The Mechanism Of PUB25 And PUB26 In Plant Immunity To Verticillium Dahliae Arabidopsis

Plants,unlike mammals,lack mobile defender cells.The immunity is activated when plants are infected by pathogens.Plants have developed a multi-layered defense system to survive from infection.There are two branches of the plant immune system:the first is called PAMPs-triggered immunity(PTI)activated by pathogen-associated molecular patterns(PAMPs),and the second is called effector-triggered immunity(ETI)activated by pathogen effectors.The two layers of immune pathways can lead to transcriptional reprogramming,which will induce stomatal closure,callose accumulation,productions of ROS and disease-resistant metabolites,which work together to stop the infection.Verticillium wilt in plants is caused by Verticillium dahlia,which belongs to filamentous fungi.However,little is known about the infection mechanisms and how plants resist the hemibiotrophic fungi.In previous research we identified a protein secreted by Verticillium dahlia,named VdAL.VdAL can bind to zinc ion in vitro.Overexpression of VdAL in Arabidopsis confers a higher resistance to Verticillium dahlia to plants.In this study,we identified two E3 ubiquitin ligases called PUB25 and PUB26 that interact with VdAL through immunoprecipitation of VdAL followed by liquid chromatography-tandem mass spectrometry(LC-MS).The interactions were confirmed by yeast two-hybrid,firefly luciferase complementation imaging(LCI)and co-immunoprecipitation(Co-IP)assays.We investigated whether PUB25 and PUB26 mediate plants9 defenses to Verticillium dahlia.We found that VdAL is an Aspf2-like protein and had no protease activity.PUB25 and PUB26 can ubiquitinate VdAL which leads to 26S proteasome-mediated degradation of VdAL.The pub25,pub26 and pub25 pub26 mutants showed higher resistance to Verticillium dahlia,similar as the VdAL-overexpression lines.The RNA-seq data revealed a total of 737 genes upregulated in both pub25 pub26 mutant and the VdAL overexpression lines when treated with Verticillium dahlia.Most of these genes are involved in defense responses and the biosynthesis of disease-resistant metabolites.We also identified some genes down-regulated in pub25 pub26 mutant and the VdAL overexpression lines which are involved in regulating plant growth and development.Together,PUB25 and PUB26 might regulated the plant immune responses by targeting VdAL for ubiquitination.To further investigate the mechanisms underlying PUB25/26-mediated resistance to Verticillium dahlia,we performed yeast two-hybrid screening for PUB25/26-interacting proteins.MYB6,a member of MYB transcription factor family,was identified.The interaction between MYB6 and PUB25/26 was confirmed by LCI and Co-IP assays.Moreover,PUB25 and PUB26 can also ubiquitinate MYB6 in vitro.Consistent with this,the stability of MYB6 in vivo was affected by the MG 132 treatment,indicating that MYB6 was degraded by the 26S proteasome pathway.The myb6 mutant was more susceptive to Verticillium dahlia than the wild type.Together,these results indicated that PUB25 and PUB26 regulate plant immune responses through targeting VdAL and MYB6 for degradation.To summarize,this study reveals that PUB25 and PUB26 function as negative regulators of plants'resistance to Verticillium dahlia by targeting VdAL and MYB6 for 26S proteasome-mediated degradation.Whether VdAL works together with MYB6 awaits further investigation.Together,this study provides novel insights into plants' immune responses to dahlia.