AGROtools: A comprehensive modular cloning toolset for Agrobacterium strain engineering

Agrobacterium-mediated transformation is a cornerstone of plant genetic engineering, yet many crops remain recalcitrant to transformation and its efficiency is highly species- and genotype-dependent. The limited diversity of disarmed agrobacterial strains available for biotechnology, and the technically challenging workflows typically required for strain modification, constrain efforts to develop improved plant transformation platforms. Here, we describe the development of AGROtools-a versatile, expandable, and user-friendly modular cloning plasmid collection comprising more than 75 individual components, that is designed to simplify genetic engineering of agrobacteria. Constructs assembled with this toolkit couple visual reporters with selectable and counterselectable markers to facilitate identification of recombinant clones, thereby reducing reliance on PCR-based screening. We constructed and tested reporter cassettes that are functional across diverse agrobacterial strains and genomic contexts. By linking a visual reporter combinatorially with various antibiotic resistance markers and orthogonal site-specific recombinase recognition sequences, AGROtools can enable marker removal and recycling for up to four successive genomic modifications. We demonstrate the utility of AGROtools by disarming 10 novel wild-type strains, and accomplishing serial modifications within individual strain backgrounds. In addition to conventional recA-mediated homologous recombination, the collection incorporates components for applying contemporary strain engineering approaches, including CRISPR/dCas9 base editing, INTEGRATE-mediated targeted insertion, and phage-derived recombineering systems. Constructs can be assembled and deployed rapidly, enabling isolation of strains carrying an intended modification in less than two weeks. AGROtools provides a broadly applicable resource for accelerating strain development and genetic studies in agrobacteria, with potential to advance plant biotechnology by aiding improvement of disarmed strains.