The response of Alnus glutinosa to Frankia alni ACN14a is driven by several sequential physiological events from calcium spiking and root hair deformation to the development of the nodule. Early stages of actinorhizal symbiosis were monitored at the transcriptional level to observe plant host responses to Frankia. Forty-two genes were significantly upregulated in inoculated compared to non-inoculated roots. Most of these genes encode proteins involved in biological processes induced during microbial infection such as oxidative stress or response to stimuli but a large part of them are not differentially modulated or downregulated later in the process of nodulation. In contrast, several of them remained upregulated in mature nodules, and this included the gene most upregulated, which encodes a non-specific lipid transfer protein (nsLTP). Classified as an antimicrobial peptide, this nsLTP, was immunolocalized on the deformed root hair surfaces that are points of contact for Frankia during infection. Later in nodules, it binds to the surface of Frankia’s vesicles, which are the specialized cells for nitrogen fixation. This nsLTP, named AgLTP24, was biologically produced in a heterologous host and purified for assay on F. alni ACN14a to identify physiological effects. Thus, the activation of the plant immunity response occurs upon first contact, while the recognition of Frankia switches off part of the defense system during nodulation. AgLTP24 constitutes a part of the defense system that is maintained all along the symbiosis with potential functions such as the formation of infection threads or nodule primordia to the control of Frankia proliferation.