Fusarium graminearum (Fg) interaction with wheat in an infected spikelet alters core plant immunity via modulating resistance and susceptibility genetic factors and allows opportunistic expression of Fg’s effectors and mycotoxin-producing genes, adding to the already ongoing complex and asymmetric gene expression in developing grain. This interaction can result in alternative host responses depending on pathogen virulence and host resistance status. Fhb1 is the most effective and stable source of deployed Fusarium head blight (FHB) resistance in wheat and primarily follows FHB Type II resistance, which prevents the spread of infection from affected spikelets. However, the resistance conundrum includes varied Fhb1 effectiveness in diverse genetic backgrounds and the confounding spatiotemporal activation and/or suppression of more than one functional gene family that contribute to Fhb1 resistance phenotype. Thus, this genetic complexity piqued the interest of our collaborative group to create an unbiased, spatially resolved transcriptomics cell atlas during wheat-Fg interaction and answer a few of above-mentioned questions. Spatially resolved tissue specific transcriptomics and targeted spatial gene expression approaches are powerful gene network discovery approaches that can simultaneously capture inaccessible view of tissue biology to truly explore the conserved transcriptional reprogramming or genetic factors driving FHB resistance, which are often overlooked in bulk analysis techniques. Our group has taken initial steps and is currently utilizing spring wheat varieties, i.e., the Fhb1-carrying variety ‘Brick’ and a susceptible control ‘Boost’, inoculated with fluorescently tagged Fg strain PH-1 that derives GFP expression through Tri5 promoter to optimize the 10x Genomics Visium spatial assay. A wild-type PH-1 strain was also initially used for assay optimization. We collected fresh-frozen, 10 µm-thick ‘spikelet-rachis-spikelet’ mega tissue sections at 5 days post-point inoculation and achieved fitting it in a 5mm fiducial frame on Visium gene expression slide. This fiducial frame contains ~5000 barcoded capture spots for sequencing. At this stage, we have also optimized the balance between the complexity of microscopy and RNA stabilization, a major bottleneck in capturing the plant-pathogen interaction simultaneously during a spatial expression assay. We are currently processing these tissue sections for RNA release optimization and sequencing library preparation. We aim to spatially resolve the intertwined and complex orchestra of gene expression interaction between diverse pathogens and plant tissues in infection court leveraging wheat’s cellular defense against FHB as a prioritized model.