The novel elicitorAsES, a fungal subtilisin protein, does not require its enzymatic activity for the activation of the defence response in Arabidopsis

CARO MA. DEL PILAR; HOLTIN, NICK; ZIPFEL, CYRIL ; ASURMENDI, SEBASTIAN; DÍAZ RICCI, JUAN C.

Ciego de Avila

Congreso; 11º Congreso Internacional de Biología Vegetal y Agricultura; 2017

Centro Bioplantas - Cuba

AsES (Acremonium strictum elicitor subtilisin) is an extracellular protease produced by the avirulent isolate of the fungus Acremoniumstrictum, a strawberry pathogen. It has been previously shown that AsES produces a defence response that is characterized by ROS production, callose deposition, defence-related gene expression, NO accumulation and protection against Colletotrichum acutatum, the causal agent of anthracnose disease in strawberry, and against Botrytis cinerea in Arabidopsis.The aim of this work was to obtain AsES protein (full length, I9+S8 domains, AsESfl), as well as a mutantaffected in its enzymatic activity by heterologous expression in orderto determine whether the protease activity is required for the defence response.The CDS for AsES full length and AsESfull length mutantwerecloned into the expression vectorpMAL-p5X (Novagen) that allow exporting the protein to the periplasm, and expressed in Escherichia coli BL21(DE3). Both proteins wereextracted by osmotic shock and purified by affinity chromatography. The capacity of these proteins to induce a PTI response in Arabidopsis was evaluated by RT-qPCR and seedling growth inhibition (SGI).The results showed that AsESfl, inhibited in its enzymatic activity by PMSF and the inactive mutant, are capable of up-regulate defencerelated genes such as PR1, FRK1, WRKY70 and WRKY53 at 2 and 4 hours post treatment (h.p.t.) in 5 weeks old Arabidopsis plants. For the SGI assay, Col-0 and also FLS2, EFR and CERK1 triple mutant (Fec) seedlings, showed a significant reduction in growth (fresh weight) compared to mock plants (MS) when treated with AsESfl and AsESfl mutant 60 nM.This work provides new insights into AsES mechanism of action and potential uses as an alternative biocontrol agent in orderto activate innate immunity against microbial infections in crops.