The Non-classical Secretion Characteristics And Pathogenic Function Of The Extracellular Protein VdSOD1 Of Verticillium Dahliae

Verticillium dahliae is a plant pathogen of soil-borne vascular fungus.V.dahliae can infect hundreds of dicotyledon plants,including many economically important crops such as cotton,potato,eggplant.To elucidate the pathogenic mechanism,the identification of V.dahliae pathogenicity related genes has become a hotspot of current reasearch.The accumulation of reactive oxygen species?ROS?is a widespread defense mechanism in higher plants against pathogen attack.Although the superoxide dismutase?SOD?-based ROS scavenging system in pathogens constitutes the frontline of defense against intra-and extracellular ROS generated as part of the defense response by the host,the roles of SODs in V.dahliae are unclear.Our previous study showed the exoproteome of V.dahliae induced in a cotton tissue powder containing C'zapek-Dox medium contained an abundance of secreted proteins.Among the 271pathogen proteins that were induced,a Cu/Zn-SOD1?VdSOD1?without signal-peptide was up-regulated 3.95-fold,suggesting that it may be functional during V.dahliae-host interactions and thus play a role in pathogenicity.Here,this research constructed deletion mutants of VdSOD1 and investigated its function in scavenging ROS and promoting pathogen virulence on the host.The main results are as follows:1.The the yeast signal trap assay demonstrated the VdSOD1 N-terminal peptide is unable to mediate the protein secretion to the extracellular space in a classical pathway.Despite lacking a signal peptide,the extracellular secretion of VdSOD1 was detected in vitro by western blot and in vivo by confocal microscopy.The results indicated signal-less protein VdSOD1 can be secreted.2.?VdSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione.These results demonstrated that VdSOD1 contributes to scavenging of intracellular superoxide radicals.Cotton roots infected with?VdSOD1 accumulated less H₂O₂ and more O-₂than those infected with the wild-type strain.The results demonstrated that VdSOD1plays a role in scavenging extracellular ROS produced by the host.3.Absence of VdSOD1 significantly impaired symptom severity as well as pathogen colonization in both cotton and Nicotiana benthamiana.Therefore,VdSOD1is required for full virulence of V.dahliae on susceptible cotton and N.benthamiana.In conclusion,these results suggested that the signal-less Cu-Zn superoxide dismutase VdSOD1 is an unconventional secretion protein.VdSOD1,detoxifies both intracellular ROS and host-generated extracellular ROS facilitating infection.While VdSOD1 is nonessential for growth or viability,its presence contributes significantly to virulence in V.dahliae.