Generation of semi-dwarf rice (Oryza sativa L.) lines by CRISPR/Cas9-directed mutagenesis of OsGA20ox2 and proteomic analysis of unveiled changes caused by mutations

Han, Yue; Teng, Kaichong; Nawaz, Gul; Feng, Xuanjun; Usman, Babar; Wang, Xin; Luo, Liang; Zhao, Neng; Liu, Yao-Guang; Li, Rongbai

doi:10.1007/s13205-019-1919-x

Cited by 36 publications

(32 citation statements)

References 77 publications

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“…The inoculation experiments proved that the mutants were highly resistant to M. oryzae, thus can be useful for future breeding programs. The mutant lines were obtained without any off-target mutations, the previously reported work also suggests that the Cas9 rarely tempts off-target mutations in rice [54,55]. The transgene-free plants were screened in a frequency ranging between 18-34% in different homozygous, heterozygous, and biallelic lines with stable transmission of mutations (Supplementary file 1, Table S6).…”

Section: Discussionmentioning

confidence: 99%

Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line

Nawaz

Usman

Peng

et al. 2020

Genes

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Rice blast (Magnaporthe oryzae) is a devastating disease affecting rice production globally. The development of cultivars with host resistance has been proved to be the best strategy for disease management. Several rice-resistance genes (R) have been recognized which induce resistance to blast in rice but R gene-mediated mechanisms resulting in defense response still need to be elucidated. Here, mutant lines generated through CRISPR/Cas9 based targeted mutagenesis to investigate the role of Pi21 against blast resistance and 17 mutant plants were obtained in T0 generation with the mutation rate of 66% including 26% bi-allelic, 22% homozygous, 12% heterozygous, and 3% chimeric and 17 T-DNA-free lines in T1 generation. The homozygous mutant lines revealed enhanced resistance to blast without affecting the major agronomic traits. Furthermore, comparative proteome profiling was adopted to study the succeeding proteomic regulations, using iTRAQ-based proteomic analysis. We identified 372 DEPs, among them 149 up and 223 were down-regulated, respectively. GO analysis revealed that the proteins related to response to stimulus, photosynthesis, carbohydrate metabolic process, and small molecule metabolic process were up-regulated. The most of DEPs were involved in metabolic, ribosomal, secondary metabolites biosynthesis, and carbon metabolism pathways. 40S ribosomal protein S15 (P31674), 50S ribosomal protein L4, L5, L6 (Q10NM5, Q9ZST0, Q10L93), 30S ribosomal protein S5, S9 (Q6YU81, Q850W6, Q9XJ28), and succinate dehydrogenase (Q9S827) were hub-proteins. The expression level of genes related to defense mechanism, involved in signaling pathways of jasmonic acid (JA), salicylic acid (SA), and ethylene metabolisms were up-regulated in mutant line after the inoculation of the physiological races of M. oryzae as compared to WT. Our results revealed the fundamental value of genome editing and expand knowledge about fungal infection avoidance in rice.

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Section: Discussionmentioning

confidence: 99%

Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line

Nawaz

Usman

Peng

et al. 2020

Genes

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“…To ensure the fidelity of biological conclusions, it is important to confirm the success of any gene editing at the protein level. Recently, researchers have successfully edited OsGA20ox2 in rice and studied the proteome-wide changes that were induced [38]. Here, the iTRAQ-based proteomic approach was applied both to confirm protein level changes induced by gene editing and to measure the proteome-wide changes that occur in response to the system perturbation in a single experiment.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-Induced Mutagenesis of Semi-Rolled Leaf1,2 Confers Curled Leaf Phenotype and Drought Tolerance by Influencing Protein Expression Patterns and ROS Scavenging in Rice (Oryza sativa L.)

et al. 2019

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Rice leaf morphology is an essential agronomic trait to develop drought-tolerant genotypes for adequate and stable crop production in drought-prone areas. Here, rolled leaf mutant plants were acquired by CRISPR/Cas9-based mutagenesis of Semi-rolled leaf1,2 (SRL1 and SRL2) genes, and isobaric tags for relative and absolute quantification (iTRAQ) based proteomic analysis was performed to analyze the subsequent proteomic regulation events. Homozygous mutants exhibit decreased chlorophyll content, transpiration rate, stomatal conductance, vascular bundles (VB), stomatal number, and agronomic traits with increased panicle number and bulliform cells (BCs). Under drought stress, mutant plants displayed lower malondialdehyde (MDA) content while higher survival rate, abscisic acid (ABA) content, superoxide dismutase (SOD), catalase (CAT) activities, and grain filling percentage compare with their wild type (WT). Proteomic results revealed that 270 proteins were significantly downregulated, and 107 proteins were upregulated in the mutant line compared with WT. Proteins related to lateral organ boundaries’ (LOB) domain (LBD) were downregulated, whereas abiotic stress-responsive proteins were upregulated in the CRISPR mutant. LBD proteins (Q5KQR7, Q6K713, Q7XGL4, Q8LQH4), probable indole-3-acetic acid-amido synthetase (Q60EJ6), putative auxin transporter-like protein 4 (Q53JG7), Monoculm 1 (Q84MM9) and AP2 (Apetala2) domain-containing protein (Q10A97) were found to be hub-proteins. The hybrids developed from mutant restorers showed a semi-rolled leaf phenotype with increased panicle number, grain number per panicle, and yield per plant. Our findings reveal the intrinsic value of genome editing and expand the knowledge about the network of proteins for leaf rolling and drought avoidance in rice.

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“…To explore the new genes responsible for the regulation of cellular growth and division and promoting grain size is worthwhile in rice breeding. Previous studies proved that the desirable mutations in yield-associated loci under natural conditions are favorable [ 3 , 4 , 5 , 6 , 15 , 16 , 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

Generation of High Yielding and Fragrant Rice (Oryza sativa L.) Lines by CRISPR/Cas9 Targeted Mutagenesis of Three Homoeologs of Cytochrome P450 Gene Family and OsBADH2 and Transcriptome and Proteome Profiling of Revealed Changes Triggered by Mutations

Usman

Nawaz

Zhao

et al. 2020

Plants

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The significant increase in grain yield and quality are often antagonistic but a constant demand for breeders and consumers. Some genes related to cytochrome P450 family are known for rice organ growth but their role in controlling grain yield is still unknown. Here, we generated new rice mutants with high yield and improved aroma by simultaneously editing three cytochrome P450 homoeologs (Os03g0603100, Os03g0568400, and GL3.2) and OsBADH2 with the CRISPR/Cas9 system, and RNA-sequencing and proteomic analysis were performed to unveil the subsequent changes. High mutation efficiency was achieved in both target sites of each gene and the mutations were predominantly only deletions, while insertions were rare, and no mutations were detected in the five most likely off-target sites against each sgRNA. Mutants exhibited increased grain size, 2-acetyl-1-pyrroline (2AP) content, and grain cell numbers while there was no change in other agronomic traits. Transgene-DNA-free mutant lines appeared with a frequency of 44.44% and homozygous mutations were stably transmitted, and bi-allelic and heterozygous mutations followed Mendelian inheritance, while the inheritance of chimeric mutations was unpredictable. Deep RNA sequencing and proteomic results revealed the regulation of genes and proteins related to cytochrome P450 family, grain size and development, and cell cycle. The KEGG and hub-gene and protein network analysis showed that the gene and proteins related to ribosomal and photosynthesis pathways were mainly enriched, respectively. Our findings provide a broad and detailed basis to understand the role of CRISPR/Cas9 in rice yield and quality improvement.

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Generation of semi-dwarf rice (Oryza sativa L.) lines by CRISPR/Cas9-directed mutagenesis of OsGA20ox2 and proteomic analysis of unveiled changes caused by mutations

Cited by 36 publications

References 77 publications

Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line

Knockout of Pi21 by CRISPR/Cas9 and iTRAQ-Based Proteomic Analysis of Mutants Revealed New Insights into M. oryzae Resistance in Elite Rice Line

CRISPR/Cas9-Induced Mutagenesis of Semi-Rolled Leaf1,2 Confers Curled Leaf Phenotype and Drought Tolerance by Influencing Protein Expression Patterns and ROS Scavenging in Rice (Oryza sativa L.)

Generation of High Yielding and Fragrant Rice (Oryza sativa L.) Lines by CRISPR/Cas9 Targeted Mutagenesis of Three Homoeologs of Cytochrome P450 Gene Family and OsBADH2 and Transcriptome and Proteome Profiling of Revealed Changes Triggered by Mutations

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