Excessive nitrogen (N) application may induce powdery mildew (PM) in perennial grasses, but the resistance mechanisms to PM remain unclear. This study evaluated the physiological and molecular mechanisms of PM resistance affected by N supplies in Kentucky bluegrass (Poa pratensis L.). Cultivar ‘Bluemoon’ (N tolerant) and ‘Balin’ (N sensitive) were treated with low N (0.5 mM), normal N (15 mM), and high N (30 mM) for 21 d in a greenhouse. With increasing N levels, the disease growth was more severe in ‘Balin’ than in ‘Bluemoon’. RNA-seq and weighted gene coexpression network analysis revealed that the PpGS1.1 gene encoding glutamine synthetase was a potential hub gene for PM resistance after comparisons across cultivars and N treatments. The N metabolism pathway was connected with the plant–pathogen interaction pathway via PpGS1.1. The expression of PpGS1.1 in rice protoplasts indicated that the protein was located in the nucleus and cytoplasm. Overexpression of PpGS1.1 in wild-type Kentucky bluegrass increased carbon and N contents, and the transgenic plants became more susceptible to PM with a lower wax density. The most differentially expressed genes (DEGs) for N metabolism were upregulated and DEGs for fatty acid metabolism pathway were downregulated in the overexpression lines. The results elucidated mechanisms of PM resistance in relation to N metabolism in Kentucky bluegrass.

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