Biotechnology
Deciphering protein‐protein interactions (PPIs) is fundamental for understanding signal transduction pathways in plants. The split firefly luciferase (Fluc) complementation (SLC) assay has been widely used for analyzing PPIs. However, concern has risen about the bulky halves of Fluc interfering with the functions of their fusion partners. Nano luciferase (Nluc) is the smallest substitute for Fluc with improved stability and luminescence. Here, we developed a dual‐use system enabling the detection of PPIs through the Nluc‐based SLC and co‐immunoprecipitation assays. This was realized by coexpression of two proteins under investigation in fusion with the HA‐ or FLAG‐tagged Nluc halves, respectively. We validated the robustness of this system by reproducing multiple previously documented PPIs in protoplasts or Agrobacterium‐transformed plants. We next applied this system to evaluate the homodimerization of Arabidopsis CERK1, a coreceptor of fungal elicitor chitin, and its heterodimerization with other homologs in the absence or presence of chitin. Moreover, split fragments of Nluc were fused to two cytosolic ends of Arabidopsis calcium channels CNGC2 and CNGC4 to help sense the allosteric change induced by the bacterial elicitor flg22. Collectively, these results demonstrate the usefulness of the Nluc‐based SLC assay for probing constitutive or inducible PPIs and protein allostery in plant cells.
Fruit crops, including apple, orange, grape, banana, strawberry, watermelon, kiwifruit and tomato, not only provide essential nutrients for human life but also contribute to the major agricultural output and economic growth of many countries and regions in the world. Recent advancements in genome editing provides an unprecedented opportunity for the genetic improvement of these agronomically important fruit crops. Here, we summarize recent reports of applying CRISPR/Cas9 to fruit crops, including efforts to reduce disease susceptibility, change plant architecture or flower morphology, improve fruit quality traits, and increase fruit yield. We discuss challenges facing fruit crops as well as new improvements and platforms that could be used to facilitate genome editing in fruit crops, including dCas9‐base‐editing to introduce desirable alleles and heat treatment to increase editing efficiency. In addition, we highlight what we see as potentially revolutionary development ranging from transgene‐free genome editing to de novo domestication of wild relatives. Without doubt, we now see only the beginning of what will eventually be possible with the use of the CRISPR/Cas9 toolkit. Efforts to communicate with the public and an emphasis on the manipulation of consumer‐friendly traits will be critical to facilitate public acceptance of genetically engineered fruits with this new technology.