The second messenger nitric oxide (NO), phosphatidic acid (PA) and reactive oxygen species (ROS) are involved in the plant defense response during plant-pathogen interactions. NO has been shown to participate in PA production in response to the pathogen-associated molecular pattern xylanase in tomato cell suspensions. Defense responses downstream of PA include ROS production. The goal of this work was to study the signaling mechanisms involved in PA production during the defense responses triggered by xylanase and mediated by NO in the suspension-cultured tomato cells. We analyzed the participation of protein kinases, guanylate cyclase and the NO-mediated posttranslational modification S-nitrosylation, by means of pharmacology and biochemistry. We showed that NO, PA and ROS levels are significantly diminished by treatment with the general protein kinase inhibitor staurosporine. This indicates that xylanase-induced protein phosphorylation events might be the important components leading to NO formation, and hence for the downstream regulation of PA and ROS levels. When assayed, a guanylate cyclase inhibitor or a cGMP analog did not alter the PA accumulation. These results suggest that a cGMP-mediated pathway is not involved in xylanase-induced PA formation. Finally, the inhibition of protein S-nitrosylation did not affect NO formation but compromised PA and ROS production. Data collectively indicate that upon xylanase perception, cells activate a protein kinase pathway required for NO formation and that, S-nitrosylation-dependent mechanisms are involved in downstream signaling leading to PA and ROS.