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

CARO, MARIA DEL PILAR; ASURMENDI, SEBASTIÁN; ZIPFEL, CYRIL; HOLTON, NICK; DÍAZ-RICCI, JUAN CARLOS

Vardero

Congreso; Biotechnology Havana 2017: Agricultural Biotechnology in the XXI Century; 2017

ICGEB

The novel elicitor AsES, a fungalsubtilisin protein, does not require its enzymatic activity for the activationof the defence response in Arabidopsis. Caro,María del Pilar 1, Nick Holton 2 , Cyril Zipfel 2,Sebastian Asurmendi 3and Juan Carlos Diaz- Ricci 1 1 INSIBIO-CONICET-UNT, Tucumán, Argentina; 2The Sainsbury Laboratory, Norwich, UK; 3 Instituto de Biotecnología,CICVyA, Buenos Aires, Argentina AsES (Acremoniumstrictum elicitor subtilisin)is an extracellular protease produced by the avirulent isolate of the fungus Acremonium strictum, a pathogen instrawberry. It has been previously shown that AsES produces a defence responsethat is characterized by ROS production, callose deposition, defence -relatedgene expression, NO accumulation and protection against Colletotrichum acutatum, the causal agent of anthracnose disease instrawberry and against Botrytis cinereain Arabidopsis.In order todetermine if the protease activity is required for the defence response and ifthe protein is recognised by a receptor in a classical PAMP/PRR interaction,the aim of this work was to express AsES protein (full length, I9+S8 domains), aswell as a mutant version affected in its enzymatic activity using anheterologous system that allow us to obtain high yields and purity.The CDS for AsESfull length and AsES full length mutant were cloned into the expression vector pMAL-p5X(Novagen) and later expressed in Escherichiacoli BL21 (DE3). Both proteins were exported to the periplasm, extracted byosmotic shock and purified by affinity chromatography. The capacity of inducinga PTI response in Arabidopsis was evaluated by RT-qPCR, seedling growth inhibition(SGI) and challenging assays against Botrytiscinerea.The results showedthat treatments with recombinant AsESfl (34 kDa), inhibited in its enzymaticactivity by PMSF and the enzymatically inactive mutant, resulted in theup-regulation of Arabidopsis defence related genes such as PR1, FRK1, WRKY70 andWRKY 53 at 2 and 4 h.p.t. For the SGI assay, Col-0 and also FLS2, EFR and CERK1triple mutant (Fec) seedlings, showed a significant reduction in growth (freshweight) compared to mock plants (MS) when treated with AsESfl and AsESfl mutant60 nM at 8 d after treatment. Arabidopsis plants, pre-treated with the inactiveproteins and later infected with a spore suspension of Botrytis cinerea, showed a reduction in the lesion size comparedwith mock plants.This work providesnew insights into AsES mechanism. AsES protein has the potential to be used asa biocontrol agent in order to enhance immunity to microbial infections incrops.