Screening And Identification Of Host Targetsof VmPRlc,a CAP Superfamily Proteinin Valsa Mali

Apple valsa canker caused by the necrotrophs fungus Valsa mali has become a major disease in various areas of China.V.mali mainly infects the trunk,causing necrosis on apple bark and even the death of apple trees,which greatly limits apple production and fruit industry.At present,this disease prevention and treatment have not achieved desired goals,due to limited knowledge for the infection mechanism of V.mali,and particularly,few virulence factors have been identified thus far.In animals,plants and microorganisms,the CAP proteins affect a variety of biological processes,such as growth and immunity.However,the function of CAP proteins in phytopathogenic fungal has not been reported.Our previous study found that a CAP protein Vm PR1c of V.mali can cause necrosis in Nicotiana benthamiana,and it plays an important role in V.mali virulencce.However,the mechanism how Vm PR1c influences pathogen virulence is not clear.Targets identification of a certain protein is crucial for functional research,and this study aims to determine the host targets of Vm PR1c.This research will contribute to explore the infection mechanism of V.mali,and will lay a good foundation for the exploration of fungal CAP proteins.In this study,Yeast two-hybrid（Y₂H）was used to screen the interaction targets of Vm PR1c;Y₂H,Bimolecular fluorescence complementation（Bi FC）and Co-immunoprecipitation（Co-IP）techniques were used to validate the targets of Vm PR1c;the function of the Vm PR1c targets were analyzed using virus-induced gene silencing（VIGS）;the binding proteins of Vm PR1c targets in N.benthamiana were screened by mass spectrometry（MS）.The results obtained are as follows:1.Yeast two-hybrid screening revealed that Md VQ29 protein in the apple host is a potential target of Vm PR1c.VQ protein is a class of proteins containing a conserved VQ-motif,which mainly mediates transcriptional regulation and protein interaction.Md VQ29 protein has another homolog in apple（named Md VQ12）and three homologs in N.benthamiana（named Nb VQ1,Nb VQ2 and Nb VQ3,respectively）.Md VQ12,Nb VQ1,Nb VQ2 and Nb VQ3 were validated to interact with Vm PR1c,by Y₂H,Bi FC and Co-IP.2.Western blotting analysis showed that the molecular weight（MW）of Vm PR1c-Flag was around 200 k Da and 48 KDa,which is much larger than the predicted.It is speculated that,in addition to ubiquitination and glycosylation modifications,Vm PR1c may also be modified by SUMOylation.By adding SUMOylation inhibitor 2-D08 during transient expression of Vm PR1c,we found no significant changes for Vm PR1c MW by WB analysis,indicating that Vm PR1c may not be modified by SUMOylation.Previous study has found that Vm PR1c can cause weak necrosis of N.benthamiana.However,after silencing of Nb VQ1,Nb VQ2,and Nb VQ3 in N.benthamiana,Vm PR1c caused similar necrosis,indicating the Md VQ29 target have no influence on cell death-inducing activity of Vm PR1c.3.It is speculated that the target VQ may act by binding other proteins.To further study the interaction mechanism,the targets of Md VQ29 and Md VQ12 were screened by MS technology and 49 candidate target proteins in N.benthamiana were identified.Of which,Nb WRKR17 and Nbe IF4A were further validated to be the mutual targets of both Md VQ29and Md VQ12 using Bi FC.However,western blotting analysis showed that Md VQ29,Md VQ12 and Nb WRKR17 were all degraded.By adding inhibitors of different degradation pathways,we found that Md VQ29 and Md VQ12 were not degraded through lysosomal and proteasome pathway.The degradation of Nb WRKR17 could be effectively inhibited by PS-341,whereas,Nb WRKR17 degradation was more seriously after chloroquine treatment.Besides,when co-expressing Md VQ29 and Nb WRKR17（or Md VQ12 and Nb WRKR17）,the protein degradation was more severe than expression of Md VQ29,Md VQ12 alone.In summary,Vm PR1c interacts with host VQ protein Md VQ29 and Md VQ12,as well as the VQ homologs in N.benthamiana（Nb VQ1,Nb VQ2 and Nb VQ3）.Vm PR1c may not be modified by SUMOylation,and its interaction with Md VQ29 in N.benthamiana does not affect the cell death-inducing activity.Both Md VQ29 and Md VQ12 can interact with Nb WRKR17 and Nbe IF4A in N.benthamiana.Furthermore,Md VQ29 and Nb WRKR17,Md VQ12 and Nb WRKR17 can promote the degradation of each other.Nb WRKR17 may be degraded by proteasome pathway,while the degradation of Md VQ29 and Md VQ12 are not dependent on the lysosomal or proteasome pathway.