The basic helix-loop-helix transcription factors MYC1 and MYC2 have a dual role in the regulation of constitutive and stress-inducible specialized metabolism in tomato.

Plants produce specialized metabolites to protect themselves from biotic enemies. Members of the Solanaceae family accumulate phenylpropanoid-polyamine conjugates (PPCs) in response to attackers while also maintaining a chemical barrier of steroidal glycoalkaloids (SGAs). Across the plant kingdom, biosynthesis of such defense compounds is promoted by jasmonate signaling in which clade IIIe basic helix-loop-helix (bHLH) transcription factors play a central role. By characterizing hairy root mutants obtained through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) genome editing, we show that the tomato clade IIIe bHLH transcription factors, MYC1 and MYC2, redundantly control jasmonate-inducible PPC and SGA production, and are also essential for constitutive SGA biosynthesis. Double myc1 myc2 loss-of-function tomato hairy roots displayed suppressed constitutive expression of SGA biosynthesis genes, and severely reduced levels of the main tomato SGAs α-tomatine and dehydrotomatine. In contrast, basal expression of genes involved in PPC biosynthesis was not affected. CRISPR-Cas9(VQR) genome editing of a specific cis-regulatory element, targeted by MYC1/2, in the promoter of a SGA precursor biosynthesis gene led to decreased constitutive expression of this gene, but did not affect its jasmonate inducibility. Our results demonstrate that clade IIIe bHLH transcriptional regulators have evolved under the control of distinct regulatory cues to specifically steer constitutive and stress-inducible specialized metabolism.

SlKIX8 and SlKIX9 are negative regulators of leaf and fruit growth in tomato.

Plant organ size and shape are major agronomic traits that depend on cell division and expansion, which are both regulated by complex gene networks. In several eudicot species belonging to the rosid clade, organ growth is controlled by a repressor complex consisting of PEAPOD (PPD) and KINASE-INDUCIBLE DOMAIN INTERACTING (KIX) proteins. The role of these proteins in asterids, which together with the rosids constitute most of the core eudicot species, is unknown. We used Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 genome editing to target SlKIX8 and SlKIX9 in the asterid model species tomato (Solanum lycopersicum) and analyzed loss-of-function phenotypes. Loss-of-function of SlKIX8 and SlKIX9 led to the production of enlarged, dome-shaped leaves and these leaves exhibited increased expression of putative Solanum lycopersicum PPD (SlPPD target genes. Unexpectedly, kix8 kix9 mutants carried enlarged fruits with increased pericarp thickness due to cell expansion. At the molecular level, protein interaction assays indicated that SlKIX8 and SlKIX9 act as adaptors between the SlPPD and SlTOPLESS co-repressor proteins. Our results show that KIX8 and KIX9 are regulators of organ growth in asterids and can be used in strategies to improve important traits in produce such as thickness of the fruit flesh.

Give CRISPR a Chance: the GeneSprout Initiative.

Did you know that a group of early-career researchers launched an initiative enabling open dialog on new plant breeding techniques, such as genome editing? We developed a wide-ranging initiative that aims to facilitate public engagement and provide a platform for young plant scientists to encourage participation in science communication.

CRISPR-Cas-Mediated Gene Knockout in Tomato.

Loss-of-function mutants are crucial for plant functional genomics studies. With the advent of CRISPR-Cas genome editing, generating null alleles for one or multiple specific gene(s) has become feasible for many plant species including tomato (Solanum lycopersicum). An easily programmable RNA-guided Cas endonuclease efficiently creates DNA double-strand breaks (DSBs) at targeted genomic sites that can be repaired by nonhomologous end joining (NHEJ) typically leading to small insertions or deletions that can produce null mutations. Here, we describe how to utilize CRISPR-Cas genome editing to obtain stable tomato gene knockout lines.

Metabolic editing: small measures, great impact.

Metabolic pathways are tightly regulated at the transcriptional and post-translational level, often relying on protein-protein interactions or post-translational protein modifications. Whereas these principles have been established already for a long time, the number of experimentally established cases is expected to rise exponentially in the near future as a result of recent advances in protein-based detection methods. Interactions and modifications are often dependent on only short amino-acid sequences that represent excellent targets for new gene editing technologies by which specific base pairs can be exchanged. Here, we introduce the concept of metabolic editing, which is based on identifying specific amino-acid sequences that are subsequently targeted for gene editing. The proposed workflow will serve for both applied metabolic engineering purposes and proof-of-concept studies in fundamental research.

Lessons from Domestication: Targeting Cis-Regulatory Elements for Crop Improvement.

Domestication of wild plant species has provided us with crops that serve our human nutritional needs. Advanced DNA sequencing has propelled the unveiling of underlying genetic changes associated with domestication. Interestingly, many changes reside in cis-regulatory elements (CREs) that control the expression of an unmodified coding sequence. Sequence variation in CREs can impact gene expression levels, but also developmental timing and tissue specificity of expression. When genes are involved in multiple pathways or active in several organs and developmental stages CRE modifications are favored in contrast to mutations in coding regions, due to the lack of detrimental pleiotropic effects. Therefore, learning from domestication, we propose that CREs are interesting targets for genome editing to create new alleles for plant breeding.

Change of a conserved amino acid in the MYC2 and MYC3 transcription factors leads to release of JAZ repression and increased activity.

The bHLH transcription factor MYC2, together with its paralogues MYC3 and MYC4, is a master regulator of the response to the jasmonate (JA) hormone in Arabidopsis (Arabidopsis thaliana). In the absence of JA, JASMONATE ZIM (JAZ) proteins interact with the MYC proteins to block their activity. Understanding of the mechanism and specificity of this interaction is key to unravel JA signalling. We generated mutant MYC proteins and assessed their activity and the specificity of their interaction with the 12 Arabidopsis JAZ proteins. We show that the D94N mutation present in the atr2D allele of MYC3 abolishes the interaction between MYC3 and most JAZ proteins. The same effect is observed when the corresponding conserved Asp (D105) was mutated in MYC2. Accordingly, MYC2(D105N) activated target genes in the presence of JAZ proteins, in contrast to wild-type MYC2. JAZ1 and JAZ10 were the only JAZ proteins still showing interaction with the mutant MYC proteins, due to a second MYC interaction domain, besides the classical Jas domain. Our results visualize the divergence among JAZ proteins in their interaction with MYC proteins. Ultimately, the transferability of the Asp-to-Asn amino acid change might facilitate the design of hyperactive transcription factors for plant engineering.