Visual marker-assisted gene editing of MLO confers powdery mildew resistance in Nicotiana alata.

Nicotiana alata is a high-value floricultural crop, but its widely cultivated commercial cultivar 'Jasmine' is highly susceptible to powdery mildew under low-light indoor conditions. This susceptibility results in leaf mold, yellowing, and wilting, along with a sharp decline in ornamental value. To address this issue and reduce fungicide reliance, we developed the first gene-editing system for N. alata featuring antibiotic marker-free visual identification. This system employs dual-visual markers for rapid positive screening using anthocyanin accumulation to indicate successful transformation and albino phenotypes resulting from editing of NalaPDS to signal successful gene editing. To enhance the horticultural traits of N. alata, we knocked out NalaMLO, resulting in T1 and T2 plants with significantly greater resistance to powdery mildew. Headspace solid-phase microextraction-gas chromatography-mass spectrometry identified 179 floral volatiles, with key odorants such as linalool, phenylethyl alcohol, and d-limonene remaining unaffected in the edited lines. Transcriptomics, metabolomics, and proteomics analyses revealed that the resistance phenotype may be mediated by the coordination of multiple pathways, including the MAPK signaling pathway, starch and sucrose metabolism, fructose and mannose metabolism, and phenylpropanoid biosynthesis. This approach effectively reduces disease-induced losses in the N. alata cultivar Jasmine and will accelerate the deployment of disease-resistance traits in ornamental Solanaceae crops.