Although microalgae have only recently been recognized as part of the plant and soil microbiome, their application as biofertilizers has a tradition in sustainable crop production. Under consideration of their ability to produce the plant growth-stimulating hormone cytokinin (CK), known to also induce pathogen resistance, we have assessed the biocontrol ability of CK-producing microalgae. All pro- and eukaryotic CK-producing microalgae tested were able to enhance the tolerance of tobacco against Pseudomonas syringae pv. tabaci (PsT) infection. Since Chlamydomonas reinhardtii (Cre) proved to be the most efficient, we functionally characterized its biocontrol ability. We employed the CRISPR-Cas9 system to generate the first knockouts of CK biosynthetic genes in microalgae. Specifically, we targeted Cre Lonely Guy (LOG) and isopentenyltransferase (IPT) genes, the key genes of CK biosynthesis. While Cre wild-type exhibits a strong protection, the CK-deficient mutants have a reduced ability to induce plant defence. The degree of protection correlates with the CK levels, with the IPT mutants showing less protection than the LOG mutants. Gene expression analyses showed that Cre strongly stimulates tobacco resistance through defence gene priming. This study functionally verifies that Cre primes defence responses with CK, which contributes to the robustness of the effect. This work contributes to elucidate microalgae-mediated plant defence priming and identifies the role of CKs. In addition, these results underscore the potential of CK-producing microalgae as biologicals in agriculture by combining biofertilizer and biocontrol ability for sustainable and environment-friendly crop management.

AIT Austrian Institute of Technology Center for Health and Bioresources Bioresources Unit Tulln a d Donau Austria

Charité Universitätsmedizin Berlin corporate member of Freie Universität Berlin and Humboldt Universität zu Berlin Institute for translational physiology Berlin Germany

Department of Plant and Environmental Sciences University of Copenhagen Taastrup Denmark

Global Change Research Institute of the Czech Academy of Sciences Brno Czech Republic

Humboldt Universität zu Berlin Institute of Biology Experimental Biophysics Berlin Germany

Palacký University and Institute of Experimental Botany of the Czech Academy of Sciences Olomouc Czech Republic

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Structure-function relation of cytokinins determines their differential efficiency in mediating tobacco resistance against Pseudomonas syringae