land plants.

The emergence and radiation of multicellular land plants was driven by crucial innovations to their body plans. The directional transport of the phytohormone auxin represents a key, plant-specific mechanism for polarization and patterning in complex seed plants. Here, we show that already in the early diverging land plant lineage, as exemplified by the moss Physcomitrella patens, auxin transport by PIN transporters is operational and diversified into ER-localized and plasma membrane-localized PIN proteins. Gain-of-function and loss-of-function analyses revealed that PIN-dependent intercellular auxin transport in Physcomitrella mediates crucial developmental transitions in tip-growing filaments and waves of polarization and differentiation in leaf-like structures. Plasma membrane PIN proteins localize in a polar manner to the tips of moss filaments, revealing an unexpected relation between polarization mechanisms in moss tip-growing cells and multicellular tissues of seed plants. Our results trace the origins of polarization and auxin-mediated patterning mechanisms and highlight the crucial role of polarized auxin transport during the evolution of multicellular land plants.

Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis.

BACKGROUND: The root cap is a plant organ that ensheathes the meristematic stem cells at the root tip. Unlike other plant organs, the root cap shows a rapid cellular turnover, balancing constant cell generation by specific stem cells with the disposal of differentiated cells at the root cap edge. This cellular turnover is critical for the maintenance of root cap size and its position around the growing root tip, but how this is achieved and controlled in the model plant Arabidopsis thaliana remains subject to contradictory hypotheses. RESULTS: Here, we show that a highly organized cell death program is the final step of lateral root cap differentiation and that preparation for cell death is transcriptionally controlled by ANAC033/SOMBRERO. Precise timing of cell death is critical for the elimination of root cap cells before they fully enter the root elongation zone, which in turn is important in order to allow optimal root growth. Root cap cell death is followed by a rapid cell-autonomous corpse clearance and DNA fragmentation dependent on the S1-P1 type nuclease BFN1. CONCLUSIONS: Based on these results, we propose a novel concept in plant development that recognizes programmed cell death as a mechanism for maintaining organ size and tissue homeostasis in the Arabidopsis root cap.

Gateway vectors for transformation of cereals.

Until now, the availability of vectors for transgenic research in cereal crops has been rather limited. We present a novel collection of Agrobacterium tumefaciens binary T-DNA vectors compatible with Gateway recombinational cloning that facilitate the modular assembly of genes of interest together with new regulatory sequences, such as strong constitutive or endosperm-specific Brachypodium distachyon promoters. This resource aims at streamlining the creation of vectors and transgenes designed to explore gene functions in vital monocotyledonous crops.

Transcription factor WRKY23 assists auxin distribution patterns during Arabidopsis root development through local control on flavonol biosynthesis.

Gradients of the plant hormone auxin, which depend on its active intercellular transport, are crucial for the maintenance of root meristematic activity. This directional transport is largely orchestrated by a complex interaction of specific influx and efflux carriers that mediate the auxin flow into and out of cells, respectively. Besides these transport proteins, plant-specific polyphenolic compounds known as flavonols have been shown to act as endogenous regulators of auxin transport. However, only limited information is available on how flavonol synthesis is developmentally regulated. Using reduction-of-function and overexpression approaches in parallel, we demonstrate that the WRKY23 transcription factor is needed for proper root growth and development by stimulating the local biosynthesis of flavonols. The expression of WRKY23 itself is controlled by auxin through the Auxin Response Factor 7 (ARF7) and ARF19 transcriptional response pathway. Our results suggest a model in which WRKY23 is part of a transcriptional feedback loop of auxin on its own transport through local regulation of flavonol biosynthesis.

Feeding cells induced by phytoparasitic nematodes require γ-tubulin ring complex for microtubule reorganization.

Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound γ-tubulin ring complexes. Here we investigate the requirement of γ-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that γ-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis γ-tubulin (TUBG1 and TUBG2) and two γ-tubulin complex proteins genes (GCP3 and GCP4) are upregulated in galls. Electron microscopy demonstrates association of GCP3 and γ-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both γ-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the γ-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts.

Virus-induced gene silencing as a reverse genetics tool to study gene function.

Reverse genetics has proven to be a powerful approach to elucidating gene function in plants, particularly in Arabidopsis. Virus-induced gene silencing (VIGS) is one such method and achieves reductions in target gene expression as the vector moves into newly formed tissues of inoculated plants. VIGS is especially useful for plants that are recalcitrant for transformation and for genes that cause embryo lethality. VIGS provides rapid, transient knockdowns as a complement to other reverse genetics tools and can be used to screen sequences for RNAi prior to stable transformation. High-throughput, forward genetic screening is also possible by cloning libraries of short gene fragments directly into a VIGS plasmid DNA vector, inoculating, and then looking for a phenotype of interest. VIGS is especially useful for studying genes in crop species, which currently have few genetic resources. VIGS facilitates a rapid comparison of knockdown phenotypes of the same gene in different breeding lines or mutant backgrounds, as the same vector is easily inoculated into different plants. In this chapter, we briefly discuss how to choose or construct a VIGS vector and then how to design and carry out effective experiments using VIGS.

Flexible tools for gene expression and silencing in tomato.

As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources.

Gene silencing induced by hairpin or inverted repeated sense transgenes varies among promoters and cell types.

*In transgenic calli and different tissues of Arabidopsis thaliana plants, the in trans silencing capacity of a 35S-beta-glucuronidase (GUS) hairpin RNA construct was investigated on a target GUS gene, under the control of the 35S, a WRKY or several cell cycle-specific promoters. *GUS histochemical staining patterns were analyzed in all tissues of the parental lines and supertransformants harboring the hairpin construct. Quantitative GUS activity measurements determined GUS suppression by a 35S-GUS hairpin or inverted repeated GUS transgenes in leaves and calli. *In some supertransformants, GUS-based staining disappeared in all tissues, including calli. In most supertransformants, however, a significant reduction was found in mature roots and leaves, but residual GUS activity was observed in the root tips, young leaves and calli. In leaves of most hairpin RNA supertransformants, the GUS activity was reduced by c. 1000-fold or more, but, in derived calli, generally by less than 200-fold. The silencing efficiency of inverted repeated sense transgenes was similar to that of a hairpin RNA construct in leaves, but weaker in calli. *These results imply that the tissue type, nature of the silencing inducer locus and the differential expression of the targeted gene codetermine the silencing efficiency.

Clone-based functional genomics.

Annotated genomes have provided a wealth of information about gene structure and gene catalogs in a wide range of species. Taking advantage of these developments, novel techniques have been implemented to investigate systematically diverse aspects of gene and protein functions underpinning biology processes. Here, we review functional genomics applications that require the mass production of cloned sequence repertoires, including ORFeomes and silencing tag collections. We discuss the techniques employed in large-scale cloning projects and we provide an up-to-date overview of the clone resources available for model plant species and of the current applications that may be scaled up for systematic plant gene studies.