Single-cell functional expression assay

The single-cell transient functional expression assay was conducted essentially as described⁵⁹. The cDNA coding region of TRIUR3_01037 was amplified from PI428198 and PI428322, respectively, with the primers listed in Table S9. The resultant cDNA sequences, designated as RAA and SAA, respectively, were confirmed to be error-free by DNA sequencing. They were then cloned into a plasmid vector driven by the maize polyubiquitin promoter, resulting in the expression constructs pUbi-RAA and pUbi-SAA, respectively. A β-glucuronidase (GUS) reporter gene construct (pUbi-GUS) was also used in this assay, whose expression facilitated the visualization of transformed cells⁵⁹.

The RAA and SAA constructs were each mixed with pUbi-GUS at a 1:1 molar ratio for coating the DNA microcarrier. As control for the assay, only pUbi-GUS was used for coating the DNA microcarrier. The plasmid DNA coated on the microcarrier was delivered into the leaf epidermal cells of the T. urartu accession PI428252, which was susceptible to Bgt isolate E09, through particle bombardment. The leaf segments were inoculated with E09 conidial spores at 4 h post bombardment. At 48 hpi of Bgt, the leaf segments were stained in a β-glucuronidase staining solution for 8 h at 37 °C, followed by destaining for 72 h at 25 °C. Before mounting for microscopic examination, the leaf segments were stained with Coomassie blue to visualize Bgt structures. For assessing the effects of each construct (pUbi-RAA, pUbi-SAA or pUbi-GUS), approximately 200 cells (in 6 to 8 leaf segments) with GUS signals and Bgt mycelia were examined for haustorium growth, with haustorium index calculated as the percentage of examined cells with haustorium presence⁵⁹.