Screening And Mechanism Of Mycoparasite Pythium Effectors In Inhibiting Bacteria Or Inducing Cell Death In Nicotiana Benthamiana

Pythium,genus of the family Pythiaceae(phylum Oomycota,kingdom Stramenopiles).Most members in the genus Pythium are plant pathogens,whereas Pythium oligandrum and Pythium periplocum distinguishes themselves from the pathogenic species by its ability to protect plants from biotic stresses in addition to promoting plant growth.They are non-pathogenic soil-inhabiting oomycete,colonize the root ecosystem of many crop species.The beneficial effects of P.oligandrum on plants are the result of the synergistic action of several mechanisms,including antagonism against an array of soilbome pathogens,plant growth promotion through the production of auxin precursors and plant-induced long-term resistance.So far,the research has found that the main biocontrol mechanisms of Pythium oligandrum are:the use of mycoparasitism,secretion of antimicrobial compounds,nutritional competition,promote plant growth,to trigger plant immunity through its own protein components.In recent years,some Pathogen-associated molecular patterns and apoplast effectors that can be recognized by Pattern-recognition receptors on the surface of host plant cells and trigger plant immune responses are also identified.However,as mycoparasite Pythium with outstanding biocontrol activity,do Pythium oligandrum and Pythium periplocum have potential antimicrobial activity by secreting effector proteins to suppress pathogen invasion and inducing plant resistance?Not sure for now.In this study,we conducted a screening of the effectors of Pythium oligandrum and Pythium periplocum:through the E.coli induced expression system,a series of effectors that can inhibit the growth of E.coli BL21 and have potential antibacterial activity were obtained;took advantage of transient expression system in N.benthaminany a number of apoplast effectors that can induce plant cell death and resistance were identied.The main research contents are as follows:Among a large number of oomycete effectors,only a small number of effectors interact with plants,and the functions of other effectors are still unknown.In addition to interacting with plants,can some of the effectors secreted by mycoparasite Pythium inhibited the growth of plant-associated bacteria to promote their own colonization?In the process of interaction with filamentous pathogens,Pythium oligandrum will produce a haustorium-like structure to invade pathogens.This phenomenon is similar to pathogenic oomycetes that secrete effectors into host plant cells by generating haustorium to inhibiting host immunity.Therefore,we used bioinformatics analysis to identify the effectors of Pythium oligandrum and Pythium periplocum,cloned genes and constructed them into pBAD vectors.Through induced expression of arabinose,we identified five effectors that can inhibit the growth of E.coli BL21.In addition,none of these five effectors can cause the cell death of N.benthamiana.Through further bioinformatics analysis,we found that PoAvhl9 has a repeating tandem TSP1 domain.Previous studies have shown that this domain is related to adhesion in oomycetes,which provides a mechanism for subsequent research.From a new perspective,this study explored whether oomycete effectors can play a role in the interaction with plant-associated bacteria.With the help of the E.coli induced expression system,a number of effectors with potential antibacterial activity were identified,Which broadens the biological function of effectors,provides ideas for future research on the interaction between oomycetes and microbes in the plant microenvironment,and also provides a theoretical basis for the development of noval green fungicides and the application of green prevention and control technologies to prevent and control diseases.The genomes of Pythium oligandrum and Pythium periplocum encode a large number of apoplast effectors.In order to screen for potential resistance-inducing proteins,this study combined with the literature report selected the NLP(Nep1-like proteins)family,the Elicitin family and CBEL(Cellulose-binding elicitor lectin)family as candidates.First,we conducted bioinformatics analysis on three types of apoplast effector families,and identified 27 NLP,116 Elicitin,and 50 CBEL protein genes,respectively.Next,we used the transient expression system mediated by Agrobacterium to use the induction of N.benthaminan cell death as indicator.We identified 7 apoplast effectors that can trigger cell death,6 effectors of which belong to NLP family,1 belongs to the Elicitin family.This study provided powerful support for the development and application of noval induce resistance technologies based on mycoparasite Pythium by screening immune proteins and analyzing the basic molecules mechanism.