In Arabidopsis, NPR1 and its role in SA signaling has been very well studied. NPR1-like proteins NPR3 and NPR4 bind to the same TGA factors that bind NPR1, to inhibit their ability to activate defense gene expression. NPR3 and NPR4 function counteract the ability of NPR1 to activate defenses. Knockdown of NPR3 and NPR4 results in enhanced resistance to some pathogens, confirming that they are ‘susceptibility factors. WhNPR3 and WhNPR4 are expressed in Fg infected wheat spikes. In comparison to the Arabidopsis wild-type plants, npr3/npr4 mutant exhibited HR-like symptoms and elevated levels of ROS accumulation, along with reduced fungal biomass in the infected leaves. Besides, npr3/npr4 mutant showed enhanced resistance to Fg in Arabidopsis inflorescence compared to wild-type plants. We further demonstrated that non-GMO knockdown of alleles of WhNPR3 and WhNPR4 in Kronos TILLING lines resulted in enhanced resistance against Fg compared to Kronos wild-type plants. Therefore, natural variants with missense mutation that knockdown activity of WhNPR3/WhNPR4 could provide novel genetic material for integration into FHB resistance breeding programs.