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

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.

Automated summary

The study utilized CRISPR/Cas9 system to generate loss-of-function mutations in AtWRKY3 and AtWRKY4 genes in Arabidopsis thaliana, revealing their significant up-regulation under salt and Me-JA stresses. The knockout of these genes led to inhibited growth and increased sensitivity to salt and Me-JA stresses, affecting ROS scavenging pathways.