AtNUDT7 was reported to be a negative regulator of EDS1-mediated immunity in Arabidopsis. However, the underlying molecular and genetic mechanism of the AtNUDT7-regulated defense pathway remains elusive. Here we report that AtNUDT7 and its closest paralog AtNUDT6 function as novel negative regulators of SNC1, a TIR-NB-LRR-type R gene. SNC1 is upregulated at transcriptional and possibly post-transcriptional levels in nudt6-2 nudt7. The nudt6-2 nudt7 double mutant exhibits autoimmune phenotypes that are modulated by temperature and fully dependent on EDS1. The nudt6-2 nudt7 mutation causes EDS1 nuclear accumulation shortly after the establishment of autoimmunity caused by the temperature shift. We found that a low ammonium/nitrate ratio in growth media leads to a higher level of nitrite-dependent nitric oxide (NO) production in nudt6-2 nudt7, and NO acts in a positive feedback loop with EDS1 to promote the autoimmunity. The low ammonium/nitrate ratio also enhances autoimmunity in snc1-1 and cpr1, two other autoimmune mutants in Arabidopsis. Our study indicates that Arabidopsis senses the ammonium/nitrate ratio as an input signal to determine the amplitude of the EDS1-mediated defense response, probably through the modulation of NO production. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.