CRISPR/Cas9-mediated gene editing to confer turnip mosaic virus (TuMV) resistance in Chinese cabbage (Brassica rapa)

Genome editing approaches,particularly the CRISPR/Cas9 technology,are becoming state-of-the-art for trait development in numerous breeding programs.Significant advances in improving plant traits are enabled by this influential tool,especially for disease resistance,compared to traditional breeding.One of the potyviruses,the turnip mosaic virus(TuMV),is the most widespread and damaging virus that infects Brassica spp.worldwide.We generated the targeted mutation at the eIF(iso)4E gene in the TuMV-susceptible cultivar“Seoul”using CRISPR/Cas9 to develop TuMV-resistant Chinese cabbage.We detected several heritable indel mutations in the edited T0 plants and developed T1 through generational progression.It was indicated in the sequence analysis of the eIF(iso)4E-edited T1 plants that the mutations were transferred to succeeding generations.These edited T1 plants conferred resistance to TuMV.It was shown with ELISA analysis the lack of accumulation of viral particles.Furthermore,we found a strong negative correlation(r=-0.938)between TuMV resistance and the genome editing frequency of eIF(iso)4E.Consequently,it was revealed in this study that CRISPR/Cas9 technique can expedite the breeding process to improve traits in Chinese cabbage plants.

ESR2–HDA6 complex negatively regulates auxin biosynthesis to delay callus initiation in Arabidopsis leaf explants during tissue culture

Plants exhibit an astonishing ability to regulate organ regeneration upon wounding.Excision of leaf explants promotes the biosynthesis of indole-3-acetic acid(IAA),which is polar-transported to excised regions,where cell fate transition leads to root founder cell specification to induce de novo root regeneration.The regeneration capacity of plants has been utilized to develop in vitro tissue culture technologies.Here,we report that IAA accumulation near the wounded site of leaf explants is essential for callus formation on 2,4-dichlorophenoxyacetic acid(2,4-D)-rich callus-inducing medium(CIM).Notably,a high concentration of 2,4-D does not compensate for the action of IAA because of its limited efflux;rather,it lowers IAA biosynthesis via a negative feedback mechanism at an early stage of in vitro tissue culture,delaying callus initiation.The auxin negative feedback loop in CIM-cultured leaf explants is mediated by an auxin-inducible APETALA2 transcription factor,ENHANCER OF SHOOT REGENERATION 2(ESR2),along with its interacting partner HISTONE DEACETYLASE 6(HDA6).The ESR2–HDA6 complex binds directly to,and removes the H3ac mark from,the YUCCA1(YUC1),YUC7,and YUC9 loci,consequently repressing auxin biosynthesis and inhibiting cell fate transition on 2,4-D-rich CIM.These findings indicate that negative feedback regulation of auxin biosynthesis by ESR2 and HDA6 interferes with proper cell fate transition and callus initiation.

DICER-like Proteins and Their Role in Plant-herbivore Interactions in Nicotiana attenuata

DICER-like （DCL） proteins produce small RNAs that silence genes involved in development and defenses against viruses and pathogens. Which DCLs participate in plant-herbivore interactions remains unstudied. We identified and stably silenced four distinct DCL genes by RNAi in Nicotiana attenuata （Torrey ex. Watson）, a model for the study of plant-herbivore interactions. Silencing DCL1 expression was lethal. Manduca sexta larvae performed significantly better on ir-dcl3 and ir-dcl4 plants, but not on ir-dcl2 plants compared to wild type plants. Phytohormones, defense metabolites and microarray analyses revealed that when DCL3 and DCL4 were silenced separately, herbivore resistance traits were regulated in distinctly different ways. Crossing of the lines revealed complex interactions in the patterns of regulation. Single ir-dcl4 and double ir-dcl2 ir-dcl3 plants were impaired in JA accumulation, while JA- Ile was increased in ir-dcl3 plants. Ir-dcl3 and ir-dcl4 plants were impaired in nicotine accumulation; silencing DCL2 in combination with either DCL3 or DCL4 restored nicotine levels to those of WT. Trypsin proteinase inhibitor activity and transcripts were only silenced in ir-dcl3 plants. We conclude that DCL2/3/4 interact in a complex manner to regulate anti-herbivore defenses and that these interactions significantly complicate the already challenging task of understanding smRNA function in the regulation of biotic interactions.

Circadian clock component, LHY, tells a plant when to respond photosynthetically to light in nature

The circadian clock is known to increase plant growth and fitness, and is thought to prepare plants for photosynthesis at dawn and dusk; whether this happens in nature was unknown. We transformed the native tobacco, Nicotiana attenuata to silence two core clock components, NaLHY （irLHY） and NaTOC1 （irTOC1）. We characterized growth and light- and dark-adapted photosynthetic rates （Ac） throughout a 24 h clay in empty vector-transformed （EV）, irLHY, and irTOC1 plants in the field, and in NaPhyA-and NaPhyB1-silenced plants in the glasshouse. The growth rates of irLHY plants were lower than those of EV plants in the field. While irLHY plants reduced Ac earlier at dusk, no differences between irLHY and EV plants were observed at dawn in the field, irLHY, but not EV plants, responded to light in the night by rapidly increasing Ac. Under controlled conditions, EV plants rapidly increased Ac in the day compared to dark-adapted plants at night; irLHY plants lost these time-dependent responses. The role of NaLHY in gating photosynthesis is independent of the light-depen- dent reactions and red light perceived by NaPhyA, but not NaPhyB1. In summary, the circadian clock allows plants not to respond photosynthetically to light at night by anticipating and gating red light-mediated in native tobacco.

Functional specialization of Nicotiana attenuata phytochromes in leaf development and flowering time

Phytochromes mainly function in photoau- totrophic organisms to adjust growth in response to fluctuating light signals. The different isoforms of plant phytochromes often display both conserved and divergent roles, presumably to fine-tune plant responses to environmental signals and optimize fitness. Here we describe the distinct, yet partially redundant, roles of phytochromes NaPHYA, NaPHYB1 and NaPHYB2 in a wild tobacco species, Nicotiana attenuata using RNAi-silenced phytochrome lines. Consistent with results reported from other species, silencing the expression of NaPHYA or NaPHYB2 in N. attenuata had mild or no influence on plant develop- ment as long as NaPHYBI was functional; whereas silencing the expression of NaPHYB1 alone strongly altered flowering time and leaf morphology. Thecontribution of NaPHYB2 became significant only in the absence of NaPHYB1; plants silenced for both NaPHYB1 and NaPHYB2 largely skipped the rosette- stage of growth to rapidly produce long, slender stalks that bore flowers early： hallmarks of the shade- avoidance responses. The phenotyping of phyto- chrome-silenced lines, combined with sequence and transcript accumulation independent functional analysis, suggest the diversification of the phytochromes, and a dominant role of NaPHYB1 and NaPHYB2 in N. attenuata＇s vegetative and reproductive development.

Fitness consequences of a clock pollinator filter in Nicotiana attenuata flowers in nature

Nicotiana attenuata flowers, diurnally open,emit scents and move vertically to interact with nocturnal hawkmoth and day-active hummingbird pollinators. To examine the fitness consequences of these floral rhythms, we conducted pollination trials in the plant’s native habitat with phase-shifted flowers of plants silenced in circadian clock genes. The results revealed that some pollination benefits observed under glasshouse conditions were not reproduced under natural field conditions. Floral arrhythmicity increased pollination success by hummingbirds, while reducing those by hawkmoths in the field. Thus, floral circadian rhythms may influence a plant’s fitness by filtering pollinators leading to altered seed set from outcrossed pollen.

A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes（Sp Cas9） cleaves double-stranded DNA targeted by a chimeric single-guide RNA（sg RNA）. For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sg RNAs under the control of Ca MV 35 S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19 20 bp of sg RNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an Sp Cas9 expressing cassette. Twostep cloning procedures：（1） annealing two target-specific oligonucleotides with overhangs specific to the Aar I restriction enzyme site of the binary vector; and（2） ligating the annealed oligonucleotides into the two Aar I sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the GatewayTMsystem and unique Eco RI/Xho I sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.

The circadian clock contributes to diurnal patterns of plant indirect defense in nature

The plant circadian clock regulates the rhythms of plant metabolism.Many herbivore-induced plant volatiles(HIPVs)fluctuate,diurnally,but the role of the circadian clock in the emission of HIPVs and their ecological consequences remains largely unknown.Here,we show that the timing of herbivore attack can alter the outcome of tri-trophic interactions,and this is mediated by the circadian clock,under both field and glasshouse conditions.Although most HIPV emissions did not have a circadian rhythm,the circadian clock modulated HIPV emissions in a time-dependent manner.HIPVs mediate tri-trophic interactions,and the circadian clock may affect these interactions by modulating HIPV emission in nature.

Fitness consequences of altering floral circadian oscillations for Nicotiana attenuata

Ecological interactions between flowers and pollinators are all about timing. Flower opening/closing and scent emissions are largely synchronized with pollinator activity, and a circadian clock regulates these rhythms. However, whether the circadian clock increases a plant＇s reproductive success by regulating these floral rhythms remains untested. Flowers of Nicotiana attenuata, a wild tobacco, diurnally and rhythmically open, emit scent and move vertically through a 140° arc to interact with nocturnal hawkmoths. We tethered flowers to evaluate the importanceof flower positions for Manduca flower position dramatically sexta-mediated pollinations; nfluenced pollination. We examined the pollination success of phase-shifted flowers, silenced in circadian clock genes, NaZTL, NaLHY, and NaTOCI, by RNAi. Circadian rhythms in N. attenuata flowers are responsible for altered seed set from outcrossed pollen.

Shifting Nicotiana attenuata's diurnal rhythm does not alter its resistance to the specialist herbivore Manduca sexta

Arabidopsis thaliana plants are less resistant to attack by the generalist lepidopteran herbivore Trichoplusia ni when plants and herbivores are entrained to opposite, versus identical diurnal cycles and tested under constant conditions. This effect is associated with circadian fluctuations in levels of jasmonic acid, the transcription factor MYC2, and glucosinolate contents in leaves. We tested whether a similar effect could be observed in a different plant-herbivore system： the wild tobacco Nicotiana attenuata and its co-evolved specialist herbivore, Manduca sexta. We measured larval growth on plants under both constant and diurnal conditions following identical or opposite entrainment, profiled the metabolome of attacked leaf tissue, quantified specific metabolites known to reduce M. sexta growth, and monitored A/I. sexta feeding activity under all experimental conditions. Entrainment did not consistently affect NI. sexta growth or plant defense induction. However, both were reduced under constant dark conditions,as was M. sexta feeding activity. Our data indicate that the response induced by NI. sexta in N. attenuata is robust to diurnal cues and independent of plant or herbivore entrain- ment. We propose that while the patterns of constitutive or general damage-induced defense may undergo circadian fluctuation, the orchestration of specific induced responses is more complex.

Submergence promotes auxin-induced callus formation through ethylene-mediated post-transcriptional control of auxin receptors

Plant cells in damaged tissue can be reprogrammed to acquire pluripotency and induce callus formation.However,in the aboveground organs of many species,somatic cells that are distal to the wound site become less sensitive to auxin-induced callus formation,suggesting the existence of repressive regulatory mechanisms that are largely unknown.Here we reveal that submergence-induced ethylene signals promote callus formation by releasing post-transcriptional silencing of auxin receptor transcripts in non-wounded regions.We determined that short-term submergence of intact seedlings induces auxin-mediated cell dedifferentiation across the entirety of Arabidopsis thaliana explants.The constitutive triple response 1-1(ctr1-1)mutation induced callus formation in explants without submergence,suggesting that ethylene facilitates cell dedifferentiation.We show that ETHYLENE-INSENSITIVE 2(EIN2)post-transcriptionally regulates the abundance of transcripts for auxin receptor genes by facilitating microRNA393 degradation.Submergence-induced calli in non-wounded regions were suitable for shoot regeneration,similar to those near the wound site.We also observed submergence-promoted callus formation in Chinese cabbage(Brassica rapa),indicating that this may be a conserved mechanism in other species.Our study identifies previously unknown regulatory mechanisms by which ethylene promotes cell dedifferentiation and provides a new approach for boosting callus induction efficiency in shoot explants.