Transformation and regeneration of DNA polymerase Θ mutant rice plants.

Agrobacterium T-DNA integration into the plant genome is essential for the process of transgenesis and is widely used for genome engineering. The importance of the non-homologous end-joining (NHEJ) protein DNA polymerase Θ, encoded by the PolQ gene, for T-DNA integration is controversial, with some groups claiming it is essential whereas others claim T-DNA integration in Arabidopsis and rice polQ mutant plant tissue. Because of pleiotropic effects of PolQ loss on plant development, scientists have previously had difficulty regenerating transgenic polQ mutant plants. We describe a protocol for regenerating transgenic polQ mutant rice plants using a sequential transformation method. This protocol may be applicable to other plant species.

Characterization of T-Circles and Their Formation Reveal Similarities to Agrobacterium T-DNA Integration Patterns.

Agrobacterium transfers T-DNA to plants where it may integrate into the genome. Non-homologous end-joining (NHEJ) has been invoked as the mechanism of T-DNA integration, but the role of various NHEJ proteins remains controversial. Genetic evidence for the role of NHEJ in T-DNA integration has yielded conflicting results. We propose to investigate the formation of T-circles as a proxy for understanding T-DNA integration. T-circles are circular double-strand T-DNA molecules, joined at their left (LB) and right (RB) border regions, formed in plants. We characterized LB-RB junction regions from hundreds of T-circles formed in Nicotiana benthamiana or Arabidopsis thaliana. These junctions resembled T-DNA/plant DNA junctions found in integrated T-DNA: Among complex T-circles composed of multiple T-DNA molecules, RB-RB/LB-LB junctions predominated over RB-LB junctions; deletions at the LB were more frequent and extensive than those at the RB; microhomology was frequently used at junction sites; and filler DNA, from the plant genome or various Agrobacterium replicons, was often present between the borders. Ku80 was not required for efficient T-circle formation, and a VirD2 ω mutation affected T-circle formation and T-DNA integration similarly. We suggest that investigating the formation of T-circles may serve as a surrogate for understanding T-DNA integration.

Plant DNA Repair and Agrobacterium T-DNA Integration.

Agrobacterium species transfer DNA (T-DNA) to plant cells where it may integrate into plant chromosomes. The process of integration is thought to involve invasion and ligation of T-DNA, or its copying, into nicks or breaks in the host genome. Integrated T-DNA often contains, at its junctions with plant DNA, deletions of T-DNA or plant DNA, filler DNA, and/or microhomology between T-DNA and plant DNA pre-integration sites. T-DNA integration is also often associated with major plant genome rearrangements, including inversions and translocations. These characteristics are similar to those often found after repair of DNA breaks, and thus DNA repair mechanisms have frequently been invoked to explain the mechanism of T-DNA integration. However, the involvement of specific plant DNA repair proteins and Agrobacterium proteins in integration remains controversial, with numerous contradictory results reported in the literature. In this review I discuss this literature and comment on many of these studies. I conclude that either multiple known DNA repair pathways can be used for integration, or that some yet unknown pathway must exist to facilitate T-DNA integration into the plant genome.

Agrobacterium VirE2 Protein Modulates Plant Gene Expression and Mediates Transformation From Its Location Outside the Nucleus.

Agrobacterium effector protein VirE2 is important for plant transformation. VirE2 likely coats transferred DNA (T-DNA) in the plant cell and protects it from degradation. VirE2 localizes to the plant cytoplasm and interacts with several host proteins. Plant-expressed VirE2 can complement a virE2 mutant Agrobacterium strain to support transformation. We investigated whether VirE2 could facilitate transformation from a nuclear location by affixing to it a strong nuclear localization signal (NLS) sequence. Only cytoplasmic-, but not nuclear-localized, VirE2 could stimulate transformation. To investigate the ways VirE2 supports transformation, we generated transgenic Arabidopsis plants containing a virE2 gene under the control of an inducible promoter and performed RNA-seq and proteomic analyses before and after induction. Some differentially expressed plant genes were previously known to facilitate transformation. Knockout mutant lines of some other VirE2 differentially expressed genes showed altered transformation phenotypes. Levels of some proteins known to be important for transformation increased in response to VirE2 induction, but prior to or without induction of their corresponding mRNAs. Overexpression of some other genes whose proteins increased after VirE2 induction resulted in increased transformation susceptibility. We conclude that cytoplasmically localized VirE2 modulates both plant RNA and protein levels to facilitate transformation.

Agrobacterium T-DNA integration in somatic cells does not require the activity of DNA polymerase θ.

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.

Agrobacterium-delivered VirE2 interacts with host nucleoporin CG1 to facilitate the nuclear import of VirE2-coated T complex.

Agrobacterium tumefaciens is the causal agent of crown gall disease. The bacterium is capable of transferring a segment of single-stranded DNA (ssDNA) into recipient cells during the transformation process, and it has been widely used as a genetic modification tool for plants and nonplant organisms. Transferred DNA (T-DNA) has been proposed to be escorted by two virulence proteins, VirD2 and VirE2, as a nucleoprotein complex (T-complex) that targets the host nucleus. However, it is not clear how such a proposed large DNA-protein complex is delivered through the host nuclear pore in a natural setting. Here, we studied the natural nuclear import of the Agrobacterium-delivered ssDNA-binding protein VirE2 inside plant cells by using a split-GFP approach with a newly constructed T-DNA-free strain. Our results demonstrate that VirE2 is targeted into the host nucleus in a VirD2- and T-DNA-dependent manner. In contrast with VirD2 that binds to plant importin α for nuclear import, VirE2 directly interacts with the host nuclear pore complex component nucleoporin CG1 to facilitate its nuclear uptake and the transformation process. Our data suggest a cooperative nuclear import model in which T-DNA is guided to the host nuclear pore by VirD2 and passes through the pore with the assistance of interactions between VirE2 and host nucleoporin CG1. We hypothesize that this large linear nucleoprotein complex (T-complex) is targeted to the nucleus by a "head" guide from the VirD2-importin interaction and into the nucleus by a lateral assistance from the VirE2-nucleoporin interaction.

Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco.

KEY MESSAGE: Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants. Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.

CRISPR/Cas9-mediated targeted T-DNA integration in rice.

KEY MESSAGE: Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome. Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50-80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.

RepB C-terminus mutation of a pRi-repABC binary vector affects plasmid copy number in Agrobacterium and transgene copy number in plants.

A native repABC replication origin from pRiA4b was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacterium-mediated transformation. A high copy pRi-repABC variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type pRi-repABC binary vector showed that Agrobacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated pRi-repABC binary vector was compared with the original wild-type pRi-repABC binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy pRi-repABC with the RepBY299H mutation provides no advantage in generating high frequency single copy, backbone-free transgenic plants in comparison with the single copy wild-type pRi-repABC binary vector.

The VirE-asy Way to Genetically Transform Plants.

Agrobacterium transfers T-DNA and several virulence effector proteins to plant cells. It is not known how and where T-complexes containing these components are assembled. A new study suggests that T-complexes form on the plant plasma membrane, mediated by the effector protein VirE3.

VIP1 and Its Homologs Are Not Required for Agrobacterium-Mediated Transformation, but Play a Role in Botrytis and Salt Stress Responses.

The bZIP transcription factor VIP1 interacts with the Agrobacterium virulence protein VirE2, but the role of VIP1 in Agrobacterium-mediated transformation remains controversial. Previously tested vip1-1 mutant plants produce a truncated protein containing the crucial bZIP DNA-binding domain. We generated the CRISPR/Cas mutant vip1-2 that lacks this domain. The transformation susceptibility of vip1-2 and wild-type plants is similar. Because of potential functional redundancy among VIP1 homologs, we tested transgenic lines expressing VIP1 fused to a SRDX repression domain. All VIP1-SRDX transgenic lines showed wild-type levels of transformation, indicating that neither VIP1 nor its homologs are required for Agrobacterium-mediated transformation. Because VIP1 is involved in innate immune response signaling, we tested the susceptibility of vip1 mutant and VIP1-SRDX plants to Pseudomonas syringae and Botrytis cinerea. vip1 mutant and VIP1-SRDX plants show increased susceptibility to B. cinerea but not to P. syringae infection, suggesting a role for VIP1 in B. cinerea, but not in P. syringae, defense signaling. B. cinerea susceptibility is dependent on abscisic acid (ABA) which is also important for abiotic stress responses. The germination of vip1 mutant and VIP1-SRDX seeds is sensitive to exogenous ABA, suggesting a role for VIP1 in response to ABA. vip1 mutant and VIP1-SRDX plants show increased tolerance to growth in salt, indicating a role for VIP1 in response to salt stress.

An armadillo-domain protein participates in a telomerase interaction network.

KEY MESSAGE: Arabidopsis and human ARM protein interact with telomerase. Deregulated mRNA levels of DNA repair and ribosomal protein genes in an Arabidopsis arm mutant suggest non-telomeric ARM function. The human homolog ARMC6 interacts with hTRF2. Telomerase maintains telomeres and has proposed non-telomeric functions. We previously identified interaction of the C-terminal domain of Arabidopsis telomerase reverse transcriptase (AtTERT) with an armadillo/ß-catenin-like repeat (ARM) containing protein. Here we explore protein-protein interactions of the ARM protein, AtTERT domains, POT1a, TRF-like family and SMH family proteins, and the chromatin remodeling protein CHR19 using bimolecular fluorescence complementation (BiFC), yeast two-hybrid (Y2H) analysis, and co-immunoprecipitation. The ARM protein interacts with both the N- and C-terminal domains of AtTERT in different cellular compartments. ARM interacts with CHR19 and TRF-like I family proteins that also bind AtTERT directly or through interaction with POT1a. The putative human ARM homolog co-precipitates telomerase activity and interacts with hTRF2 protein in vitro. Analysis of Arabidopsis arm mutants shows no obvious changes in telomere length or telomerase activity, suggesting that ARM is not essential for telomere maintenance. The observed interactions with telomerase and Myb-like domain proteins (TRF-like family I) may therefore reflect possible non-telomeric functions. Transcript levels of several DNA repair and ribosomal genes are affected in arm mutants, and ARM, likely in association with other proteins, suppressed expression of XRCC3 and RPSAA promoter constructs in luciferase reporter assays. In conclusion, ARM can participate in non-telomeric functions of telomerase, and can also perform its own telomerase-independent functions.

Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration.

Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

Integration of Agrobacterium T-DNA into the Plant Genome.

Agrobacterium strains transfer a single-strand form of T-DNA (T-strands) and Virulence (Vir) effector proteins to plant cells. Following transfer, T-strands likely form complexes with Vir and plant proteins that traffic through the cytoplasm and enter the nucleus. T-strands may subsequently randomly integrate into plant chromosomes and permanently express encoded transgenes, a process known as stable transformation. The molecular processes by which T-strands integrate into the host genome remain unknown. Although integration resembles DNA repair processes, the requirement of known DNA repair pathways for integration is controversial. The configuration and genomic position of integrated T-DNA molecules likely affect transgene expression, and control of integration is consequently important for basic research and agricultural biotechnology applications. This article reviews our current knowledge of the process of T-DNA integration and proposes ways in which this knowledge may be manipulated for genome editing and synthetic biology purposes.

Perturbation of H3K27me3-Associated Epigenetic Processes Increases Agrobacterium-Mediated Transformation.

Agrobacterium-mediated transformation is a core technology for basic plant science and agricultural biotechnology. Improving transformation frequency is a major goal for plant transgenesis. We previously showed that T-DNA insertions in some histone genes decreased transformation susceptibility, whereas overexpression of several Arabidopsis H2A and H4 isoforms increased transformation. Overexpression of several histone H2B and H3 isoforms had little effect on transformation frequency. However, overexpression of histone H3-11 (HTR11) enhanced transformation. HTR11 is a unique H3 variant that lacks lysine at positions 9 and 27. The modification status of these lysine residues in canonical H3 proteins plays a critical role in epigenetic determination of gene expression. We mutated histone H3-4 (HTR4), a canonical H3.3 protein that does not increase transformation when overexpressed, by replacing either or both K9 and K27 with the amino acids in HTR11 (either K9I, K27Q, or both). Overexpression of HTR4 with the K27Q but not the K9I substitution enhanced transformation. HTR4K27Q was incorporated into chromatin, and HTR4K27Q overexpression lines exhibited deregulated expression of H3K27me3-enriched genes. These results demonstrate that mutation of K27 in H3.3 is sufficient to perturb H3K27me3-dependent expression in plants as in animals and suggest a distinct epigenetic role for histone HTR11. Further, these observations implicate manipulation of H3K27me3-dependent gene expression as a novel strategy to increase transformation susceptibility.

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.

Somaclonal variation does not preclude the use of rice transformants for genetic screening.

Rice (Oryza sativa) is one of the world's most important crops. Rice researchers make extensive use of insertional mutants for the study of gene function. Approximately half a million flanking sequence tags from rice insertional mutant libraries are publicly available. However, the relationship between genotype and phenotype is very weak. Transgenic plant assays have been used frequently for complementation, overexpression or antisense analysis, but sequence changes caused by callus growth, Agrobacterium incubation medium, virulence genes, transformation and selection conditions are unknown. We used high-throughput sequencing of DNA from rice lines derived from Tainung 67 to analyze non-transformed and transgenic rice plants for mutations caused by these parameters. For comparison, we also analyzed sequence changes for two additional rice varieties and four T-DNA tagged transformants from the Taiwan Rice Insertional Mutant resource. We identified single-nucleotide polymorphisms, small indels, large deletions, chromosome doubling and chromosome translocations in these lines. Using standard rice regeneration/transformation procedures, the mutation rates of regenerants and transformants were relatively low, with no significant differences among eight tested treatments in the Tainung 67 background and in the cultivars Taikeng 9 and IR64. Thus, we could not conclusively detect sequence changes resulting from Agrobacterium-mediated transformation in addition to those caused by tissue culture-induced somaclonal variation. However, the mutation frequencies within the two publically available tagged mutant populations, including TRIM transformants or Tos17 lines, were about 10-fold higher than the frequency of standard transformants, probably because mass production of embryogenic calli and longer callus growth periods were required to generate these large libraries.

cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-Telomeric Roles of Arabidopsis Telomerase.

Telomerase-reverse transcriptase (TERT) plays an essential catalytic role in maintaining telomeres. However, in animal systems telomerase plays additional non-telomeric functional roles. We previously screened an Arabidopsis cDNA library for proteins that interact with the C-terminal extension (CTE) TERT domain and identified a nuclear-localized protein that contains an RNA recognition motif (RRM). This RRM-protein forms homodimers in both plants and yeast. Mutation of the gene encoding the RRM-protein had no detectable effect on plant growth and development, nor did it affect telomerase activity or telomere length in vivo, suggesting a non-telomeric role for TERT/RRM-protein complexes. The gene encoding the RRM-protein is highly expressed in leaf and reproductive tissues. We further screened an Arabidopsis cDNA library for proteins that interact with the RRM-protein and identified five interactors. These proteins are involved in numerous non-telomere-associated cellular activities. In plants, the RRM-protein, both alone and in a complex with its interactors, localizes to nuclear speckles. Transcriptional analyses in wild-type and rrm mutant plants, as well as transcriptional co-analyses, suggest that TERT, the RRM-protein, and the RRM-protein interactors may play important roles in non-telomeric cellular functions.

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.