Study On PAMP Activity Zone And Site Of 45th Family Glycoside Hydrolytic Enzyme In Rhizoctonia Solani

Corn plays an important role in China's national economy and agricultural production.It is an important grain and feed crop and important industrial raw material in China.Corn sheath blight has become one of the main disease of production.Corn has occurred throughout the country and make influences in the production and quality of agricultural production of China.The plant cell wall is a natural barrier against external pathogen invasion.Plant pathogenic fungi and bacteria can produce cell wall degrading enzymes,as in the plant cells and cells spread between the main pathogenic factors,such as cellulase and pectinase.Recently,more attention has been paid to cell wall degrading enzymes which play a role in fungal-plant interaction mechanisms.PAMP molecules are some conserved molecules that exist on the surface of pathogenic microorganisms and we can find in microorganisms.Pattern recognition receptors identify pathogens by activating signal pathways and induced defense response to limit the invasion of pathogens.Previous studies have shown that R.solani AG-1-IA fusion group ?-1,4-endo-cellulase EG1 is a PAMP molecule which has the activity of PAMP.It's PAMP activity and catalytic activity are independent of each other.We used a series of experiments to further determine the areas and specific sites that function.The wild type of EG1 ois named WT.Mutation of Aspartic acid(Asp)at position 32 of the EG1 amino acid sequence to alanine(Ala),resulting in the loss of its catalytic activity and the name D32 A.In order to determine the region where EG1 exerts PAMP activity,we mutated six relatively conserved regions of EG1.The results showed that the wild type WT mutant zone C6 still has a high catalytic activity and could induce the necrosis of maize and tobacco leaves,but could not induce the excessive expression of the defense response gene.However,the mutant D32 A mutant zone C6 almost no catalytic activity and could neither induce the necrosis of maize and tobacco leaves nor induced overexpression of defense response gene.Therefore,we guess that the PAMP active region of EG1 is present in the conserved region C6.We used the PVX expression system to experiment to confirm our guess.The signal peptide sequence was inserted into the 5 ' by PCR amplification,and the fragment was inserted into pGR106 empty vector to construct the PVX expression vector while Agrobacterium tumefaciens as a control(CK).GV3101 competent cells were transformed by freeze-thawing method.As a result,the WT-C6 could produce tobacco leaf lesion,while D32A-C6 could not.For the different manifestations of WT-C6 and D32A-C6,we consider it to be the effect of degradation of cell wall cellulose.These results demonstrate that the C6 conserved region(sequence GCNWRFDWF)is a key region for EG1 to exhibit PAMP activity.In order to further explore the specific activity site of EG1,we mutated all the amino acids in the C6 region and obtained the corresponding mutant engineering bacteria and protein.After adjusting the concentration of pure enzyme inoculated with maize,we fond only RA can not cause Corn leaves necrosis.To adjust the appropriate concentration of RA inoculated with corn and tobacco leaves while determine the effect of RA inoculation on the production of reactive oxygen species in maize and tobacco leaves.To determine the effect of RA inoculation on the expression of defense response gene.It was found that RA did not cause necrosis of corn and tobacco leaves,nor did it produce a large amount of reactive oxygen species,and did not cause excessive expression of defense response genes.The recombinant plasmid pGR106/pd32a-ra was not expressed by the expression of d32a-ra by PVX expression vector.These experimental results initially identified the specific activity sites of EG1.The study confirmed the key segment when R.solani ?-1,4-endo-cellulase EG1 exert PAMP activity.At the same time,the specific activity sites were explored.