A new use of Agrobacterium plant growth regulator genes for plant bioengineering

Delivery of biomolecules into plant vascular tissues remains a barrier to managing diseases caused by insect vector-borne pathogens and to modifying phenotypes of established perennial crops. Inspired by the vascularized growth of crown galls induced by <i>Agrobacterium tumefaciens</i>, we repurposed the bacterium's plant growth regulator (PGR) genes to engineer autonomously dividing, transgene-expressing plant cell structures termed symbionts. A plant transformation vector (pSYM) incorporating the IaaM, IaaH, Ipt and gene5 cassette from <i>A. tumefaciens</i> strain C58 together with a gene of interest on the same transfer DNA was delivered to stems of herbaceous and woody dicots using disarmed <i>A. tumefaciens</i> strain EHA105. Symbiont morphology, vascular differentiation, transgene expression, molecular mobility and protein secretion were evaluated using microscopy, fluorescent reporters, dye tracing, RNA silencing assays and mass spectrometry-based proteomics. pSym inoculation reproducibly generated symbionts across diverse host plant species that were vascularly integrated into their host plants and transgene expression ranging from heterogeneous niches to more uniform patterns. Small molecules moved between symbionts and host vascular tissues, whereas larger proteins exhibited more restricted mobility. Post-transcriptional gene silencing signals moved freely throughout the symbiont and slightly into adjacent stem tissue. Under tested field and greenhouse conditions in potato and tomato, respectively, gall or symbiont formation had no negative impacts on plant growth or tuber and fruit yield. <i>In vitro</i>, symbiont cultures abundantly secreted recombinant protein into surrounding media. Together, these results establish symbionts as a modular, plant bioengineering platform capable of producing and potentially delivering biomolecules without modifying the host plant genome, providing a foundation for vascular-targeted therapeutics and phenotype modulation in crops.