Apple CRISPR-Cas9-A Recipe for Successful Targeting of AGAMOUS-like Genes in Domestic Apple.

Fruit trees and other fruiting hardwood perennials are economically valuable, and there is interest in developing improved varieties. Both conventional breeding and biotechnology approaches are being utilized towards the goal of developing advanced cultivars. Increased knowledge of the effectiveness and efficiency of biotechnology approaches can help guide use of the CRISPR gene-editing technology. Here, we examined CRISPR-Cas9-directed genome editing in the valuable commodity fruit tree Malus x domestica (domestic apple). We transformed two cultivars with dual CRISPR-Cas9 constructs designed to target two AGAMOUS-like genes simultaneously. The main goal was to determine the effectiveness of this approach for achieving target gene changes. We obtained 6 Cas9 control and 38 independent CRISPR-Cas9 events. Of the 38 CRISPR-Cas9 events, 34 (89%) had gene edits and 14 (37%) showed changes to all alleles of both target genes. The most common change was large deletions, which were present in 59% of all changed alleles, followed by small deletions (21%), small insertions (12%), and a combination of small insertions and deletions (8%). Overall, a high rate of successful gene alterations was found. Many of these changes are predicted to cause frameshifts and alterations to the predicted peptides. Future work will include monitoring the floral development and floral form.

Variation in floral form of CRISPR knock-outs of the poplar homologs of LEAFY and AGAMOUS after FT heat-induced early flowering.

Plant migration and gene flow from genetically modified or exotic trees to nearby lands or by crossing with wild relatives is a major public and regulatory concern. Many genetic strategies exist to mitigate potential gene flow; however, the long delay in onset of flowering is a severe constraint to research progress. We used heat-induced FT overexpression to speed assessment of the expected floral phenotypes after CRISPR knockout of poplar homologs of the key floral genes, LEAFY and AGAMOUS. We selected events with previously characterized CRISPR-Cas9 induced biallelic changes then re-transformed them with the Arabidopsis thaliana FLOWERING LOCUS T (AtFT) gene under control of either a strong constitutive promoter or a heat-inducible promoter. We successfully obtained flowering in both a male and female clones of poplar, observing a wide range of inflorescence and floral forms among flowers, ramets, and insertion events. Overall, flowers obtained from the selected LFY and AG targeted events were consistent with what would be predicted for loss-of-function of these genes. LFY-targeted events showed small catkins with leaf-like organs, AG-targeted events had nested floral organs consistent with reduction in floral determinacy and absence of well-formed carpels or anthers. These findings demonstrate the great developmental plasticity of Populus flowers during genetically accelerated flowering, which may be of horticultural value. They also provide an informative early view of floral phenotypes and apparent sterility from knockouts of both these gene targets.

Knockout of floral and meiosis genes using CRISPR/Cas9 produces male-sterility in Eucalyptus without impacts on vegetative growth.

Eucalyptus spp. are widely cultivated for the production of pulp, energy, essential oils, and as ornamentals. However, their dispersal from plantings, especially when grown as an exotic, can cause ecological disruptions. To provide new tools for prevention of sexual dispersal by pollen as well as to induce male-sterility for hybrid breeding, we studied the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated knockout of three floral genes in both FT-expressing (early-flowering) and non-FT genotypes. We report male-sterile phenotypes resulting from knockout of the homologs of all three genes, including one involved in meiosis and two regulating early stages of pollen development. The targeted genes were Eucalyptus homologs of REC8 (EREC8), TAPETAL DEVELOPMENT AND FUNCTION 1 (ETDF1), and HECATE3 (EHEC3-like). The erec8 knockouts yielded abnormal pollen grains and a predominance of inviable pollen, whereas the etdf1 and ehec3-like knockouts produced virtually no pollen. In addition to male-sterility, both erec8 and ehec3-like knockouts may provide complete sterility because the failure of erec8 to undergo meiosis is expected to be independent of sex, and ehec3-like knockouts produce flowers with shortened styles and no visible stigmas. When comparing knockouts to controls in wild-type (non-early-flowering) backgrounds, we did not find visible morphological or statistical differences in vegetative traits, including average single-leaf mass, stem volume, density of oil glands, or chlorophyll in leaves. Loss-of-function mutations in any of these three genes show promise as a means of inducing male- or complete sterility without impacting vegetative development.

Genetic Containment for Molecular Farming.

Plant molecular farming can provide humans with a wide variety of plant-based products including vaccines, therapeutics, polymers, industrial enzymes, and more. Some of these products, such as Taxol, are produced by endogenous plant genes, while many others require addition of genes by artificial gene transfer. Thus, some molecular farming plants are transgenic (or cisgenic), while others are not. Both the transgenic nature of many molecular farming plants and the fact that the products generated are of high-value and specific in purpose mean it is essential to prevent accidental cross-over of molecular farming plants and products into food or feed. Such mingling could occur either by gene flow during plant growth and harvest or by human errors in material handling. One simple approach to mitigate possible transfer would be to use only non-food non-feed species for molecular farming purposes. However, given the extent of molecular farming products in development, testing, or approval that do utilize food or feed crops, a ban on use of these species would be challenging to implement. Therefore, other approaches will need to be considered for mitigation of cross-flow between molecular farming and non-molecular-farming plants. This review summarized some of the production systems available for molecular farming purposes and options to implement or improve plant containment.

RNAi Suppression of LEAFY Gives Stable Floral Sterility, and Reduced Growth Rate and Leaf Size, in Field-Grown Poplars.

The central floral development gene LEAFY (LFY), whose mutation leads to striking changes in flowering and often sterility, is commonly expressed in non-floral structures; however, its role in vegetative development is poorly understood. Sterility associated with suppression of LFY expression is an attractive means for mitigating gene flow by both seeds and pollen in vegetatively propagated forest trees, but the consequences of its suppression for tree form and wood production are unclear. To study the vegetative effects of RNAi suppression of LFY, we created a randomized, multiple-year field study with 30-40 trees (ramets) in each of two sterile gene insertion events, three transgenic control events, and a wild-type control population. We found that floral knock-down phenotypes were stable across years and propagation cycles, but that several leaf morphology and productivity traits were statistically and often substantially different in sterile vs. normal flowering RNAi-LFY trees. Though trees with suppressed LEAFY expression looked visibly normal, they appear to have reduced growth and altered leaf traits. LFY appears to have a significant role in vegetative meristem development, and evaluation of vegetative impacts from LFY suppression would be prudent prior to large-scale use for genetic containment.

Overexpression of SHORT VEGETATIVE PHASE-LIKE (SVL) in Populus delays onset and reduces abundance of flowering in field-grown trees.

The spread of transgenes and exotic germplasm from planted crops into wild or feral species is a difficult problem for public and regulatory acceptance of genetically engineered plants, particularly for wind-pollinated trees such as poplar. We report that overexpression of a poplar homolog of the floral repressor SHORT VEGETATIVE PHASE-LIKE (SVL), a homolog of the Arabidopsis MADS-box repressor SHORT VEGETATIVE PHASE (SVP), delayed the onset of flowering several years in three genotypes of field-grown transgenic poplars. Higher expression of SVL correlated with a delay in flowering onset and lower floral abundance, and did not cause morphologically obvious or statistically significant effects on leaf characteristics, tree form, or stem volume. Overexpression effects on reproductive and vegetative phenology in spring was modest and genotype-specific. Our results suggest that use of SVL and related floral repressors can be useful tools to enable a high level of containment for vegetatively propagated short-rotation woody energy or pulp crops.

Genetic containment in vegetatively propagated forest trees: CRISPR disruption of LEAFY function in Eucalyptus gives sterile indeterminate inflorescences and normal juvenile development.

Eucalyptus is among the most widely planted taxa of forest trees worldwide. However, its spread as an exotic or genetically engineered form can create ecological and social problems. To mitigate gene flow via pollen and seeds, we mutated the Eucalyptus orthologue of LEAFY (LFY) by transforming a Eucalyptus grandis × urophylla wild-type hybrid and two Flowering Locus T (FT) overexpressing (and flowering) lines with CRISPR Cas9 targeting its LFY orthologue, ELFY. We achieved high rates of elfy biallelic knockouts, often approaching 100% of transgene insertion events. Frameshift mutations and deletions removing conserved amino acids caused strong floral alterations, including indeterminacy in floral development and an absence of male and female gametes. These mutants were otherwise visibly normal and did not differ statistically from transgenic controls in juvenile vegetative growth rate or leaf morphology in greenhouse trials. Genes upstream or near to ELFY in the floral development pathway were overexpressed, whereas floral organ identity genes downstream of ELFY were severely depressed. We conclude that disruption of ELFY function appears to be a useful tool for sexual containment, without causing statistically significant or large adverse effects on juvenile vegetative growth or leaf morphology.

RNAi of AGAMOUS genes in sweetgum alters reproductive organ identity and decreases fruit persistence.

Sweetgums (Liquidambar), members of the family Altingiaceae (Altingiales), have inflorescences and floral organs that are distinctive in structure compared with other angiosperms in which the roles of floral homeotic genes have been studied. To begin to understand the role of AGAMOUS (AG)-a floral homeotic gene that has a major role in stamen and carpel development-in development of the monosexual flowers of sweetgum, we used RNAi to reduce the expression of two members of the AG subfamily. Because AG suppression should induce floral sterility, RNAi might also provide a tool to mitigate the risks of invasiveness-and to reduce the production of its nuisance fruits or allergenic pollen-when sweetgum is used as an exotic shade or forest tree. We tested 33 independent transgenic events and non-transgenic controls during 10 years in the field. The RNAi-AG sweetgum trees maintained normal growth, phenology, and vivid fall coloration during the 10 years of study, but 8 insertion events had highly modified inflorescence and floral morphology. The modified flowers had anthers and carpels that were converted to flat leaf-like structures lacking pollen grains and ovules, respectively. The female inflorescences developed into dry papery structures that failed to produce seeds. These infructescences were smaller than control infructescences, and lost a greater percentage of biomass in a controlled decay assay. RNAi against AG genes was highly effective at impairing fertility and modifying reproductive development without significant vegetative effects in sweetgum and gave phenotypes distinct from, but similar to, that of AG loss of function in other angiosperms.

RNA interference suppression of AGAMOUS and SEEDSTICK alters floral organ identity and impairs floral organ determinacy, ovule differentiation, and seed-hair development in Populus.

The role of the floral homeotic gene AGAMOUS (AG) and its close homologues in development of anemophilous, unisexual catkins has not previously been studied. We transformed two RNA interference (RNAi) constructs, PTG and its matrix-attachment-region flanked version MPG, into the early-flowering female poplar clone 6K10 (Populus alba) to suppress the expression of its two duplicate AG orthologues. By early 2018, six out of 22 flowering PTG events and 11 out of 12 flowering MPG events showed modified floral phenotypes in a field trial in Oregon, USA. Flowers in catkins from modified events had 'carpel-inside-carpel' phenotypes. Complete disruption of seed production was observed in seven events, and sterile anther-like organs in 10 events. Events with strong co-suppression of both the two AG and two SEEDSTICK (STK) paralogues lacked both seeds and associated seed hairs. Alterations in all of the modified floral phenotypes were stable over 4 yr of study. Trees from floral-modified events did not differ significantly (P < 0.05) from nonmodified transgenic or nontransgenic controls in biomass growth or leaf morphology. AG and STK genes show strong conservation of gene function during poplar catkin development and are promising targets for genetic containment of exotic or genetically engineered trees.

Strategies for Engineering Reproductive Sterility in Plantation Forests.

A considerable body of research exists concerning the development of technologies to engineer sterility in forest trees. The primary driver for this work has been to mitigate concerns arising from gene flow from commercial plantings of genetically engineered (GE) trees to non-GE plantations, or to wild or feral relatives. More recently, there has been interest in the use of sterility technologies as a means to mitigate the global environmental and socio-economic damage caused by the escape of non-native invasive tree species from planted forests. The current sophisticated understanding of the molecular processes underpinning sexual reproduction in angiosperms has facilitated the successful demonstration of a number of control strategies in hardwood tree species, particularly in the model hardwood tree Poplar. Despite gymnosperm softwood trees, such as pines, making up the majority of the global planted forest estate, only pollen sterility, via cell ablation, has been demonstrated in softwoods. Progress has been limited by the lack of an endogenous model system, long timescales required for testing, and key differences between softwood reproductive pathways and those of well characterized angiosperm model systems. The availability of comprehensive genome and transcriptome resources has allowed unprecedented insights into the reproductive processes of both hardwood and softwood tree species. This increased fundamental knowledge together with the implementation of new breeding technologies, such as gene editing, which potentially face a less oppressive regulatory regime, is making the implementation of engineered sterility into commercial forestry a realistic possibility.

Phenotypic Expression and Stability in a Large-Scale Field Study of Genetically Engineered Poplars Containing Sexual Containment Transgenes.

Genetic engineering (GE) has the potential to help meet demand for forest products and ecological services. However, high research and development costs, market restrictions, and regulatory obstacles to performing field tests have severely limited the extent and duration of field research. There is a notable paucity of field studies of flowering GE trees due to the time frame required and regulatory constraints. Here we summarize our findings from field testing over 3,300 GE poplar trees and 948 transformation events in a single, 3.6 hectare field trial for seven growing seasons; this trial appears to be the largest field-based scientific study of GE forest trees in the world. The goal was to assess a diversity of approaches for obtaining bisexual sterility by modifying RNA expression or protein function of floral regulatory genes, including LEAFY, AGAMOUS, APETALA1, SHORT VEGETATIVE PHASE, and FLOWERING LOCUS T. Two female and one male clone were transformed with up to 23 different genetic constructs designed to obtain sterile flowers or delay onset of flowering. To prevent gene flow by pollen and facilitate regulatory approval, the test genotypes chosen were incompatible with native poplars in the area. We monitored tree survival, growth, floral onset, floral abundance, pollen production, seed formation and seed viability. Tree survival was above 95%, and variation in site conditions generally had a larger impact on vegetative performance and onset of flowering than did genetic constructs. Floral traits, when modified, were stable over three to five flowering seasons, and we successfully identified RNAi or overexpression constructs that either postponed floral onset or led to sterile flowers. There was an absence of detectable somaclonal variation; no trees were identified that showed vegetative or floral modifications that did not appear to be related to the transgene added. Surveys for seedling and sucker establishment both within and around the plantation identified small numbers of vegetative shoots (root sprouts) but no seedlings, indicative of a lack of establishment of trees via seeds in the area. Overall, this long term study showed that GE containment traits can be obtained which are effective, stable, and not associated with vegetative abnormalities or somaclonal variation.

Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.

In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus Populus). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, LEAFY (LFY), and the two poplar orthologs of the floral organ identity gene AGAMOUS (AG). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles. Out of the 684 amplicons from events with sgRNAs, 474 had mutations in both alleles (77.5%). We sequenced both AG paralogs for 71 events in INRA clone 717-1B4 and 22 events in INRA clone 353-53, and found that 67 (94.4%) and 21 (95.5%) were double locus knockouts. Due partly to a single nucleotide polymorphism (SNP) present in the target region, one sgRNA targeting the AG paralogs was found to be completely inactive by itself (0%) but showed some activity in generating deletions when used in a construct with a second sgRNA (10.3-24.5%). Small insertion/deletion (indel) mutations were prevalent among mutated alleles of events with only one sgRNA (ranging from 94.3 to 99.1%), while large deletions were prevalent among alleles with two active sgRNAs (mean proportion of mutated alleles was 22.6% for small indels vs. 77.4% for large indels). For both LFY and AG, each individual sgRNA-gene combination had a unique mutation spectrum (p < 0.001). An AG-sgRNA construct with two sgRNAs had similar mutation spectra among two poplar clones (p > 0.05), however, a LFY-sgRNA construct with a single sgRNA gave significantly different mutation spectra among the same two clones (p < 0.001). The 49 empty vector control events had no mutations in either allele, and 310 potential "off-target" sequences also had no mutations in 58 transgenic events studied. CRISPR/Cas9 is a very powerful and precise system for generating loss-of-function mutations in poplars, and should be effective for generating reliably infertile trees that may promote regulatory, market, or public acceptance of genetic engineering technology.

Reproductive modification in forest plantations: impacts on biodiversity and society.

1000 I. 1000 II. 1001 III. 1014 IV. 1015 V. 1016 1016 References 1016 SUMMARY: Genetic engineering (GE) can be used to improve forest plantation productivity and tolerance of biotic and abiotic stresses. However, gene flow from GE forest plantations is a large source of ecological, social and legal controversy. The use of genetic technologies to mitigate or prevent gene flow has been discussed widely and should be technically feasible in a variety of plantation taxa. However, potential ecological effects of such modifications, and their social acceptability, are not well understood. Focusing on Eucalyptus, Pinus, Populus and Pseudotsuga - genera that represent diverse modes of pollination and seed dispersal - we conducted in-depth reviews of ecological processes associated with reproductive tissues. We also explored potential impacts of various forms of reproductive modification at stand and landscape levels, and means for mitigating impacts. We found little research on potential reactions by the public and other stakeholders to reproductive modification in forest plantations. However, there is considerable research on related areas that suggest key dimensions of concern and support. We provide detailed suggestions for research to understand the biological and social dimensions of containment technologies, and consider the role of regulatory and market restrictions that obstruct necessary ecological and genetic research.

Transgenic Suppression of AGAMOUS Genes in Apple Reduces Fertility and Increases Floral Attractiveness.

We investigated the ability of RNA interference (RNAi) directed against two co-orthologs of AGAMOUS (AG) from Malus domestica (domestic apple, MdAG) to reduce the risks of invasiveness and provide genetic containment of transgenes, while also promoting the attractiveness of flowers for ornamental usage. Suppression of two MdAG-like genes, MdMADS15 and MdMADS22, led to the production of trees with highly showy, polypetalous flowers. These "double-flowers" had strongly reduced expression of both MdAG-like genes. Members of the two other clades within in the MdAG subfamily showed mild to moderate differences in gene expression, or were unchanged, with the level of suppression approximately proportional to the level of sequence identity between the gene analyzed and the RNAi fragment. The double-flowers also exhibited reduced male and female fertility, had few viable pollen grains, a decreased number of stigmas, and produced few viable seeds after cross-pollination. Despite these floral alterations, RNAi-AG trees with double-flowers set full-sized fruit. Suppression or mutation of apple AG-like genes appears to be a promising method for combining genetic containment with improved floral attractiveness.

FT overexpression induces precocious flowering and normal reproductive development in Eucalyptus.

Eucalyptus trees are among the most important species for industrial forestry worldwide. However, as with most forest trees, flowering does not begin for one to several years after planting which can limit the rate of conventional and molecular breeding. To speed flowering, we transformed a Eucalyptus grandis × urophylla hybrid (SP7) with a variety of constructs that enable overexpression of FLOWERING LOCUS T (FT). We found that FT expression led to very early flowering, with events showing floral buds within 1-5 months of transplanting to the glasshouse. The most rapid flowering was observed when the cauliflower mosaic virus 35S promoter was used to drive the Arabidopsis thaliana FT gene (AtFT). Early flowering was also observed with AtFT overexpression from a 409S ubiquitin promoter and under heat induction conditions with Populus trichocarpa FT1 (PtFT1) under control of a heat-shock promoter. Early flowering trees grew robustly, but exhibited a highly branched phenotype compared to the strong apical dominance of nonflowering transgenic and control trees. AtFT-induced flowers were morphologically normal and produced viable pollen grains and viable self- and cross-pollinated seeds. Many self-seedlings inherited AtFT and flowered early. FT overexpression-induced flowering in Eucalyptus may be a valuable means for accelerating breeding and genetic studies as the transgene can be easily segregated away in progeny, restoring normal growth and form.

Recruitment of PLANT U-BOX13 and the PI4Kß1/ß2 phosphatidylinositol-4 kinases by the small GTPase RabA4B plays important roles during salicylic acid-mediated plant defense signaling in Arabidopsis.

Protection against microbial pathogens involves the activation of cellular immune responses in eukaryotes, and this cellular immunity likely involves changes in subcellular membrane trafficking. In eukaryotes, members of the Rab GTPase family of small monomeric regulatory GTPases play prominent roles in the regulation of membrane trafficking. We previously showed that RabA4B is recruited to vesicles that emerge from trans-Golgi network (TGN) compartments and regulates polarized membrane trafficking in plant cells. As part of this regulation, RabA4B recruits the closely related phosphatidylinositol 4-kinase (PI4K) PI4Kß1 and PI4Kß2 lipid kinases. Here, we identify a second Arabidopsis thaliana RabA4B-interacting protein, PLANT U-BOX13 (PUB13), which has recently been identified to play important roles in salicylic acid (SA)-mediated defense signaling. We show that PUB13 interacts with RabA4B through N-terminal domains and with phosphatidylinositol 4-phosphate (PI-4P) through a C-terminal armadillo domain. Furthermore, we demonstrate that a functional fluorescent PUB13 fusion protein (YFP-PUB13) localizes to TGN and Golgi compartments and that PUB13, PI4Kß1, and PI4Kß2 are negative regulators of SA-mediated induction of pathogenesis-related gene expression. Taken together, these results highlight a role for RabA4B and PI-4P in SA-dependent defense responses.

Arabidopsis Myosin XI-K Localizes to the Motile Endomembrane Vesicles Associated with F-actin.

Plant myosins XI were implicated in cell growth, F-actin organization, and organelle transport, with myosin XI-K being a critical contributor to each of these processes. However, subcellular localization of myosins and the identity of their principal cargoes remain poorly understood. Here, we generated a functionally competent, fluorescent protein-tagged, myosin XI-K, and investigated its spatial distribution within Arabidopsis cells. This myosin was found to associate primarily not with larger organelles (e.g., Golgi) as was broadly assumed, but with endomembrane vesicles trafficking along F-actin. Subcellular localization and fractionation experiments indicated that the nature of myosin-associated vesicles is organ- and cell type-specific. In leaves, a large proportion of these vesicles aligned and co-fractionated with a motile endoplasmic reticulum (ER) subdomain. In roots, non-ER vesicles were a dominant myosin cargo. Myosin XI-K showed a striking polar localization at the tips of growing, but not mature, root hairs. These results strongly suggest that a major mechanism whereby myosins contribute to plant cell physiology is vesicle transport, and that this activity can be regulated depending on the growth phase of a cell.