Publications

Dr. Samuel Bocobza

ד"ר שמואל בוקובזא

Optimization of T‑DNA configuration with UBIQUITIN10 promoters and tRNA–sgRNA complexes promotes highly efficient genome editing in allotetraploid tobacco: Combination of UBIQUITIN10 promoter-directed CAS9 and tRNA–gRNA complexes in gene-editing assay induces 80% mutant phenotype with a knockout of the four allelic copies in the T0 generation of allotetraploid tobaccos.

Dr. Samuel Bocobza
and Dr. Dani Eshel

ד"ר שמואל בוקובזא
Dr. Dani Eshel
Advances in protoplast transfection promote efcient CRISPR/ Cas9‑mediated genome editing in tetraploid potato: An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor.

Dr. Ziv Speigelman

Powdery mildew (PM) diseases may severely limit the production of various crops, including members of  the Cucurbitaceae family. Successful PM infection relies on the Mildew Resistance Locus O (MLO) plant gene family, which encodes susceptibility factors essential for fungus penetration into the host cell. In cucumber, natural mutations in CsaMLO8 confer resistance to the PM pathogen Podosphaera xanthii. Here, the authors successfully used CRISPR/Cas9-mediated mutagenesis to generate PM resistance in the susceptible cucumber cv. ‘Ilan’.

Prof. Avraham Levy

פרופ' אברהם לוי

CRISPR/Cas9-mediated genome editing relies on error-prone repair of targeted DNA double-strand breaks (DSBs). Understanding CRISPR/Cas9-mediated DSB induction and subsequent repair dynamics requires measuring the rate of cutting and that of precise repair, a hidden-variable of the repair machinery. Here, we present a molecular and computational toolkit for multiplexed quantification of DSB intermediates and repair products by single-molecule sequencing. Using this approach, we characterized the dynamics of DSB induction, processing and repair at endogenous loci along a 72-hour time-course in tomato protoplasts. Combining this data with kinetic modeling reveals that indel accumulation is not an accurate reflection of DSB induction efficiency due to prominent precise re-ligation, accounting for 40-70% of all repair events. Altogether, this system exposes previously unseen flux in the DSB repair process, decoupling induction and repair dynamics, and suggesting an essential role of high-fidelity repair in limiting CRISPR editing efficiency in somatic cells.

Dr. Guy Mechrez

This research presents gene expression after DNA delivery into intact plant cells by protein nanoparticles. The DNA delivery is carried out by casein nanoparticles (CNPs). A plasmid harboring the red fluorescent protein DsRed sequence is absorbed to the CNPs surface by electrostatic interaction and served as a model DNA in this study, and its expression is monitored by the fluorescence of the DsRed protein. The zeta potential of the CNPs is tuned by altering the pH to obtain sufficient electrostatic interaction between the CNPs and the DsRed plasmid for successful DNA delivery into the cells of the model plant Nicotiana benthamiana. The CNPs are covalently modified with the green fluorescent dye 6-Aminofluorescein (6-AF) to determine their location in the plant. To assess the ability of the CNPs to deliver DNA into the cells, CNP/6-AF/DsRed plasmid electrostatic conjugates are infiltrated into N. benthamiana leaves. Confocal fluorescence microscopy results showed successful intracellular and nucleus uptake of the conjugates at pH 4.5 and a concentration of 2 mg mL−1 at CNPs: DsRed plasmid ratio of 1:0.01. The successful gene expression is confirmed by RT-PCR and qRT-PCR. The first appearance of the emitted red signal of the DsRed protein is observed 24 h post-infiltration.

Prof. Alexander Vainstein


Emission of scent volatiles by flowers is important for successful pollination and consequently, reproduction. Petunia (Petunia hybrida) floral scent is formed mainly by volatile products of the phenylpropanoid pathway. We identified and characterized a regulator of petunia scent production: the GRAS protein PHENYLPROPANOID EMISSION-REGULATING SCARECROW-LIKE (PES). Its expression increased in petals during bud development and was highest in open flowers. Overexpression of PES increased the production of floral volatiles, while its suppression resulted in scent reduction. We showed that PES upregulates the expression of genes encoding enzymes of the phenylpropanoid and shikimate pathways in petals, and of the core regulator of volatile biosynthesis ODORANT1 by activating its promoter. PES is an ortholog of Arabidopsis (Arabidopsis thaliana) PHYTOCHROME A SIGNAL TRANSDUCTION 1, involved in physiological responses to far-red (FR) light. Analyses of the effect of nonphotosynthetic irradiation (low-intensity FR light) on petunia floral volatiles revealed FR light as a scent-activating factor. While PHYTOCHROME A regulated scent-related gene expression and floral scent production under FR light, the influence of PES on volatile production was not limited by FR light conditions.

 

Prof. Alexander Vainstein


In moth-pollinated petunias, production of floral volatiles initiates when the flower
opens and occurs rhythmically during the day, for optimal flower–pollinator
interaction. To characterize the developmental transcriptomic response to time
of day, we generated RNA-Seq databases for corollas of floral buds and mature
flowers in the morning and in the evening. Around 70% of transcripts accumulating
in petals demonstrated significant changes in expression levels in response to the
flowers’ transition from a 4.5-cm bud to a flower 1 day postanthesis (1DPA).
Overall, 44% of the petal transcripts were differentially expressed in the morning vs.
evening. Morning/evening changes were affected by flower developmental stage,
with a 2.5-fold larger transcriptomic response to daytime in 1DPA flowers
compared to buds. Analyzed genes known to encode enzymes in volatile
organic compound biosynthesis were upregulated in 1DPA flowers vs. buds—in
parallel with the activation of scent production. Based on analysis of global
changes in the petal transcriptome, PhWD2 was identified as a putative scent-related
factor. PhWD2 is a protein that is uniquely present in plants and has a threedomain
structure: RING–kinase–WD40. Suppression of PhWD2 (termed UPPER –
Unique Plant PhEnylpropanoid Regulator) resulted in a significant increase in the
levels of volatiles emitted from and accumulated in internal pools, suggesting that
it is a negative regulator of petunia floral scent production.

Prof. Avraham Levy

פרופ' אברהם לוי

CRISPR/Cas9-mediated genome editing relies on error-prone repair of targeted DNA double-strand breaks (DSBs). Understanding CRISPR/Cas9-mediated DSB induction and subsequent repair dynamics requires measuring the rate of cutting and that of precise repair, a hidden-variable of the repair machinery. Here, we present a molecular and computational toolkit for multiplexed quantification of DSB intermediates and repair products by single-molecule sequencing. Using this approach, we characterized the dynamics of DSB induction, processing and repair at endogenous loci along a 72-hour time-course in tomato protoplasts. Combining this data with kinetic modeling reveals that indel accumulation is not an accurate reflection of DSB induction efficiency due to prominent precise re-ligation, accounting for 40-70% of all repair events. Altogether, this system exposes previously unseen flux in the DSB repair process, decoupling induction and repair dynamics, and suggesting an essential role of high-fidelity repair in limiting CRISPR editing efficiency in somatic cells.