CRISPR-finder: A high throughput and cost-effective method to identify successfully edited Arabidopsis thaliana individuals

Genome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to mutagenesis. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. We successfully identified an ISOCHORISMATE SYNTHASE 1 mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.

ATG4 Mediated Psm ES4326/AvrRpt2-Induced Autophagy Dependent on Salicylic Acid in Arabidopsis Thaliana

Psm ES4326/AvrRpt2 (AvrRpt2) was widely used as the reaction system of hypersensitive response (HR) in Arabidopsis. The study showed that in npr1 (GFP-ATG8a), AvrRpt2 was more effective at inducing the production of autophagosome and autophagy flux than that in GFP-ATG8a. The mRNA expression of ATG1, ATG6 and ATG8a were more in npr1 during the early HR. Based on transcriptome data analysis, enhanced disease susceptibility 1 (EDS1) was up-regulated in wild-type (WT) but was not induced in atg4a4b (ATG4 deletion mutant) during AvrRpt2 infection. Compared with WT, atg4a4b had higher expression of salicylic acid glucosyltransferase 1 (SGT1) and isochorismate synthase 1 (ICS1); but less salicylic acid (SA) in normal condition and the same level of free SA during AvrRpt2 infection. These results suggested that the consumption of free SA should be occurred in atg4a4b. AvrRpt2 may trigger the activation of Toll/Interleukin-1 receptor (TIR)-nucleotide binding site (NB)-leucine rich repeat (LRR)—TIR-NB-LRR—to induce autophagy via EDS1, which was inhibited by nonexpressor of PR genes 1 (NPR1). Moreover, high expression of NPR3 in atg4a4b may accelerate the degradation of NPR1 during AvrRpt2 infection.

CRISPR-finder: A high throughput and cost effective method for identifying successfully edited A. thaliana individuals

BackgroundGenome editing with the CRISPR/Cas9 system allows the user to mutate a targeted region of the genome using an endonuclease (Cas9) and an artificial single-guide RNA (sgRNA). Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to CRISPR/Cas9 mutagenesis. This process can be laborious, expensive and inefficient with conventional methods such as the T7E1 assay or Sanger sequencing. An alternative comprises methods for amplicon sequencing, but most available protocols do not include a facile way for high throughput generation of the samples for sequencing.ResultsIn this study we provide a full pipeline based on amplicon sequencing, CRISPR-finder. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. For example, we were able to analyze in one sequencing reaction over 900 Arabidopsis thaliana individuals whose genomes had been targeted with the CRISPR/Cas9 system.ConclusionsIn order to validate the potential of CRISPR-finder, we targeted the ISOCHORISMATE SYNTHASE 1 gene in A. thaliana using the CRISPR/Cas9 system. We successfully identified a mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and -efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.

PBS3 and EPS1 complete salicylic acid biosynthesis from isochorismate in Arabidopsis

Salicylic acid (SA) is an important phytohormone mediating both local and systemic defense responses in plants. Despite over half a century of research, how plants biosynthesize SA remains unresolved. In Arabidopsis, a major part of SA is derived from isochorismate, a key intermediate produced by the isochorismate synthase (ICS), which is reminiscent of SA biosynthesis in bacteria. Whereas bacteria employ an isochorismate pyruvate lyase (IPL) that catalyzes the turnover of isochorismate to pyruvate and SA, plants do not contain an IPL ortholog and generate SA from isochorismate through an unknown mechanism. Combining genetic and biochemical approaches, we delineated the SA biosynthetic pathway downstream of isochorismate in Arabidopsis. We show that PBS3, a GH3 acyl adenylase-family enzyme important for SA accumulation, catalyzes ATP- and Mg2+-dependent conjugation of L-glutamate primarily to the 8-carboxyl of isochorismate and yields the key SA biosynthetic intermediate isochorismoyl-glutamate A. Moreover, EPS1, a BAHD acyltransferase-family protein with previously implicated role in SA accumulation upon pathogen attack, harbors a noncanonical active site and an unprecedented isochorismoyl-glutamate A pyruvoyl-glutamate lyase (IPGL) activity that produces SA from the isochorismoyl-glutamate A substrate. Together, PBS3 and EPS1 form a two-step metabolic pathway to produce SA from isochorismate in Arabidopsis, which is distinct from how SA is biosynthesized in bacteria. This study closes a major knowledge gap in plant SA metabolism and would help develop new strategies for engineering disease resistance in crop plants.