Study On The Interaction Mechanism Between Two Seereted Proteins Of Fusarium Oxysporum And SlPR1 Protein In Tomato

Phytopathogenic fungi secrete many enzymes,such as cell wall degrading enzymes,glycosylhydrolases,proteases,peptidases and ribonucleases,which play an important role in the pathogenicity during the infection.Host plants would active the plant defense responses to improve the resistance to pathogens.Fusarium oxysporum is an important wold-widely distributed soil-borne fungal pathogen,and has a broad host range.F.oxysporum infects from host root and colonized in the plant vascular system,which causes the Fusarium wilt disease and has a great threat to agricultural production.In this study,we have identified two proteins,FoRnt2 and FoAPY1,from the F.oxysporum secretome,and then the biological functions of those two secreted proteins were analyzed.In addition,we used the yeast two-hybrid library screening approach to find the Sl PR1 protein from tomato that associated with the secreted FoRnt2 proteins.And the interaction mechanism between the F.oxysporum and the host plnats was further analyzed.The mainly results obtained in this study are as follows:(1)FoRnt2 contains the signal peptides at its N-terminus,and could be secreted from F.oxysporum.FoRnt2 belongs to the ribonuclease T2 family and exhibited ribonuclease activity on the tomato tatol RNA in vitro.Further study indicated FoRnt2 performed its enzyme activity depending on the active site residues H80 and H142.Deletion of the FoRnt2 gene significantly decreased fungal virulence on tomato,however,the mycelial growth and conidial production were not affected.Furthermore,the over expression of FoRnt2 in tomato significantly enhanced plant susceptibility to pathogens and promoted the infection of pathogens.(2)FoAPY1 was identified to be secreted from F.oxysporum depending on its N-terminal signal peptide of the protein.As the member of the peptidase M28 family,FoAPY1 could hydrolyze the specific substrate Lys-p NA in vitro,which indicated its peptidase activity.Furthermore,the FoAYP1 gene knockout strain displayed the reduced virulence to tomato plants,but its mycelial growth and conidia production were not affected.Moreover,the level of resistance related protein was decreased when overexpression FoAYP1 in tomato,leading to tomato susceptibility upon F.oxysporum infection.(3)Using the yeast two-hybrid(Y2H)library screening system,a tomato Sl PR1 was identified to interact with FoRnt2.Their interaction between Sl PR1 and FoRnt2 was further confirmed by Y2 H,bimolecular fluorescence complementation(Bi Fc)and pull-down assays.Further studies showed that amino acid residue E98 and H117 of Sl PR1 play an important role in the interaction with FoRnt2.Furthermore,the purified Sl PR1 protein can inhibit the accumulation of FoRnt2 protein in planta and can degrade FoRnt2 in vitro.Similarly,we found that the Sl PR1 has the ability to degrade the FoAPY1 in vitro.In addition,Sl PR1 overexpression transgenic tomato enhances the resistance to F.oxysporum and inhibits the pathogens infection.These results suggested Sl PR1 could promote the protease activity to pathogen secreted virulence protein to enhance tomato resistance.