Amy L. Klocko

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.

Virus-Induced Flowering: An Application of Reproductive Biology to Benefit Plant Research and Breeding

Virus-induced flowering combines fundamental research in reproductive biology with efficient tools for manipulating gene expression in nonmodel systems to accelerate discovery and breeding.

Containment of transgenic trees by suppression of LEAFY

VOLUME 34 NUMBER 9 SEPTEMBER 2016 NATURE BIOTECHNOLOGY To the Editor: Field studies and commercial use of genetically engineered (GE) trees have been limited, in large part owing to concerns over transgene flow into wild or feral tree populations1–4. Unlike other crops, trees are long-lived, weakly domesticated and their propagules can spread over several kilometers5. Although male sterility has been engineered in pine, poplar, and eucalyptus trees grown under field conditions by expression of the barnase RNase gene in anther tapetal cells6,7, barnase can reduce rates of genetic transformation and vegetative growth6. Furthermore, barnase expression may not be fully stable8. Bisexual sterility would allay concerns over seed dispersal, could be used to control invasive exotic trees, and might increase wood production9. We report the use of RNA interference (RNAi) to suppress expression of the single-copy LEAFY (LFY) gene to produce sterility in poplar. RNAi has been used to reduce gene expression in many plant species10,11, and the reduction in gene expression that RNAi confers is highly stable in trees under field conditions12. LFY is required for the early stages of male and female floral organ formation in plants, and encodes a transcription factor that promotes floral meristem identity13,14. In Arabidopsis thaliana, loss of LFY function results in the formation of vegetative structures instead of floral meristems, whereas reduction of LFY expression decreases floral abundance and results in partial conversion of floral organs to leaf-like structures13,14. We selected LFY studies might benefit from continuous, rather than ordinal, risk response designs. Self-reporting error is another limitation of our study. This issue is a well-known challenge in survey design. We limited our medical usage outcomes to survey questions asking about discrete events that clearly linked action to PGT results to minimize vague interpretations. But self-reporting error cannot be eliminated under this research design. Furthermore, the 6-month risk perception changes and medical usage choices are only a snapshot of an individual’s beliefs and behavior. Longerterm tracking and longer follow-up surveys would be needed to understand how these perceptions and actions evolve over time. Finally, the study can measure only the increases in healthcare usage as a result of increased risk perceptions and does not provide conclusions about the overall health value of PGT. Decreased risk or good news PGT results might give a consumer false reassurance and lead to a reduction in healthcare usage or health-enhancing behaviors. An ideal study design might include an individual’s entire medical history as well as measures of health behaviors both before and after PGT. However, the time horizon and survey limitations of the PGen Study did not allow this. Whereas the study provides a step toward better empirical understanding of the psychological and behavioral impact of PGT, the data do not allow us to measure the long-term health benefits (or costs) of PGT. We hope this study will encourage future attempts for linking information interventions with medical records and long-term behavioral tracking, as well as qualitative data on risk perception. A frequently cited concern regarding the regulation of DTC genomics is a lack of understanding about how individuals respond to the information presented in these tests. Our results provide early evidence of how customers adjust their perceptions and engage with their health providers as a result of different types of PGT results. Though we found good news– bad news asymmetry in risk perception changes, these changes appeared to be moderate and congruent with test results. Furthermore, extreme perception changes drove much of the follow-up medical appointments and procedures. Taken together, our results suggest that DTC consumers learn from their PGT results and update their beliefs, but they primarily seek additional medical actions in response to large and unexpected risks. Behavior & Health Education, University of Michigan School of Public Health, Ann Arbor, Michigan, USA. 3Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Partners HealthCare Personalized Medicine, the Broad Institute and Harvard Medical School, Boston, Massachusetts, USA. e-mail: jkrieger@mit.edu

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.

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.

Lessons from Two Decades of Field Trials with Genetically Modified Trees in the USA: Biology and Regulatory Compliance

We summarize the many field trials that we have conducted in the USA beginning in 1995 and continuing to this day. Under USDA APHIS federal regulatory notifications and permits, we have planted nearly 20,000 trees derived from approximately 100 different constructs in more than two dozen field experiments. The large majority of the trials were in Populus and included hybrid white poplars (P. tremula × alba INRA 717-1B4 and P. tremula × tremuloides INRA 353-53), but also included diverse hybrid cottonwoods such as P. trichocarpa × deltoides and P. deltoides × nigra. One field trial used transgenic sweetgum ( Liquidambar ). Most trials were conducted on Oregon State University (OSU) land, but several were also conducted on the land of industry collaborators in Oregon, Washington, and other states. The main traits we have studied are floral sterility and flowering time modification; size and growth rate modification by gibberellin perturbation; activation-based gene tagging; stability of reporter gene expression and RNAi suppression; herbicide and pest resistance gene impacts on plantation productivity; lignin modification and its impacts on physiological processes; and effects of isoprene reduction on growth and stress tolerance. The most significant lessons from these years of trials are: (1) Visual abnormalities in form or growth rate due to the transformation and in vitro regeneration (somaclonal variants) have been observed in several experiments, but are extremely rare (below 1 % of events produced). (2) Gene expression and RNAi-induced gene suppression have been highly stable—with a virtual absence of transgene silencing —over many years for virtually all transgenic trees whether assayed by a visual phenotype (reporter gene, flowering time, sexual sterility, herbicide or pest tolerance), or by molecular measures of transgene expression (e.g., quantitative RT-PCR). (3) The regulatory process has largely been efficient and workable, though it imposes significant biological constraints, costs, and risks that are very difficult for an academic laboratory to bear when trials span several years. It is most difficult where flowering is needed. (4) Field environments invoke complex and largely unpredictable changes to expression and associated phenotypes when studying physiology-modifying transgenes, including those affecting wood properties, suggesting the need to study several field sites, genetic backgrounds, and gene insertion events over many years, similar to common practices of conventional breeding. However, regulatory requirements make this very difficult to do for transgenic trees. (5) Collaborative field trials with industry have shown that common transgenic traits, such as herbicide and insect resistance, can have large productivity benefits in near-operational plantation conditions (e.g., two-year volume growth improvements of ~20 %)—suggesting that it could be highly beneficial to incorporate transgenic traits into production programs. Regulatory reforms to focus on product benefits as well as risks, and that do not assume harm from the use of recombinant DNA methods, are needed if transgenic technology is to provide significant benefits in forestry.

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.

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.