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CRISPR/Cas9-mediated mutagenesis of WRKY3 and WRKY4 function decreases salt and Me-JA stress tolerance in Arabidopsis thaliana

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Abstract

Background

WRKY transcription factor is involved in regulation of plant growth and development, response to biotic and abiotic stresses, including homologous WRKY3 and WRKY4 genes which play a vital role in regulating plants defense response to pathogen and drought stress.

Methods and results

To investigate the function of AtWRKY3 and AtWRKY4 genes in regulating salt and Me-JA stresses, the loss-of-function mutations were generated by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system in Arabidopsis thaliana. Several independent transgenic lines with single or double mutations were obtained via Agrobacterium-mediated transformation. The knockout lines of AtWRKY3 and AtWRKY4 genes were successfully achieved and confirmed by qRT-PCR technology. Expression analysis showed that AtWRKY3 and AtWRKY4 genes had significantly up-regulated under salt and Me-JA stresses. The growth of double mutant plants under salt or Me-JA stresses were significantly inhibited compared with corresponding wild type (WT) plants, especially their root lengths. Moreover, the double mutant plants displayed salt and Me-JA sensitivity phenotypic characteristics, such as the increased relative electrolyte leakage (REL) and a substantial reduce in the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities.

Conclusion

Taken together, these data suggested that the simultaneous modification of homologous gene copies of WRKY are established using CRISPR/Cas9 system in A. thaliana and the loss of AtWRKY3 and AtWRKY4 has an effect on ROS scavenging pathways to reduce stress tolerance.

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Abbreviations

CRISPR:

Clustered regularly interspaced short palindromic repeat

Cas9:

CRISPR-associated 9

WT:

Wide type

SOD:

Superoxide dismutase

POD:

Peroxidase

CAT:

Catalas

REL:

Relative electrolyte leakage

TFs:

Transcription factors

AP2:

APETALA2

ERF:

Ethylene-responsive factor

NAM:

No apical meristem

ATAF1/2:

Arabidopsis thaliana transcription activation factor

CUC2:

Cup-shaped cotyledon

DBD:

DNA binding domains

SPF1:

SWEET POTATO FACTOR1

SA:

Salicylic acid

JA:

Jasmonic acid

ABA:

Abscisic acid

ROS:

Reactive oxygen species

sgRNA:

Single guide RNA

PAM:

Protospacer adjacent motif

DSB:

Double-strand breaks

Clo-0:

Columbia-0

qRT-PCR:

Quantitative real-time PCR

MS:

Murashige and Skoog

PAM:

Protospacer adjacent motif

ZFNs:

Zinc finger nucleases

TALENs:

Transcription activator-like effector nucleases

MLO:

MILDEW-RESISTANCE LOCUS

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Acknowledgements

We are grateful to Dr. Qi Xie (Institute of Genetics and Developmental Biology, VAS) for providing us with the vector pYAO:hSpCas9-target-sgRNA.

Funding

This research was supported by the Shanghai Sailing Program (19YF1414800). The funding body had no role in study design, analysis, decision to publish, or preparation of the manuscript.

Author information

Author notes

  1. Peng Li and Xiwen Li have contributed equally to this work.

Authors and Affiliations

  1. Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, Institute of Biodiversity Science, School of Life Sciences, Fudan University, Shanghai, 200438, China

    Min Jiang

  2. Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai, 201602, China

    Peng Li, Xiwen Li & Min Jiang

Authors
  1. Peng Li
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  2. Xiwen Li
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Contributions

MJ conceived, designed and funded the research. MJ, PL and XL performed the experiments, analyzed the data and wrote the manuscript. MJ revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Min Jiang.

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Supplementary Information

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Supplementary file1 The primer list of all genes used in this study (DOCX 15 kb)

11033_2021_6541_MOESM2_ESM.tif

Supplementary file2 CRISPR/Cas9-mediated the mutations of AtWRKY3 and AtWRKY4 genes in T1 generation of different transgenic plant lines. (A-B) Identification of PCR products digested with restriction enzyme NdeI by gel electrophoresis. NdeI, PCR products digested with NdeI. The length of PCR products of NdeI digested are marked by red arrow heads. The number on the lanes represents the different transgenic plant lines. e Sign “N” represents the no enzyme digestion, while other lanes represents enzyme digestion. M, DNA marker. (C-D) Comparisons and analyses of the sequencing results. The gRNA sequence is labeled with light yellow and the PAM sequence is marked with a box. (E-F) Detailed mutation information of the AtWRKY3 and AtWRKY4 transgenic line (T1), respectively. DNA fragments around the target sequences were amplified by PCR and then subjected to sequencing analysis. The PAM and the sgRNA target sequences are indicated by blue and red lines, respectively. Green arrows indicated the insertion of an alanine (A) nucleotide. The blue arrow indicated the position of a deleted A or threonine (T) nucleotide (TIF 1808 kb)

11033_2021_6541_MOESM3_ESM.jpg

Supplementary file3 CRISPR/Cas9-mediated the single mutations of AtWRKY3 (A) or AtWRKY4 (B) gene in T2 generation of different transgenic plant lines. The number plus pound sign represents the different transgenic plant lines (JPG 130 kb)

11033_2021_6541_MOESM4_ESM.tif

Supplementary file4 Relative expression levels of AtWRKY3 and AtWRKY4 in mutants by qRT-PCR. The asterisk indicates the fold changes of expression levels is less 0.5  than WT plants (TIF 3041 kb)

11033_2021_6541_MOESM5_ESM.jpg

Supplementary file5 Physiological indies measurement under salt stress. (A) Phenotypes of wrky3 plants under salt stress. (B) Root lengths. (C) SOD activity. (D) POD activity. (E) CAT activity. (F) REL contents. Control represents a normal growth condition. The number plus pound sign represents the different transgenic plant lines. WT, wild type; 24#, wrky3-24; 25#, wrky3-25; 28#, wrky3-28; 23#, wrky4-23; 32#, wrky4-32; 35#, wrky4-35; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; REL, relative electrolyte leakage. Mean and S.D. values were obtained from three independent experiments (JPG 127 kb)

11033_2021_6541_MOESM6_ESM.jpg

Supplementary file6 Physiological indies measurement under Me-JA stress. (A) Phenotypes of wrky4 plants under Me-JA stress. (B) Root lengths. (C) SOD activity. (D) POD activity. (E) CAT activity. (F) REL contents. Control represents a normal growth condition. The number plus pound sign represents the different transgenic plant lines. WT, wild type; 24#, wrky3-24; 25#, wrky3-25; 28#, wrky3-28; 23#, wrky4-23; 32#, wrky4-32; 35#, wrky4-35; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase; REL, relative electrolyte leakage. Mean and S.D. values were obtained from three independent experiments (JPG 129 kb)

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Li, P., Li, X. & Jiang, M. CRISPR/Cas9-mediated mutagenesis of WRKY3 and WRKY4 function decreases salt and Me-JA stress tolerance in Arabidopsis thaliana. Mol Biol Rep 48, 5821–5832 (2021). https://doi.org/10.1007/s11033-021-06541-4

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