Liew, Y. Y. | Search bioRxiv

bioRxiv2024

Structure determinants of DANGEROUS MIX 3, an alpha/beta hydrolase, for triggering NLR-mediated genetic incompatibility in plants

Genetic incompatibility occurs when a mismatched pair of plant immune components mounts autoimmune responses in hybrids. Highly diversified NLR receptors are main culprits of the genetic conflict, recognizing host proteins from different origin as immune trigger. Here, we report the molecular mechanism underlying a DANGEROUS MIX (DM) autoimmunity, comprising DM2h/RPP1 NLR and its incompatible partner DM3, an alpha/beta hydrolase. Cryo-electron microscopy reveals the oligomeric nature of two natural DM3 variants in a trimer of dimer configuration. The polymorphism triggering autoimmunity is located at the dimer interface, resulting in drastic structural differences such that dimerizing helix and loop reinforcing the interface is lost and disordered. Structure-function analysis shows that integrity of the dimer interface, but neither maintenance of hexamer nor its enzymatic activity, is the key factor contributing to autoimmunity. Our finding pinpoints checkpoints embedded in the oligomeric configuration of a host enzyme that controls the switching mechanism of NLR activity.

plant biology↗

bioRxiv2022

Generation of inheritable A-to-G transitions using adenine base editing and NG-PAM Cas9 in Arabidopsis thaliana

CRISPR/Cas9 technology is an important tool for functional genomics and crop improvement. It can be used to generate mutations at precise positions in the genome. Base editors consist of deaminase components and Cas9 to specify the type of mutation, such as C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. Available adenine base editor vectors usually make use of canonical Cas9, which limits their use to 5-NGG-3 containing targets. We combined a relaxed variant of SpCas9 that uses 5-NG-3 containing targets with the adenine base editor containing TadA7.10 or TadA8e to make a set of vectors. By using a phenotype-based screen, we found that our vectors efficiently induce A-to-G somatic mutations in a 5-NGG-3 PAM context in Arabidopsis thaliana up to 81% efficiency. Such mutations are inheritable at the homozygous stage in T2. Among tested vectors, pECNUS4 (Addgene #184887), which carries TadA8e, showed highest efficiency at generating a stable A-to-G transition in a 5-NGH-3 PAM context in the gene DM3. Using this vector, we were able to recreate a naturally occurring allele of DM3 in two generations without the transgene. pECNUS4 is a new component of the CRISPR toolbox to be used for introducing desired adenine base transitions with an expanded target window for functional genomic research and trait improvement.

plant biology↗