| Sheath blight,one of the three major diseases of rice,occurs in all rice producing areas of the world.Rhizoctonia solani Kühn,the pathogen of rice sheath blight,is a strong saprophytic fungus that destroys the structure of host cells by secreting cell wall degrading enzymes and toxinsn killing the host cells and providing nutrients for its growth,development,and expanding in the host.Previous studies have shown that OsPGIP1(Polygalacturonase-inhibiting protein 1 from Oryza sativa)could inhibit the activity of RsPG1(Polygalacturonases 1 from R.solani).Overexpression of OsPGIPl in rice could significantly increase its resistance to sheath blight,but few study has been reported on the molecular mechanism of this gene to increase rice resistance to this disease.In order to clarify the role of the gene in the resistance to rice sheath blight,eukaryotic expression,transient expression,site mutation,yeast double-hybrid and deletion mutation had been use to in this paper.The results were as follows:RsPGl gene was constructed into the eukaryotic expression vector pPIC9K and then transformed into Pichia pastoris GS115.After methanol induction,the the enzyme activity of fermentation supernatant reached 388.79 U/mL on the 4th day.The expression production was extracted and purified with a size of about 40 kDa,consistent with the predicted molecular weight of RsPG1.Enzyme activity of the expression production was the highest at 50℃ and pH 5.0.Organic solvents such as methanol,formaldehyde,ethanol,ethyl acetate and acetone had no significant effects on the activity of RsPG1,and short-time UV irradiation also had no effect on its activity of RsPG1.Which means that RsPG1 protein is relatively stable.Similarly,when OsPGIP1 gene was constructed into eukaryotic expression vector pPIC9K,also positive transformants were obtained,the expression product could not be extracted,and the fermentation supernatant,the cell fragment supernatant and the precipitate of GS115 had no inhibitory effect on enzyme activity of RsPG1.OsPGIPl and RsPG1 genes were constructed into transient expression vector pMD1-35S-GFP and transformed into Agrobacterium tumefaciens GV3101.When the solutions of GV3101 containing the vectors harbored the target genes were injected into tobacco leaves,the tobacco leaves with RsPG1 gene transient expression vector could produce water-stained lesions after 2 days,while the leaves injected with GV3101-OsPGIP1 or co-injected with GV3101-RsPG1/GV3101-OsPGIP1 had no change.The results indicated that the transient expression product of OsPGIP1 gene in tobacco could inhibit the activity of RsPG1.In order to identify the interaction sites of OsPGIPl and RsPG1,six amino acids with a frequency more than 0.5 were screened for site mutaitons through homology modeling and surface free energy analysis,and the mutants were named D60A,Q77A,R175A,R204A,R227A,and D269A,respectively.Yeast double hybrid showed that wild-type OsPGIPl and its 6 mutants could interact with RsPG1,indicating that single amino acid mutation in these sites did not affect the interaction between OsPGIP1 and RsPG1.The six mutants were co-expressed with RsPG1 in the tobacco leaves,all the co-expression leaves had no water-stained lesions,indicating that these sites had nothing to do with OsPGIP1’s activity to inhibit RsPG1.PGIP is a kind of cell wall binding proteins.In order to define the key regions for extracellular transport of OsPGIP1,gradual fragment deletion on its LRR region was performed,and it was found that OsPGIP1Δ09 OsPGIP1Δ8-9,OsPGIP1Δ7-9,OsPGIP1Δ6-9,OsPGIP1Δ5-9,OsPGIP1Δ4-9,OsPGIP1Δ3-9,OsPGIP1Δ2-9 and OsPGIP1Δ1-9,which were partial or total delection of the LRR region of OsPGIP1,had no effect on OsPGIP1 extracellular transport.Signal peptide secretion tests showed that signal peptide of OsPGIP1 had strong out-secretion function.This may be the reason why OsPGIPl mutants are able to transport extracellular by losing the LRR region but retaining the N-terminus,C-terminus and signal peptide regions. |
