Assessment of nitric oxide signaling functions during the plant hypersensitive disease resistance response

In plants, nitric oxide (NO) plays a crucial role in mediating defense signal in response to infection by bacterial pathogens. NO signal can be mediated by its direct interaction with target molecules, via post-translational modifications for instance, but it is believed that, like in animal cells, NO signal can also be relay by the second messenger cGMP (cyclic guanosine 3',5'-monophosphate) in particular to regulate defense gene expression. Despite the demonstration of its role during the HR, the detection of NO is still highly debated, mainly because its measurement methods are not fully specific. In the same way, cGMP detection in plants until now has been based on methods that often display a low sensitivity and a low throughput. In addition, due to the lack of identification of the enzymes responsible for cGMP metabolism, until now, the role of cGMP has been investigated only with the use of pharmacological compounds that sometimes raise the question about response specificity. In this study, we thus confirmed that NO is produced specifically during the incompatible interaction thanks to a chemiluminescence-based method and that NO production involves mainly nitrite as substrate and requires NR as well as other source(s) still unidentified. Moreover, a highly sensitive and with high throughput method based on AlphaScreen technology was successfully applied in plants to demonstrate the NO-dependent local and distal increase of cGMP level in response to avirulent pathogens. The characterization of transgenic Arabidopsis thaliana plants expressing the mammalian soluble guanylate cyclase (GC) showed that these plants accumulate high constitutive levels of cGMP compared with WT plants and display an altered expression of salicylic acid-dependent defence genes correlated with the loss of systemic acquired resistance establishment, while they show a normal resistance and hypersensitive cell death at local level. Moreover, in GC lines, SA-dependent pathway seems to be compromised in favour of jasmonate-dependent responses, suggesting a role for cGMP in regulating the hormonal switch.