Knockdown of<i>STAYGREEN</i>in Perennial Ryegrass (<i>Lolium perenne</i>L.) Leads to Transcriptomic Alterations Related to Suppressed Leaf Senescence and Improved Forage Quality

Xu, Bin; Yu, Guohui; Li, Hui; Xie, Zihao; Wen, Wuwu; Zhang, Jing; Huang, Bingru

doi:10.1093/pcp/pcy203

Cited by 34 publications

(40 citation statements)

References 40 publications

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“…For phenotype assessment, we targeted the alfalfa MsSGR gene as a candidate to test the effectiveness, because mutants of the stay-green gene can be easily identified by dark-induced senescence. As reported in previous studies, stay-green gene knockdown or knockout mutants displayed greenness in leaves, stems, pods and seeds of the plants in Arabidopsis thaliana , Medicago truncatula, Medicago sativa (alfalfa) and Lolium perenne after induced senescence by dark treatment ( Park et al, 2007 ; Ren et al, 2007 ; Zhou et al, 2011 ; Xu et al, 2018 ). After targeting four sites across the first, second, and third exons of the MsSGR gene, homozygous mutants were identified through rigorous genotypic and phenotypic analyses in the T0 generation.…”

Section: Discussionsupporting

confidence: 68%

“…To decide whether the MsSGR mutants identified by genotyping screening could show a true stay-green phenotype, leaves collected from the mutants were exposed to dark-induced senescence treatment assay ( Zhou et al, 2011 ). Typically the phenotype of a sgr mutant can easily be identified by its chlorophyll retention capacity using detached-leaf dark treatment or whole-plant shade treatment ( Ren et al, 2007 ; Zhou et al, 2011 ; Xu et al, 2018 ). Replications of four to five leaves of MsSGR mutants and controls were harvested from the middle part of each plant and incubated at room temperature in darkness.…”

Section: Methodsmentioning

confidence: 99%

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Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa)

et al. 2020

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Alfalfa ( Medicago sativa ) is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the Medicago sativa stay-green ( MsSGR ) gene. The replacement of CaMV35S promoter by the Arabidopsis ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.

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Section: Discussionsupporting

confidence: 68%

Section: Methodsmentioning

confidence: 99%

Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa)

et al. 2020

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“…RNA-Seq enables us to analyse transcriptomes, the comprehensive expression profile of the genome, and has been used for a variety of analyses, such as the effects of mutations 1,2 , stress responses [3][4][5] , chemical biosynthesis pathways 6 and plant-pathogen interactions 7,8 . However, large scale experiments have been limited due to the large costs required for library preparation and sequencing.…”

Section: Rna-seq Is a Whole-transcriptome Analysis Methods Used To Resmentioning

confidence: 99%

Lasy-Seq: a high-throughput library preparation method for RNA-Seq and its application in the analysis of plant responses to fluctuating temperatures

Kamitani

Kashima

Tezuka

et al. 2019

Sci Rep

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Uniquely mapped reads with a mapping quality value of ≥4 were generated using SAMtools and 5.0 × 10 5 reads were used for the following analysis. The rates of false-assignment caused by pooled-PCR or sequencing steps were calculated from the numbers of ERCC-control reads in samples with and without ERCC-control (Supplementary Fig. S2). Briefly, ERCC reads detected in the late-pooled samples (without ERCC addition) were regarded as false-assignments caused by the sequencing of each sample. Therefore, the rate of total false-assignment reads in all eight samples against total ERCC reads in the lane was estimated to be the false-assignment rate caused by sequencing (Supplementary Fig. S2). The false-assignment rate caused by pooled-PCR was estimated from the ERCC-reads number detected in early-pooled samples (without ERCC addition), as explained in Supplementary Fig. S2. Estimate deviation between technical replicates in Lasy-Seq. Correlation coefficient between the early-pooled samples were calculated using rpm except for ERCC-controls to estimate deviation between technical replicates. Pearson's correlation coefficient was calculated with cor function in R version 3.5.0 52 .

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“…We also found that plant trichomes play a key role in altering the abundance of leaf chewing herbivores, but there were unclear correlations between insect abundance and laboratory-measured GSL profiles (Sato et al 2018). To link insect abundance and plant physiological status in the field, we employed transcriptomics, which were widely used to reveal comprehensive pictures of gene expression (Sun et al 2017; Xu et al 2018; Wang et al 2018; Lin et al 2018; Want et al 2018). Here we adopted our previously established protocol of cost-effective RNA-Seq (Nagano et al 2015; Kamitani et al 2016; Ishikawa et al 2017) to the field-grown A. thaliana.…”

Section: Introductionmentioning

confidence: 99%

Transcriptional variation in glucosinolate biosynthetic genes and inducible responses to aphid herbivory on field-grownArabidopsis thaliana

Sato

Tezuka

Kashima

et al. 2019

Preprint

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25Recently, increasing attempts have been made to understand how plant genes function in 26 natura studies. To determine whether plant defense genes are activated under multiple biotic 27 stimuli, we combined a high-throughput RNA-Seq with insect survey data on 19 accessions 28 of Arabidopsis thaliana growing on the field site of Switzerland. We found that genes with 29 GO annotations "glucosinolate biosynthetic process" and "response to insects" were the most 30 significantly enriched, exhibiting largely variable expression among plant accessions. Nearly 31 half of the total expression variation in glucosinolate biosynthetic genes, AOPs, ESM1, ESP, 32 and TGG1, was explained by among-accession variance. Combined with the field RNA-Seq 33 data, bioassays confirmed that AOP3 was up-regulated in response to the mustard aphid 34 Lipaphis erysimi. In addition, we also found that the expression of a major cis-jasmone 35 activated gene CYP81D11 was positively correlated with the number of the flea beetles 36 Phyllotreta spp. The combined results from RNA-Seq and insect surveys suggested that 37 plants can activate their defenses even when they are exposed to multiple biotic stimuli in 38 natura. 39

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Knockdown ofSTAYGREENin Perennial Ryegrass (Lolium perenneL.) Leads to Transcriptomic Alterations Related to Suppressed Leaf Senescence and Improved Forage Quality

Cited by 34 publications

References 40 publications

Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa)

Development of a Highly Efficient Multiplex Genome Editing System in Outcrossing Tetraploid Alfalfa (Medicago sativa)

Lasy-Seq: a high-throughput library preparation method for RNA-Seq and its application in the analysis of plant responses to fluctuating temperatures

Transcriptional variation in glucosinolate biosynthetic genes and inducible responses to aphid herbivory on field-grownArabidopsis thaliana

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