Single-cell RNA sequencing profiles reveal cell type-specific transcriptional regulation networks conditioning fungal invasion in maize roots.

Stalk rot caused by Fusarium verticillioides (Fv) is one of the most destructive diseases in maize production. The defence response of root system to Fv invasion is important for plant growth and development. Dissection of root cell type-specific response to Fv infection and its underlying transcription regulatory networks will aid in understanding the defence mechanism of maize roots to Fv invasion. Here, we reported the transcriptomes of 29 217 single cells derived from root tips of two maize inbred lines inoculated with Fv and mock condition, and identified seven major cell types with 21 transcriptionally distinct cell clusters. Through the weighted gene co-expression network analysis, we identified 12 Fv-responsive regulatory modules from 4049 differentially expressed genes (DEGs) that were activated or repressed by Fv infection in these seven cell types. Using a machining-learning approach, we constructed six cell type-specific immune regulatory networks by integrating Fv-induced DEGs from the cell type-specific transcriptomes, 16 known maize disease-resistant genes, five experimentally validated genes (ZmWOX5b, ZmPIN1a, ZmPAL6, ZmCCoAOMT2, and ZmCOMT), and 42 QTL or QTN predicted genes that are associated with Fv resistance. Taken together, this study provides not only a global view of maize cell fate determination during root development but also insights into the immune regulatory networks in major cell types of maize root tips at single-cell resolution, thus laying the foundation for dissecting molecular mechanisms underlying disease resistance in maize.

Enhancing plant growth and biomass production by overexpression of GA20ox gene under control of a root preferential promoter.

Overexpression of GA20 oxidase gene has been a recent trend for improving plant growth and biomass. Constitutive expression of GA20ox has successfully improved plant growth and biomass in several plant species. However, the constitutive expression of this gene causes side-effects, such as reduced leaf size and stem diameter, etc. To avoid these effects, we identified and employed different tissue-specific promoters for GA20ox overexpression. In this study, we examined the utility of At1g promoter to drive the expression of GUS (ß-glucuronidase) reporter and AtGA20ox genes in tobacco and Melia azedarach. Histochemical GUS assays and quantitative real-time-PCR results in tobacco showed that At1g was a root-preferential promoter whose expression was particularly strong in root tips. The ectopic expression of AtGA20ox gene under the control of At1g promoter showed improved plant growth and biomass of both tobacco and M. azedarach transgenic plants. Stem length as well as stem and root fresh weight increased by up to 1.5-3 folds in transgenic tobacco and 2 folds in transgenic M. azedarach. Both tobacco and M. azedarach transgenic plants showed increases in root xylem width with xylem to phloem ratio over 150-200% as compared to WT plants. Importantly, no significant difference in leaf shape and size was observed between At1g::AtGA20ox transgenic and WT plants. These results demonstrate the great utility of At1g promoter, when driving AtGA20ox gene, for growth and biomass improvements in woody plants and potentially some other plant species.

Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean.

BACKGROUND: CRISPR/Cas9 gene editing is now revolutionizing the ability to effectively modify plant genomes in the absence of efficient homologous recombination mechanisms that exist in other organisms. However, soybean is allotetraploid and is commonly viewed as difficult and inefficient to transform. In this study, we demonstrate the utility of CRISPR/Cas9 gene editing in soybean at relatively high efficiency. This was shown by specifically targeting the Fatty Acid Desaturase 2 (GmFAD2) that converts the monounsaturated oleic acid (C18:1) to the polyunsaturated linoleic acid (C18:2), therefore, regulating the content of monounsaturated fats in soybean seeds. RESULTS: We designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of GmFAD2-1A and GmFAD2-1B. In order to test whether the Cas9 and gRNAs would perform properly in transgenic soybean plants, we first tested the CRISPR construct we developed by transient hairy root transformation using Agrobacterium rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and characterized ten, randomly selected T0 events. Genotyping of CRISPR/Cas9 T0 transgenic lines detected a variety of mutations including large and small DNA deletions, insertions and inversions in the GmFAD2 genes. We detected CRISPR- edited DNA in all the tested T0 plants and 77.8% of the events transmitted the GmFAD2 mutant alleles to T1 progenies. More importantly, null mutants for both GmFAD2 genes were obtained in 40% of the T0 plants we genotyped. The fatty acid profile analysis of T1 seeds derived from CRISPR-edited plants homozygous for both GmFAD2 genes showed dramatic increases in oleic acid content to over 80%, whereas linoleic acid decreased to 1.3-1.7%. In addition, transgene-free high oleic soybean homozygous genotypes were created as early as the T1 generation. CONCLUSIONS: Overall, our data showed that dual gRNA CRISPR/Cas9 system offers a rapid and highly efficient method to simultaneously edit homeologous soybean genes, which can greatly facilitate breeding and gene discovery in this important crop plant.

Advancing Crop Transformation in the Era of Genome Editing.

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.

Transcriptome Profiling of Plant Genes in Response to Agrobacterium tumefaciens-Mediated Transformation.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. During infection of the host plant, Agrobacterium transfers T-DNA from its Ti plasmid into the host cell, which can then be integrated into the host genome. This unique genetic transformation capability has been employed as the dominant technology for producing genetically modified plants for both basic research and biotechnological applications. Agrobacterium has been well studied as a disease-causing agent. The Agrobacterium-mediated transformation process involves early attachment of the bacterium to the host's surface, followed by transfer of T-DNA and virulence proteins into the plant cell. Throughout this process, the host plants exhibit dynamic gene expression patterns at each infection stage or in response to Agrobacterium strains with varying pathogenic capabilities. Shifting host gene expression patterns throughout the transformation process have effects on transformation frequency, host morphology, and metabolism. Thus, gene expression profiling during the Agrobacterium infection process can be an important approach to help elucidate the interaction between Agrobacterium and plants. This review highlights recent findings on host plant differential gene expression patterns in response to A. tumefaciens or related elicitor molecules.

Transcriptomic Analysis of Arabidopsis Seedlings in Response to an Agrobacterium-Mediated Transformation Process.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. This pathogen is capable of transferring the T-DNA from its Ti plasmid to the host cell and, then, integrating it into the host genome. To date, this genetic transformation ability has been harnessed as the dominant technology to produce genetically modified plants for both basic research and crop biotechnological applications. However, little is known about the interaction between Agrobacterium tumefaciens and host plants, especially the host responses to Agrobacterium infection and its associated factors. We employed RNA-seq to follow the time course of gene expression in Arabidopsis seedlings infected with either an avirulent or a virulent Agrobacterium strain. Gene Ontology analysis indicated many biological processes were involved in the Agrobacterium-mediated transformation process, including hormone signaling, defense response, cellular biosynthesis, and nucleic acid metabolism. RNAseq and quantitative reverse transcription-polymerase chain reaction results indicated that expression of genes involved in host plant growth and development were repressed but those involved in defense response were induced by Agrobacterium tumefaciens. Further analysis of the responses of transgenic Arabidopsis lines constitutively expressing either the VirE2 or VirE3 protein suggested Vir proteins act to enhance plant defense responses in addition to their known roles facilitating T-DNA transformation.

Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2.

KEY MESSAGE: Discriminatory co-expression of maize BBM and WUS transcriptional factor genes promoted somatic embryogenesis and efficient Agrobacterium -mediated transformation of recalcitrant maize inbred B73 and sorghum P898012 genotypes without use of a selectable marker gene. The use of morphogenic regulators to overcome barriers in plant transformation is a revolutionary breakthrough for basic plant science and crop applications. Current standard plant transformation systems are bottlenecks for genetic, genomic, and crop improvement studies. We investigated the differential use of co-expression of maize transcription factors BABY BOOM and WUSCHEL2 coupled with a desiccation inducible CRE/lox excision system to enable regeneration of stable transgenic recalcitrant maize inbred B73 and sorghum P898012 without a chemical selectable marker. The PHP78891 expression cassette contains CRE driven by the drought inducible maize RAB17M promoter with lox P sites which bracket the CRE, WUS, and BBM genes. A constitutive maize UBI M promoter directs a ZsGreen GFP expression cassette as a reporter outside of the excision sites and provides transient, transgenic, and developmental analysis. This was coupled with evidence for molecular integration and analysis of stable integration and desiccation inducible CRE-mediated excision. Agrobacterium-mediated transgenic introduction of this vector showed transient expression of GFP and induced somatic embryogenesis in maize B73 and sorghum P898012 explants. Subjection to desiccation stress in tissue culture enabled the excision of CRE, WUS, and BBM, leaving the UBI M::GFP cassette and allowing subsequent plant regeneration and GFP expression analysis. Stable GFP expression was observed in the early and late somatic embryos, young shoots, vegetative plant organs, and pollen. Transgene integration and expression of GFP positive T0 plants were also analyzed using PCR and Southern blots. Progeny segregation analysis of primary events confirmed correlation between functional GFP expression and presence of the GFP transgene in T1 plants generated from self pollinations, indicating good transgene inheritance. This study confirms and extends the use of morphogenic regulators to overcome transformation barriers.

Agrobacterium T-DNA integration into the plant genome can occur without the activity of key non-homologous end-joining proteins.

Non-homologous end joining (NHEJ) is the major model proposed for Agrobacterium T-DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA-PKcs, DNA ligase IV (LIG4), Ataxia telangiectasia mutated (ATM), and ATM- and Rad3-related (ATR), play an important role in 'classical' (c)NHEJ. Other proteins, including histone H1 (HON1), XRCC1, and PARP1, participate in a 'backup' (b)NHEJ process. We examined transient and stable transformation frequencies of Arabidopsis thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, and the plant-specific DNA Ligase VI (LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild-type plants, suggesting enhanced T-DNA integration in these mutants. These results were confirmed using a promoter-trap transformation vector that requires T-DNA integration into the plant genome to activate a promoterless gusA (uidA) gene, by virus-induced gene silencing (VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T-DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, lig4, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T-DNA integration and transgene methylation. A double mutant (ku80/parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T-DNA integration. Thus, T-DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration.

Expression of ZmGA20ox cDNA alters plant morphology and increases biomass production of switchgrass (Panicum virgatum L.).

Switchgrass (Panicum virgatum L.) is considered a model herbaceous energy crop for the USA, for its adaptation to marginal land, low rainfall and nutrient-deficient soils; however, its low biomass yield is one of several constraints, and this might be rectified by modulating plant growth regulator levels. In this study, we have determined whether the expression of the Zea mays gibberellin 20-oxidase (ZmGA20ox) cDNA in switchgrass will improve biomass production. The ZmGA20ox gene was placed under the control of constitutive CaMV35S promoter with a strong TMV omega enhancer, and introduced into switchgrass via Agrobacterium-mediated transformation. The transgene integration and expression levels of ZmGA20ox in T0 plants were analysed using Southern blot and qRT-PCR. Under glasshouse conditions, selected transgenic plants exhibited longer leaves, internodes and tillers, which resulted in twofold increased biomass. These phenotypic alterations correlated with the levels of transgene expression and the particular gibberellin content. Expression of ZmGA20ox also affected the expression of genes coding for key enzymes in lignin biosynthesis. Our results suggest that the employment of ectopic ZmGA20ox and selection for natural variants with high level expression of endogenous GA20ox are appropriate approaches to increase biomass production of switchgrass and other monocot biofuel crops.

Novel constructs for efficient cloning of sRNA-encoding DNA and uniform silencing of plant genes employing artificial trans-acting small interfering RNA.

KEY MESSAGE: TAS atasiRNA-producing region swapping used one-step, high efficiency, and high fidelity directional TC-cloning. Uniform silencing was achieved without lethality using miRNA trigger- TAS overexpression fusion cassettes to generate 21-nt atasiRNA. Plant transgenic technologies are very important for basic plant research and biotechnology. Artificial trans-acting small interfering RNA (atasiRNA) represents an attractive platform with certain advantages over other silencing approaches, such as hairpin RNA, artificial microRNA (amiRNA), and virus-induced gene silencing (VIGS). In this study, we developed two types of constructs for atasiRNA-mediated gene silencing in plants. To functionally validate our constructs, we chose TAS1a as a test model. Type 1 constructs had miR173-precursor sequence fused with TAS1a locus driven by single promoter-terminator cassette, which simplified the expression cassette and resulted in uniform gene silencing. Type 2 constructs contained two separate cassettes for miR173 and TAS1a co-expression. The constructs in each type were further improved by deploying the XcmI-based TC-cloning system for highly efficient directional cloning of short DNA fragments encoding atasiRNAs into TAS1a locus. The effectiveness of the constructs was demonstrated by cloning an atasiRNA DNA into the TC site of engineered TAS1a and silencing of CHLORINA 42 (CH42) gene in Arabidopsis. Our results show that the directional TC-cloning of the atasiRNA DNA into the engineered TAS1a is highly efficient and the miR173-TAS1a fusion system provides an attractive alternative to achieve moderate but more uniform gene silencing without lethality, as compared to conventional two separate cassettes for miR173 and TAS locus co-expression system. The design principles described here should be applicable to other TAS loci such as TAS1b, TAS1c, TAS2, or TAS3, and cloning of amiRNA into amiRNA stem-loop.

Rapid and efficient Agrobacterium-mediated transformation of sorghum (Sorghum bicolor) employing standard binary vectors and bar gene as a selectable marker.

KEY MESSAGE: A rapid and efficient Agrobacterium -mediated transformation system in sorghum has been developed employing standard binary vectors and bar gene as a selectable marker. Sorghum (Sorghum bicolor) is an important food and biofuel crop worldwide, for which improvements in genetic transformation are needed to study its biology and facilitate agronomic and commercial improvement. Here, we report optimization of regeneration and transformation of public sorghum genotype P898012 using standard binary vectors and bar gene as a selectable marker. The tissue culture regeneration time frame has been reduced to 7-12 weeks with a yield of over 18 plants per callus, and the optimized transformation system employing Agrobacterium tumefaciens strain AGL1 and the bar with a MAS promoter achieved an average frequency over 14 %. Of randomly analyzed independent transgenic events, 40-50 % carry single copy of integrated T-DNA. Some independent transgenic events were derived from the same embryogenic callus lines, but a 3:1 Mendelian segregation ratio was found in all transgenic events with single copy as estimated by Southern blots. The system described here should facilitate studies of sorghum biology and agronomic improvement.

Effect of lipo-chitooligosaccharide on early growth of C4 grass seedlings.

Although lipo-chitooligosaccharides (LCOs) are important signal molecules for plant-symbiont interactions, a number of reports suggest that LCOs can directly impact plant growth and development, separate from any role in plant symbioses. In order to investigate this more closely, maize and Setaria seedlings were treated with LCO and their growth was evaluated. The data indicate that LCO treatment significantly enhanced root growth. RNA-seq transcriptomic analysis of LCO-treated maize roots identified a number of genes whose expression was significantly affected by the treatment. Among these genes, some LCO-up-regulated genes are likely involved in root growth promotion. Interestingly, some stress-related genes were down-regulated after LCO treatment, which might indicate reallocation of resources from defense responses to plant growth. The promoter activity of several LCO-up-regulated genes using a ß-glucuronidase reporter system was further analysed. The results showed that the promoters were activated by LCO treatment. The data indicate that LCO can directly impact maize root growth and gene expression.

Artificial trans-acting small interfering RNA: a tool for plant biology study and crop improvements.

Completion of whole genome sequencing in many plant species including economically important crop species not only opens up new opportunities but also imposes challenges for plant science research community. Functional validation and utilization of these enormous DNA sequences necessitate new or improved tools with high accuracy and efficiency. Of various tools, small RNA-mediated gene silencing platform plays an important and unique role in functional verification of plant genes and trait improvements. Artificial trans-acting small interfering RNA (atasiRNA) has emerged as a potent and specific gene silencing platform which overcomes major limitations of other small RNA silencing approaches including double-stranded RNA, artificial microRNA (amiRNA), and microRNA-induced gene silencing. To best utilize atasiRNA platform, it is essential to be able to test candidate atasiRNAs efficiently through either in vivo or in vitro validation approach. Very recently, a breakthrough has been made in developing a new method for in vitro screen of amiRNA candidates, named "epitope-tagged protein-based amiRNA screens". Such a screen can be readily employed to validate atasiRNA candidates and thus accelerate the deployment of atasiRNA technology. Therefore, atasiRNA as an emerging tool shall accelerate both plant biology study and crop genetic improvements including trait stacking.

Establishment and optimization of a hemp (Cannabis sativa L.) agroinfiltration system for gene expression and silencing studies.

Industrial hemp (Cannabis sativa L.) is a high-yielding annual crop primarily grown for fiber, seeds, and oil. Due to the phytochemical composition of hemp, there has been an increased interest in the market for nutraceuticals and dietary supplements for human health. Recent omics analysis has led to the elucidation of hemp candidate genes involved in the syntheses of specialized metabolites. However, a detailed study of these genes has not been undertaken due to the lack of a stable transformation system. We report for the first time an agroinfiltration system in hemp utilizing vacuum infiltration, which is an alternative method to stable transformation. A combination of 0.015% Silwett L-77, 5 mM ascorbic acid, and thirty second sonication followed by a 10-minute vacuum treatment resulted in the highest ß-glucuronidase expression in the leaf, male and female flowers, stem, and root tissues. The phytoene desaturase gene was silenced with a transient hairpin RNA expression, resulting in an albino phenotype in the leaves and the male and female flowers. This agroinfiltration system would be useful for overexpression and silencing studies of target genes to regulate the yield of specialized metabolites in hemp.

The intergenic region of Arabidopsis thaliana cab1 and cab2 divergent genes functions as a bidirectional promoter.

Genetic engineering plays a unique role in fundamental plant biology studies and in improving crop traits. These efforts often necessitate introduction and expression of multiple genes using promoters from a very limited repertoire. Current common practice of expressing multiple genes is the repeated use of the same or similar promoters. This practice causes more frequent transgene silencing due to a high degree of sequence homology and a greater chance of rearrangement among repeatedly used promoter sequences. Therefore, availability and use of natural bidirectional promoters to minimize gene silencing and achieve desirable expression pattern of transgenes is a critical issue in the field of plant genetic engineering. Here we describe the use of a single natural bidirectional promoter to drive the expression of two reporter genes in onion epidermal cells and in transgenic tobacco plants. We show that (1) the promoter drives the simultaneous expression of GUS and GFP reporter genes after transient expression and stable transformation, (2) the transcription is equally strong in both directions, (3) immediate upstream regions in each direction control transcription independently from each other, and (4) the reporter genes are expressed in leaves and stems but not in roots, as expected from the fact that the endogenous promoter controls the expression of two photosynthetic genes in Arabidopsis. Hence, use of bidirectional promoters in heterologous background provides a means to express multiple genes in transgenic plants and aids genetic engineering-based crop improvement.

Edit at will: Genotype independent plant transformation in the era of advanced genomics and genome editing.

The combination of advanced genomics, genome editing and plant transformation biology presents a powerful platform for basic plant research and crop improvement. Together these advances provide the tools to identify genes as targets for direct editing as single base pair changes, deletions, insertions and site specific homologous recombination. Recent breakthrough technologies using morphogenic regulators in plant transformation creates the ability to introduce reagents specific toward their identified targets and recover stably transformed and/or edited plants which are genotype independent. These technologies enable the possibility to alter a trait in any variety, without genetic disruption which would require subsequent extensive breeding, but rather to deliver the same variety with one trait changed. Regulatory issues regarding this technology will predicate how broadly these technologies will be implemented. In addition, education will play a crucial role for positive public acceptance. Taken together these technologies comprise a platform for advanced breeding which is an imperative for future world food security.

Rapid and Efficient Genetic Transformation of Sorghum via Agrobacterium-Mediated Method.

Genetic transformation via Agrobacterium-mediated methodology has been used in many sorghum studies. However, the transformation efficiency still varies significantly due to high dependence on sorghum genotypes and technical expertise. In this article, we describe a sorghum transformation procedure in sufficient detail using a public genotype, P898012. This system utilizes a standard binary transgenic vector carrying the bar gene as a selectable marker and immature embryos as starting explants. Glufosinate is employed as the selective agent during callus and shoot induction. This procedure is relatively rapid, efficient, highly reproducible, and should be applicable for many other sorghum genotypes. © 2018 by John Wiley & Sons, Inc.

Morphogenic Regulator-Mediated Transformation of Maize Inbred B73.

Maize B73 is a reference genome and has long been a major resource for genetics and molecular biology research. We have developed an efficient B73 transformation protocol by enabling somatic embryogenesis through differential co-expression of maize morphogenic regulators BBM and WUS2. We describe a successful protocol that utilizes Agrobacterium tumefaciens strain AGL1 harboring binary vector PHP78891 that comprises a BBM and WUS2 expression cassette as well as a green fluorescent protein (GFP) reporter cassette. The PHP78891 vector also contains, within the T-DNA region, a CRE/lox recombination system flanking the CRE/BBM/WUS2 co-expression cassette driven by the desiccation inducible RAB17 promoter that allows removal of the BBM/WUS2 cassette. Introduction and co-expression of BBM and WUS2 induced direct somatic embryogenesis (SE) in non-regenerable maize B73 from immature embryo explants. Removal of the CRE/BBM/WUS2 cassette is essential to allow regeneration to fertile plants. The GFP expression cassette outside the lox excision sites is retained in the transgenic plant genome, allowing subsequent phenotypic analysis of calli and regenerated transgenic events. This transformation system enables a selectable marker-free transformation process by taking advantage of BBM/WUS2-induced SE as a developmental selection system. © 2018 by John Wiley & Sons, Inc.

Transformation of Recalcitrant Sorghum Varieties Facilitated by Baby Boom and Wuschel2.

Most reliable transformation protocols for cereal crops, including sorghum (Sorghum bicolor L. Moench), rely on the use of immature embryo explants to generate embryogenic callus cells that are then transformed using Agrobacterium- or particle-bombardment-mediated DNA delivery. Subsequent to DNA transfer, most protocols rely on selectable markers for the recovery of stably transformed callus that is then regenerated to produce T0 plants. However, these protocols require specific genotypes that are innately capable of efficient embryogenic callus initiation. Here, we describe a system that makes use of the differential expression of the morphogenic regulators Baby Boom (Bbm) and Wuschel2 (Wus2) to achieve transformation in varieties of sorghum typically recalcitrant to standard transformation methods. © 2018 by John Wiley & Sons, Inc.