| Phytophthora blight of pepper caused by Phytophthora capsici.is one of the main diseases of pepper and other vegetable.It has more than 20 hosts,including eggplant,cucumber,tomato,which is widely distributed throughout the world and is a devastating vegetable disease.Moreover,it can infect various parts such as leaves and stems,causing soft rot and lodging.With the latest research,Phytophthora capsici.secretes a large number of effectors into the apoplast and cytoplasm of host plants and destroys the defense system to promote the infection.Protein to protein interactions are the heart of almost biological processes in a cell.This study explores the interaction between P.capsici.effector PcRxLR121504 and the host plant,screened and identified the interaction protein of PcRxLR121504,and demonstrates the pathogenic pathway of PcRxLR121504,finds key interaction targets and provides a theoretical basis for prevention the infestation of Phytophthora capsici.The main research are as follows:1.Find the interacting protein of PcRxLR121504 by co-immunoprecipitation and tandem mass spectrometry.PcRxLR121504 was constructed to the pBIN-GFP vector and transgenic Agrobacterium was expressed in Nicotiana benthamiana for 48-72h.Sampling and lysing are performed according to the experimental steps of co-IP.After the sample is loaded and run,the band runs to the separation gel to start timing,and then runs for ten minutes to stop the electrophoresis.Decoloration,cut rubber strips and send samples for testing.Through mass spectrometry analysis of peptides of interaction proteins drawn by effector molecules,and search for disease resistance pathways on the KEGG website.We screened 6 more meaningfμLtarget proteins,RPM1 interacting protein 4(RIN4),Serine/Threonine protein kinase(STPK),pathogen-related protein(PR1),transcription factors VIP1,MKK2,and MKK5 to further research.2.Screening the interaction protein of PcRxLR121504 in pepper by yeast two-hybrid technology(Y2H):First,the pepper cDNA library synthesized by company was transferred to yeast strain Y187,and the quality of the yeast library was identified.The titer was 2.39X10~6CFU/m L,and the library capacity was 3.59X10~7CFU.PGBKT7-121504 and PGBT9-121504 bait vectors were constructed,and PGBKT7-121504 was found to be more toxic to yeast growth and breeding by coating a defective Trp-deficient yeast medium,while PGBT9-121504 was less toxic to yeast.Therefore,PGBT9-121504 can be used for the next step of yeast two-hybrid technology to screen the interaction proteins in the pepper.The addition of 20 mmol of Aba can inhibit the self-activation of PGBT9-121504.After decoy fusion of bait protein PGBT9-121504 with the library,9 suspected interaction proteins were obtained,which are COP9 signal complex subunit 5b-like and peptidyl-prolyl cis-trans isomerase Cyp20-1,Fructose diphosphate aldolase cytoplasmic isoenzyme(FBA),catalase(CAT),lysine-rich arabinogalactan protein 19(AGP19),capsaicin defensin c-AMP-D1-like,VQ9 Motif,VOZ1 transcription factor,MOB1A protein kinase.At the same time,the interaction protein screened by co-immunoprecipitation and tandem mass spectrometry analysis and yeast two-hybrid were subjected to yeast two-hybrid back-to-back verification,which proved that PcRxLR121504 interacted with STPK,CAT and MOB1A.3.Co-immunoprecipitation(co-IP)indicated that PcRxLR121504 interacted with STPK,MKK5.The plant expression recombinant vectors PcRxLR121504,STPK,MKK2,MKK5,RIN4,,VIP1,PR1,CAT,and MOB1A were transiently expressed in the leaves of N.benthamiana for 48-72 h,and the total protein of the leaves was extracted for co-IP.4.The Bimolecular Fluorescence Complementation Experiment(BIFC)proved that PcRxLR121504 interacted with STPK,MKK5,CAT,and MOB1A in vivo,respectively.The constructed expression vector transgenic Agrobacterium was expressed into N.benthamiana for 48-72 h.5.In order to investigate whether there are upstream and downstream or synergistic effects between interacting proteins,we constructed STPK into bait vectors PGBKT7 and MKK2、MKK5、RIN4、VIP1、PR1 were constructed into prey vectors PGADT7 for yeast two-hybrid experiments.It was found that STPK and RIN4 are interacted,and we infer that there is an upstream and downstream relationship between the two interaction proteins or a key target protein on the pathogenic pathway.Through the above experimental studies,most of the suspected interaction proteins of the P.capsici.effector PcRxLR121504 that we screened are mostly related to the disease resistance pathway or directly related to the function of the infectious molecules.Investigate how these suspected proteins interact with effector during the infection of P.capsici.,which can explain the function of PcRxLR121504 that can cause necrosis of host plants and suppress necrosis caused by elicitor INF1.The mechanism provides a key theoretical basis for scientific control of P.capsici. |
