Nitrate and ammonia deferentially modulate primary metabolism during the hypersensitive response in tobacco. In this study, tobacco RNAi lines with low nitrite reductase (NiR r) levels were used to investigate the roles of nitrite and nitric oxide (NO) in this process. The lines accumulate NO 2 -, with increased NO generation, but allow sufficient reduction to NH 4 + to maintain plant viability. For wild-type (WT) and NiR r plants grown with NO 3 -, inoculation with the non-host biotrophic pathogen Pseudomonas syringae pv. phaseolicola induced an accumulation of nitrite and NO, together with a hypersensitive response (HR) that resulted in decreased bacterial growth, increased electrolyte leakage, and enhanced pathogen resistance gene expression. These responses were greater with increases in NO or NO 2 - levels in NiR r plants than in the WT under NO 3 - nutrition. In contrast, WT and NiR r plants grown with NH 4 + exhibited compromised resistance. A metabolomic analysis detected 141 metabolites whose abundance was differentially changed as a result of exposure to the pathogen and in response to accumulation of NO or NO 2 -. Of these, 13 were involved in primary metabolism and most were linked to amino acid and energy metabolism. HR-associated changes in metabolism that are often linked with primary nitrate assimilation may therefore be influenced by nitrite and NO production.