The Screening And Verification Of The Rhizoctonia.solani Effectors

Effector is a kind of protein synthesized by the plant pathogen and secreted into the host so that achieve the purpose to control and interfere the host to enhance its infection.It Include the traditional concept of non-toxic protein(Avr),elicitor(elicitor),toxic protein and some degradation enzyme etc.The effector of fungal pathogen secreted into host by the endoplasmic reticulum-gorky protein secreting system.Its differente from III secretion system of bacterial.However,the main characteristics of the fungal pathogen effector is that there is about 15-30 hydrophobic amino acids at N-terminal of effector.Our lab sequencing and analysis the rice sheath blight fungus with the ILLUMINA platform.after the primary assembly and analysis.we found that the AG1-IA code 965 secreted proteins(accounting for 9.17%of the total protein),after homologous BLAST to the database,we get the candidate effectors.This study conducted the screening and preliminary verification of candidate effector and we get the following conclusion.1.The prokaryotical expression of effector:We design primers with the CDS sequence of effector IA008560 as template.After reverse transcriptase and sanger verification,we constructed the prokaryotic expression vector pGEX-4t-1-008560 and transfer into the competent escherichia coli(DE3),At last,we obtained the protein.2.The cell and molecular detection of host PCD triggered by effector:Inductived the prokaryotic expression of effctor 008560.after ultrasonic disruption,we get the protein and infect the rice leaf with it.We found the leaf show yellowing 48-72h later.After the DAB staining and taking off green we found there is brown spot on the leaf.It demonstrate the level of hydrogen peroxide become getting up.Extracting the DNA of leaf,it shows ladder degradation in the electrophoresis.DNA electrophoresis results showed that rice is gradient degradation,description the rice leaf cells chromosome DNA degradation in nucleosome.At the same time,we infect rice leaf with puring protein,it show similar allergic reactions too.Incubating the rice protoplast with puring effector protein,we found that the rice protoplast with incubation deads by the time,only a small number of cells survival and the control group almost no change after 5 h;Continuous sampling and with DAPI and H2DCFDA respectively for protoplast dyeing found its irregular nucleus and gradually apart;The cytoplasm peroxide level than the control group.Extraction of protoplast the degradation of DNA found a similar situation,shows that the effect of protein caused a programmed death(PCD)of rice cells,it shows the result as same as the infected leaf.3.The screening and preliminary positioning of effect factor of IA-11269:In this way,we respectively with the candidate effect factor sequence primers for template design and construction of prokaryotic expression vector,will induce expression of crude protein in the above method into the rice leaf,look at whether PCD blade organization phenomenon,select the new effect factor IA-11269,fluorescent protein carrier and build fusion to get rice protoplast found its location and the cell membrane.Database than found in IA-11269 lfi-6-16 domain,the domain belongs to prothymosin superfamily.While prothymosin superfamily is often associated with insect metamorphosis.In SMART description of the domain is interferon induced proteins(IFI6/IFI27),6/27 IFI27 promote cell death,at the same time by controlling the release of mitochondrial cytochrome C and adjust the BAX and protease activity to adjust interferons(door caused by cell apoptosis.4.The detection of host specificity with different hostWhen the effector inducing allergic reaction,it shows host specialization.This experiment with the effect factor of IA-11269 prokaryotic expression protein infect different rice varieties and observe its hypersensitivity,found the different of the effect factors of the sensitivity of different rice varieties.5.The detection of the effectors from different isolates protein:We amplifying the effector sequences from 10 isolates in t10 provinces and cities.after comparing the amino acid change was observed.Among them,we found three SNPS loci,two for the C/T,A type of A/G.After translation comparison found that the three SNPS loci are synonymous mutations.Does not affect its amino acid sequence and function.And the effect of amplification factor in separation of FY sequence of a period of 12 bases of insert.The inserted into the sequence effect on protein function remains to be further research.