Establishing VIGS and CRISPR/Cas9 techniques to verify RsPDS function in radish

Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.

RsERF40 contributes to cold stress tolerance and cell expansion of taproot in radish (Raphanus sativus L.)

The growth and development of taproots are inhibited by cold stress in radish(Raphanus sativus L.).Ethylene-responsive element binding factors(ERF)are key participators in the cold stress response and growth regulation of plants.However,the function of ERF genes in cold tolerance and root development in radish remains elusive.Here,we showed that the secondary growth of radish taproots was inhibited by cold stress.Comparative transcriptome analysis demonstrated that the RsERF40 gene is an important regulator of the cold stress response and root growth regulation.The cold tolerance of transgenic Arabidopsis plants overexpressing the RsERF40 gene was significantly improved.Overexpressing RsERF40 in the cold-sensitive radish genotype and silencing RsERF40 in the cold-tolerant radish genotype indicated that RsERF40 was beneficial for alleviating oxidative damage under cold stress in radish.Transgenic Arabidopsis seedlings showed an increase in the elongation and radial growth of dark-grown roots.RT-qPCR analysis showed that the expression of the cold-related genes(CORs)RsCOR78 and RsCOR413PM1 and the cell wall strengthening-related genes RsCESA6 and RsEXPB3 was upregulated in transgenic Arabidopsis seedlings.Yeast one-hybrid(Y1H)and dual-luciferase reporter assays(DLA)revealed that RsERF40 directly regulates RsCOR78,RsCOR413PM1,RsCESA6 and RsEXPB3 expression,illustrating that RsERF40 enhances cold tolerance and taproot growth by modulating osmotic adjustment and cell wall mechanical strength in radish.In this study,the RsERF40-regulon was firstly found to be a new cold response pathway independent of the CBF-COR pathway conferring cold stress tolerance with increasing radish taproot growth.These results provided novel insight into the molecular mechanism underlying cold stress response and would facilitate the genetic improvement of cold tolerance in radish and other root vegetable crops.