Subjects: Biology >> Botany >> Botany research, experiment, plant evolution & development submitted time 2016-05-03
Hyeran Kim
Sang-Tae Kim
Jahee Ryu
Min Kyung Choi
Jiyeon Kweon
Beum-Chang Kang
Hyo-Min Ahn
Suji Bae
Jungeun Kim
Jin-Soo Kim
Sang-Gyu Kim
Abstract：
CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19−20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI 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 GatewayTM system and unique EcoRI/XhoI 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.
Peer Review Status:
Awaiting Review
Subjects: Biology >> Botany >> Plant physiology submitted time 2016-05-03
Jasmin Herden
Stefan Meldau
Sang-Gyu Kim
Grit Kunert
Youngsung Joo
Ian T. Baldwin and Meredith C. Schuman
Abstract：
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 M. sexta feeding activity under all experimental conditions. Entrainment did not consistently affect M. 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 M. sexta in N. attenuata is robust to diurnal cues and independent of plant or herbivore entrainment. 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.
Peer Review Status:Awaiting Review
Hits
Hits
Downloads
Comment