Directed evolution is a laboratory technique that mimics natural selection and allows scientists to evolve genes and the proteins they encode. Traditionally, this technique has been used in microbes, mammalian cells, or in test tubes.
Now, researchers led by Prof. GAO Caixia from the Institute of Genetics and Developmental Biology (IGDB) of the Chinese Academy of Sciences (CAS) and Prof. QIU Jinlong from the Institute of Microbiology of CAS have developed a new system that enables rapid and scalable directed evolution of diverse genes directly in plant cells.
This new platform, called Geminivirus Replicon-Assisted in Planta Directed Evolution (GRAPE), was reported in Science on October 2.
Modern agricultural production requires abundant genetic resources. Directed evolution can rapidly generate genetic variants with new and enhanced properties. However, efficient platforms for performing such evolution directly in plant cells have been lacking. A key challenge is the slow cell division rate in plants, which limits the speed of selection cycles and the enrichment of functional variants.
To address this issue, the researchers harnessed geminiviruses—plant DNA viruses that replicate DNA rapidly in plant cells via rolling circle replication (RCR), a fast way of copying circular DNA. Specifically, they tied the replication of artificial geminivirus replicons (circular DNA capable of replicating through RCR) to targeted functions of gene variants in plant cells. Variants possessing a targeted function prompted the replicon to replicate, creating more DNA copies, thereby selectively amplifying those variants.
Building on this approach, the researchers developed GRAPE. In this platform, genes of interest (GOIs) were first mutagenized in vitro and the resulting variants were inserted into artificial geminivirus replicons. These replicon libraries were then delivered into the leaves of Nicotiana benthamiana, where the desired gene activity was linked to viral replication. Variants that promoted replication were enriched, while those that inhibited replication were depleted. A full selection cycle could be completed on a single leaf within four days.
Using GRAPE, the researchers evolved the nucleotide-binding domain leucine-rich repeat-containing (NLR) immune receptor NRC3 to evade inhibition by the nematode effector SPRYSEC15 while maintaining its immune activity. Iterative evolution of the rice NLR immune receptor Pikm-1 yielded variants that respond to six alleles of the Magnaporthe oryzae effector AVR-Pik, significantly expanding its recognition range. This strategy enables the development of valuable genetic resources for breeding disease-resistant crops.
Compared with previous microbe-based systems, GRAPE offers distinct advantages: It excels at evolving GOIs responsible for plant-specific phenotypes (such as disease resistance) or requiring plant-specific regulation. Furthermore, it works directly within plant cells, eliminating the need for re-optimization. GRAPE can potentially evolve any gene functionally coupled to RCR. Beyond plant biology, GRAPE also holds promise for broader applications, such as evolving proteases to cleave specific targets for plant and pharmaceutical research.
GRAPE provides a rapid, efficient, and versatile platform that can accelerate plant synthetic biology and molecular breeding, opening up new avenues for crop engineering and enhancing the sustainability of agriculture.
