Phytaspases: Negative regulators of AM symbiosis through proteolysis of signaling peptides

• Applicant: Dr. Javier Lidoy Logroño, Ph.D.
• Subject Area: Organismic Interactions, Chemical Ecology and Microbiomes of Plant Systems
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 572884401

Arbuscular mycorrhizal (AM) symbiosis plays an important role in plant nutrition and health. Due to its significant nutritional cost to the plant, this symbiosis is tightly regulated by the host plant, particularly in the arbuscule-containing cells where the metabolite exchange occurs. Plant subtilases - a large family of extracellular serine proteases - perform multiple functions, including modulating beneficial interactions with microorganisms. Subtilases are critically involved in the proteolytic maturation of peptide hormones and growth factors from their inactive precursors. However, the role of subtilases in mycorrhizal symbiosis remains largely unknown. Through transcriptomic and single nuclei RNA seq (snRNA seq) analyses I have identified three subtilases, the phytaspases Phyt3, Phyt4 and Phyt5, that are highly induced in tomato (Solanum lycopersicum) roots colonized by Rhizophagus irregularis, with expression enriched in arbusculated cells. CRISPR/Cas9 triple knock out (phyt3 5) lines exhibit excessive intraradical colonization and reduced biomass, demonstrating that Phyt3 5 act as negative regulators of AM to maintain mutualism in the plant-microbe interaction. In this project, I address two hypotheses for the mode of action of phytaspases in regulating AM symbiosis. Hypothesis 1 – Phytaspases activate phytosulfokine (PSK) as a negative regulator of AM symbiosis. snRNA seq data, AlphaFold3 predictions and preliminary in vitro assays indicate that Phyt3 5 cleave the PSK precursor, releasing the bioactive PSK pentapeptide that accelerates arbuscule turnover. Preliminary results show that PSKR1/2 knock out lines phenocopy the phytaspase mutant. To substantiate hypothesis 1, I aim to (i) confirm the contribution of PSK signaling to phytaspase-mediated regulation of AM, (ii) map co localization and secretion of Phyt3 5 and PSK precursors in arbusculated cells; (ii) confirm PSK precursor cleavage by each protease in vitro; and (iv) determine whether exogenous PSK promotes arbuscule degeneration and complements the phyt3-5 loss-of-function phenotype. Hypothesis 2 – Phytaspase activates a GH18 B chitinase to promote arbuscule degeneration. AlphaFold3 predicts that Phyt3 and Phyt5 interact with a GH18 B chitinase co-expressed in arbusculated cells. We hypothesize that this interaction either enhances chitinase mediated fungal cell wall degradation, promoting arbuscule turnover, or modulates the activity of chitin derived signaling molecules to trigger immunity rather than symbiosis. To substantiate this hypothesis, I aim to (i) validate the predicted Phyt3,5–chitinase interaction experimentally, and (ii) test whether the interaction with Phyt3-5 can modulate chitinase activity.

• DFG Programme: WBP Position