CRISPR/Cas9-mediated mutagenesis of the susceptibility gene OsHPP04 in rice confers enhanced resistance to rice root-knot nematode.

CRISPR crops carrying a mutation in susceptibility (S) genes provide an effective strategy for controlling plant disease, because they could be 'transgene-free' and commonly have more broad-spectrum and durable type of resistance. Despite their importance, CRISPR/Cas9-mediated editing of S genes for engineering resistance to plant-parasitic nematode (PPN) disease has not been reported. In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of the S gene rice copper metallochaperone heavy metal-associated plant protein 04 (OsHPP04), and successfully obtained genetically stable homozygous rice mutants with or without transgenic elements. These mutants confer enhanced resistance to the rice root-knot nematode (Meloidogyne graminicola), a major plant pathogenic nematode in rice agriculture. Moreover, the plant immune responses triggered by flg22, including reactive oxygen species burst, defence-related genes expression and callose deposition, were enhanced in the 'transgene-free' homozygous mutants. Analysis of rice growth and agronomic traits of two independent mutants showed that there are no obvious differences between wild-type plants and mutants. These findings suggest that OsHPP04 may be an S gene as a negative regulator of host immunity and genetic modification of S genes through the CRISPR/Cas9 technology can be used as a powerful tool to generate PPN resistant plant varieties.

Metabolome and transcriptome analysis of eleutheroside B biosynthesis pathway in Eleutherococcus senticosus.

Eleutheroside B (syringin) is a medicinal active ingredient extracted from Eleutherococcus senticosus (Ruper. et Maxim.) Maxim with high clinical application value. However, its synthesis pathway remains unknown. Here, we analyzed the eleutheroside B biosynthesis pathway in E. senticosus. Consequently, metabolomic and transcriptomic analyses identified 461 differentially expressed genes (DEGs) and 425 metabolites. Further, we identified 7 DEGs and 67 metabolites involved in the eleutheroside B biosynthetic pathway in the eleutheroside B high and low plants. The correlation between the gene and metabolites was explored using the pearson correlation coefficient (PCC) analysis. Caffeoyl-CoA O-methyltransferase, caffeic acid-O-methyltransferase, ß-amyrin synthase (ß-AS) genes, NAC5, and HB5 transcription factors were identified as candidate genes and transcription factors related to the eleutheroside B synthesis. Eleutheroside B content was the highest at the young stage of the leaves both in the high and low eleutheroside B plants. Quantitative real-time polymerase chain reaction revealed that phenylalanine ammonia-lyase1, cinnamate 4-hydroxylase, ß-AS, and leucoanthocyanidin reductase gene had higher expression levels at the young stage of the leaves in the low eleutheroside B plants but lower expression levels in the high eleutheroside B plants. In the present study, we complemented the eleutheroside B biosynthetic pathway by analyzing the expression levels of relevant genes and metabolite accumulation patterns.