Microalgae play an essential role in agriculture as bio-fertilizers and biostimulants, but some also produce various plant hormones, such as cytokinins (CK). CK are a class of natural plant hormones and there is evidence that exogenous or bacterial CK can modulate plant defense. However, there is a lack of knowledge about the effect of specific microalgae or microalgae-produced cytokinins on this response. In our study, we have evaluated the cytokinins-producing microalgae Chlamydomonas reinhardtii (Cri) on its ability to prime Nicotiana tabacum plant defenses against its natural pathogen Pseudomonas syringae pv. tabaci. Our work functionally verifies that Cri primes plant defense response, with cytokinins being a crucial component. We have used the CRISPR/Cas9 system to generate Cri LOG and IPT gene knockouts, which are major points of the CK biosynthesis pathways. While Cri shows strong protection potential, the CK-deficient mutants have a reduced ability to affect plant defense, where the degree of protection correlates with the CK levels - the IPT mutants show less protection than the LOG mutants. Additionally, by measuring plant defense gene expression by RTq-PCR we have shown that Cri treatment stimulates tobacco defense response by priming.